Cisplatin-Induced Nucleosome and RNA Polymerase II Modifications Mediate Cellular
Response to the Drug
by
Dong Wang
B. S. Chemistry
Peking University, China, 1998
SUBMITTED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN BIOLOGICAL CHEMISTRY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 2004
C Massachusetts Institute of Technology, 2004
All rights reserved
7
Signature of Author:
(
-.,,
Departmfnt of hemistry
-- Augus',4004
Certified By:
_
__ __~~~~~~~~, __
-~
Stephen J.Lippa&
Arthur Amos Noyes Professor of Chemistry
Thesis Supervisor
j -
Accepted by:
MASSACHUSETTS INSTITUTE
OF TECHNOLOGY
Roberi W. Field
Chairman, Departmental Committee on Graduate Studies
SEP 1BRARIE5
2004
LIBRARIES
'ARCHIVES
A-
..
-
_'
2
This doctoral thesis has been examined by a committee of the Department of
Chemistry as follows:
Alexander M. Klibanov
Committee Chairman
Professor of Chemistry and Bioengineering
V
Sepher. Lippard
Arthur Amos Noyes Professor of Chemistry
Thesis Supervisor
Catherine L. Drennan
Cecil and Ida Green Career Development Assistant Professor of Chemistry
3
Cisplatin-Induced Nucleosome and RNA Polymerase II Modifications Mediate
Cellular Response to the Drug
by
Dong Wang
Submitted to the Department of Chemistry on August 18, 2004, in partial
Fulfillment of the requirements for the Degree of Doctor of Philosophy
Abstract
Chapter 1. Cellular Processing of Platinum Anti-Cancer Drugs - Identifying
Pathways for Chemogenotherapeutic Drug Design
Cisplatin, carboplatin, and oxaliplatin are widely used in cancer
chemotherapy. Platinum-DNA adducts, formed following uptake of the drug
into the nucleus of cells, activate several cellular processes that mediate the
cytotoxicity of these platinum drugs. This review focuses on recently discovered
cellular pathways that are activated in response to cisplatin, including those
involved in regulating drug uptake, the signaling of DNA damage, cell cycle
checkpoints and arrest, DNA repair, and cell death. Such knowledge of the
cellular processing of cisplatin adducts with DNA provides valuable clues for the
rational design of more efficient platinum-based drugs as well as the
development of new therapeutic strategies.
Chapter 2. Nucleotide
Excision Repair from a Site-Specifically
Platinum-
Modified Nucleosome
Nucleotide excision repair is a major cellular defense mechanism against
the toxic effects of the anticancer drug cisplatin and other platinum based
chemotherapeutic agents. In this study, mononucleosomes were prepared
containing either a d(GpG) or a d(GpTpG) intrastrand cross-link. Comparison of
the extent of repair by mammalian cell extracts of free and nucleosomal DNA
containing the same platinum-DNA adduct reveals that the nucleosome
significantly inhibits nucleotide excision repair. The effects of post-translational
modification of histones on excision of platinum damage from nucleosomes were
investigated by comparing native and recombinant nucleosomes containing the
same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is
- 2-fold higher than the level observed with recombinant material. The in vitro
system established in this study will facilitate the investigation of platinum-DNA
damage by DNA repair processes and help elucidate the role of specific post-
4
translational modification in NER of platinum-DNA
physiologically relevant nucleosome level.
adducts
at the
Chapter 3. Nucleotide Excision Repair of Site-Specifically Platinum-Modified
Tetrasomes
The nucleosome, the basic structural unit of chromatin, is composed of a
histone (H3/H4)2 tetramer flanked by two H2A/H2B dimers, around which is
wrapped 146 base pairs (bp) of DNA. The (H3/H4) 2 tetramer plays a central role
in the structural integrity and positioning of the nucleosome core particle. Sitespecifically platinated tetrasomes were prepared to investigate the modulating
effects of histone tetramers on excision repair of cisplatin-intrastrand cross-links.
In addition, {Pt(DACH))2+-modified tetrasome was prepared to investigate the
effect of spectator ligands on excision repair of platinated tetrasomes. The NER
results reveal that the (H3/H4)2 tetramer is sufficient to block excision of both
cisplatin-DNA and Pt(DACH))2+-DNA adduct in vitro. The efficiency of excision
of cisplatin-modified tetrasomal DNA is about half (53%) that of the
{Pt(DACH)}2+-modified tetrasome.
Chapter 4. Cisplatin Adducts Change the Rotational Positioning of DNA on
the Nucleosome
Understanding the interaction of cisplatin-DNA adducts with
nucleosomes and the effect of cisplatin-DNA adduct formation on nucleosome
structure is important for improving the cytotoxicity of this drug. Here we use
hydroxyl radical and DNAse I footprinting analysis to characterize sitespecifically cisplatin-modified nucleosomes. Preexisting cisplatin-DNA adducts
change the rotational setting of DNA with respect to the histone core particle
surface during nucleosome formation.
Chapter 5. Cisplatin-Induced Post-Translational Modification of Histones H3
and H4
The anticancer drug cisplatin kills cells by damaging DNA and inducing
apoptosis. Understanding the detailed mechanisms by which cancer cells
respond to cisplatin has the potential to improve substantially platinum-based
therapy. Post-translational modification of histones alters chromatin structure,
facilitating the binding of nuclear factors that mediate DNA repair, transcription
and other processes. In the present study, we have investigated the effects of
cisplatin treatment on histone post-translational modification in cancer cells. We
discovered that specific phosphorylation of histone H3 at Ser 10, mediated by the
p38 MAPK pathway, is induced in response to cisplatin treatment. In addition,
hyperacetylation of histone H4 is caused by the drug treatment. These findings
reveal a link between cisplatin administration and chromosomal structural
alterations, providing mechanistic information about how cells respond to
platinum-induced stress.
Chapter 6. The Modulating Effect of DNA Methylation on the Cellular
Processing of Cisplatin-Damaged DNA
Epigenomic regulation of genes is essential for proper cellular function.
One major way of conveying epigenetic information in mammalian cells is DNA
methylation, with 5-methylcytosine within the CpG dinucleotide being the major
component. DNA methylation has profound effects on the mammalian genome.
Cancer cells frequently exhibit altered genomic methylation patterns, mostly
exhibiting global hypomethylation with region-specific hypermethylated
sequences that occur within the promoter region of certain genes in these cells.
Here we investigate the role of DNA methylation on the processing of cisplatinmodified DNA, specifically, the role of DNA methylation in the formation of
DNA-platinum adducts as well as on protein-DNA interactions and on the repair
of platinum damage. The rates and extent of platination of DNA probes
containing 5-methylcytosine residues adjacent to the platinum binding site were
compared to similar properties of non-methylated DNA. The effects of DNA
methylation on the interaction between a protein and platinated DNA were
investigated by studying HMGB1 domA binding with methylated and nonmethylated probes. Finally, the effect of DNA-methylation on the NER of
cisplatin-damaged DNA was examined with 195mer substrates containing sitespecific cisplatin-DNA cross-links with flanking or base-paired 5-methylcytosine
residues.
Chapter
7.
Transcription-Coupled
and
DNA
Damage-Dependent
Ubiquitination of RNA Polymerase II In Vitro
Transcription-coupled repair (TCR) is essential for the rapid, preferential
removal of DNA damage in active genes. The large subunit of RNA polymerase
(Pol) II is ubiquitinated in cells following UV-irradiation or cisplatin treatment,
which induces DNA damage preferentially repaired by TCR. Several human
mutations, such as Cockayne syndrome CSA and CSB, are defective in TCR and
incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate, for
the first time, a correlation between ubiquitination of RNA Pol II and arrest of
transcription in vitro. Ubiquitination of Pol II is significantly induced by alphaamanitin, an amatoxin which blocks Pol II elongation and causes its degradation
in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin
6
adducts that arrest transcription. Stimulation of ubiquitination requires the
addition of template DNA, and it does not occur in the presence of an antibody
to the general transcription factor TFIIB,indicating the transcription dependence
of the reaction. We propose that components of the reaction recognize
elongating polymerase-DNA complexes arrested by alpha-amanitin or cisplatin
lesions, triggering ubiquitination.
In the second part, site-specifically platinated-DNA probes for in vitro
transcription studies were synthesized by both primer-extension and enzymatic
ligation methods. In vitro study revealed that the presence of cisplatin-DNA
adducts on the template strand significantly block the RNA polymerase.
Chapter 8. Synthesis and Characterization of Platinated Dumbbell DNAs for
In Vitro Transcription and Repair Studies
Site-specifically platinated double-stranded DNA probes were prepared
as valuable templates for in vitro studies of cellular processing of cisplatin,
indicating the formation protein-DNA complexes, transcription, and repair.
However, the blunt-ends of double-stranded probes permit non-specific
transcription, protein-binding, and exonuclease degradation. Here we report the
synthesis of site-specifically platinated dumbbell DNA probes from a pair of
hairpins with 3' overhangs and three or four central fragments using enzymatic
ligation. Formation of the modified dumbbell DNA probes were confirmed by
exonuclease and restriction enzyme digestion assays. The dumbbell DNA probes
were then used as templates for in vitro nucleotide excision repair and
transcription studies.
Appendix Al. The Interaction of Human SWI/SNF with Platinated
Nucleosomes and Its Effect on NER
In eukaryotic cells, DNA is packaged into chromatin. Two major classes of
factors have been identified to alter DNA accessibility in chromatin: histone
modified complexes and ATP-dependent remodeling complexes. SWI/SNF, one
of best characterized members of the latter class, has been linked to nucleotide
excision repair stimulation. The stimulatory effect of SWI/SNF on NER depends
upon the nature of the DNA lesion. SWI/SNF stimulates the excision of AAFadducts and the (6-4) photoproduct from nucleosomal DNA, whereas it has a
negligible effect on the repair of TT dimers. These differential effects of SWI/SNF
on the repair of UV photoproducts and AAF adducts promoted our interest in
investigating the effect of SWI/SNF complexes on repair of cisplatin damage
within the nucleosome. In this section, the effect of SWI/SNF on the repair of
cisplatin-DNA adducts in a mononucleosome will be addressed. A moderate
7
stimulatory effect of SWI/SNF on the repair from cisplatin-DNA adducts was
observed.
Thesis Supervisor: Stephen J. Lippard
Title: Arthur Amos Noyes Professor of Chemistry
8
For my mother, my farther, and my sister,
who taught me to love, to care,
and many other things.
And for Francis Harry Compton Crick (1916-2004),
who revealed such a perfect structure,
and inspired millions of people.
9
Acknowledgments
First and foremost I must acknowledge my thesis advisor, Stephen J.
Lippard. His great insights and guidance throughout the course of this work will
be always appreciated. His constant enthusiasm and dedication for science
always inspire me. I really appreciate his unconditional support and scientific
freedom he gave to me. His excellent style for doing science has transplanted
into my mind as a great "template."
I thank Prof. Philip A. Sharp and Dr. Keng-Boon Lee for their close
collaboration on the Pol II ubiquitination project. I really appreciate those
insightful discussions with Phil regarding the present and future of science,
which I am certainly touched by his amazing love for science and his friendly
personality. I also thank K.B. who taught me a lot of hardcore biological
techniques. She is a wonderful and quiet person. I wish her and her family well.
Prof. Aziz Sancar, Dr. Ryujiro Hara, and Dr. J. T. Reardon from Univ of
North Carolina gave me tremendous help on the repair project. I went to Aziz's
lab for about one month to learn the techniques of nucleosome assembly and
purification. I really enjoyed my stay there. Aziz's sharp insight and stimulating
discussion have benefited me a lot. I really missed those wonderful afternoons at
his office and of course, the world cup of soccer.
I also thank Prof. Thomas D. Tullius and Mr. Qun Wang (Boston Univ.)
for the collaboration of footprinting project; Dr. Hua-Ying Fan, Prof. Robert E.
Kingston (Harvard Medical School), Prof. Danny Reinberg (Univ. of Medicine
and Dentistry of New Jersey), and Prof. Karolin Luger (Colorado State Univ) for
providing me materials for experiment or technical help. I have also benefited a
lot from several members of MIT faculty. I thank my thesis chair Prof. Alexander
M. Klibanov, Prof. Catherine L. Drennan, Prof. Alice Y. Ting, Prof. John
Essigmann, Prof. JoAnne Stubbe, Prof. Jianzhu Chen, Prof. Alexander Rich, and
Dr. Shuguang Zhang for thoughtful discussion of my project and general
scientific matters.
I have been extremely lucky to involve in such an excellent research
group, which I really enjoyed. I appreciated their help and contribution. In
particular, I thank Dr. Christa Kneip, Dr. Qing He, Dr. Sudhakar Marla, Dr. Yuji
Mikata, and Ms. Amanda Yarnell who taught me experimental skills in my early
days in the lab. I also thank Dr. Chuan He, Dr. Min Wei, Dr. Christiana Xin
Zhang, Dr. Juan Carlos Mareque-Rivas, Dr. Edna Ambundo, Dr. Edit Y. Tshuva,
Dr. Christopher J. Chang, Dr. Seth Cohen, Dr. Elizabeth R. Jamieson, Olga
Burenkova, Alyssia Dangel, Dr. Jane Kuzelka, Dr. Douglas A. Whittington, Dr.
Daniel Kopp for their support and friendship. As for current members, I
especially thank Ms. Katie Barnes for her help in tissue culture and her patience
10
to read most of my manuscripts. I also thank Andrew Danford, Dr. Christian
Goldsmith and Andrew Tennyson for proofreading some chapters of my thesis.
In addition, I thank Yongwon Jung, Matthew H. Sazinsky, Sungho Yoon,
Carolyn C. Woodroofe, Lisa L. Chatwood, Elizabeth Nolan, Dr. Elisabeth R.
Cadieux, Dr. Jessica Blazyk, Dr. Dong Xu, Evan Guggenheim, Mi-Hee Lim, Dr.
Sumitra Mukhopadhyay, Richard C. Girardi, Emily C. Carson, Dr. Laurance
Beauvais, Amy Kelly, Leslie Murray, Michael McCormick, Dr. Jeremy Kodanko,
Dr. Rayane Moreira, Rachel Niehuus, Sonya Tang, Annie Won, Cindy Yuan,
Caroline Saouma, Pamela Chang, and Cindy Liang for their friendship. Finally,
but not least, I want thank my three great UROPs, Andrew Danford, Gita Singh,
and Tim Davenport, for their excellent work, especially for Andrew. We have
been worked together for over three years. We shared a lot of ups and downs
during the progress of project and we are happy to get through and overcome
those difficulties. We also have a lot of interesting talks beyond chemistry. He is
a really great and fun person to know. I hope for his great success on whatever
he pursues.
On a more personal level, I thank my Mom, Dad, my dear sister for their
support, encouragement, and love throughout the years. Also special thanks go
to Q.C. for her support, love and understanding.
11
Table of Contents
Abstract ................................ ...........................................................................................................
3
D edication ......................................................................................................................................
8
Acknowledgments.................................................................................................
9
Table of Contents .................................................................
11
List of Tables ................................................................
21
List of Figures ................................................................
22
Chapter 1. Cellular Processing of Platinum Anti-CancerDrugs -Identifying
27
Pathways for Chemogenotherapeutic Drug Design ...........................................................
Introduction
................................................................
28
Mechanisms of Cisplatin Transport into Cells ................................................................ 30
Cisplatin-DNA Damage Recognition Proteins - A Postulated Key Role for HMGB1 .....32
Cell Cycle Checkpoints and Arrest ................................................................
36
p53 and p73 Pathways .................................................................
37
MAPK and Other Related Pathways ................................................................
42
Akt Pathway ................................................................
42
c-Abland Bcr-Abl.
43
...............................................................
MAPK/JNK/ERK Pathways ................................................................
DNA Repair Pathways ................................................................
46
50
Cell Death Pathways: Apoptosis and Necrosis ................................................................55
Cisplatin and Nucleosomes................................................................
57
Future Directions................................................................
60
12
A cknowledgm ents .......................................................................................................... 61
References .....................................................
61
Chapter 2. Nucleotide Excision Repair from a Site-Specifically
Platinum-Modified Nucleosome ......................................................
101
A bbreviations ................................................................................................................ 102
Introduction
......................................................
103
Materials and Methods ......................................................
105
Materials
......................................................
105
105
Synthesis of Platinated Oligonucleotides .........................................................................
Characterization of Oligonucleotides ......................................................
106
Preparation of Site-SpecificallyModified Platinum-DNA Probes ...............................
106
Cell-Free Extract Preparation ......................................................
107
107
Native Histone Octamer Preparation ...............................................................................
107
Expression and Purification of Recombinant Histone Proteins ....................................
Nucleosome Reconstitution and Purification ......................................................
107
Excision Repair Assay ......................................................
108
Results .....................................................
109
109
Synthesis and Purification of DNA 199GG-Pt and 199GTG-PtProbes .......................
Reconstitution and Purification of Nucleosomes ....................................................... 109
Excision Assay on Nucleosomal DNA......................................................
Discussion .....................................................
110
111
Comparison of NER from Free vs Nucleosomal DNA ...................................................112
13
Comparison of NER from Nucleosomes with Native vs Recombinant Histones ........114
Conclusion ..............................................................................................................
117
Acknowledgment......................................................................................................................117
References ...........................................................
118
Chapter 3. Nucleotide Excision Repair of Site-Specifically
Platinum-Modified Tetrasomes ............................................................
129
Introduction .............................................................................................................130
Method and Materials ............................................................
Materials ...............................................................................................................
132
132
Synthesis of Platinated Oligonucleotides .........................................................................
132
Preparation of Site-SpecificallyModified Platinum-DNA Probes .................................
132
Cell-Free Extract Preparation ............................................................
133
Expression and Purification of Recombinant Histone Proteins ...................................
133
Reconstitution and Purification of Tetrasome and Nucleosome ..................................
134
Excision Repair Assay .........................................................................................................
134
Results and Discussion............................................................
135
Acknowledgment
............................................................
139
References ...........................................................
139
Chapter 4. Cisplatin Adducts Changethe Rotational Positioning of
DNA on the Nucleosome ............................................................
150
Introduction ...............................................................................................................................
151
Materials and Method ..................................
152
14
Materials ................................................................................................................. 152
Synthesis of Platinated Oligonucleotides .........................................................
153
Preparation of 5'-End Radiolabeled Site-SpecificallyPlatinum-Modified
D NA Probes .......................................................................................................
153
Nucleosome Reconstitution .........................................................
153
Restriction Enzyme Digestion .........................................................
154
DNase I Footprinting Assay .........................................................
154
Hydroxyl Radical Footprinting Assay .........................................................
154
Results and Discussion .........................................................
155
Synthesis of 5'-End Radiolabeled Site-Specifically
Platinum-Modified DNA Probes .........................................................
Restriction Enzyme Assay ........................................................
155
155
156
DNAse I Footprinting Characterization of Nucleosome.................................................
157
Characterization of Nucleosome by Hydroxyl Radical Footprinting ..........................
Acknow ledgm ents .....................................................................................................
159
References ........................................................
160
Chapter 5. Cisplatin-Induced Post-TranslationalModification of Histones
H3 and H4 .........................................................
171
.........................................................
Introduction
172
Methods and Materials .........................................................
173
Reagents
.........................................................
173
Cell Culture ........................................
................
74......................
15
Preparation of Nuclear Extracts...................................................................................174
Immunoblotting
...........................................................
175
Results
...........................................................
175
Cisplatin Strongly Induces Histone H3 Ser 10 Phosphorylation in HeLa Cells .........175
Cisplatin Induces Histone H3 Phosphorylation in Other Cell Lines ...........................
176
SKF86002Inhibits Phosphorylation of H3 at Ser 10 Activated by Cisplatin ...............176
Cisplatin Induces Other Histone Modifications ..........
................................ 176
Discussion ..........................................................
177
Acknowledgment
...........................................................
180
References .................................................................................................................
181
Chapter 6. The Modulating Effect of DNA Methylation on
the Cellular Processing of Cisplatin-Damaged DNA ...............................................
190
Introduction ..............................................................................................................191
Materials and Methods ...........................................................
192
Materials ................................................................................................................
192
Synthesis of Oligonucleotide Fragments ...........................................................
193
Preparative-Scale Platination of 16mer Fragments ..............
............................................
193
Time Course of Platination of Double-Stranded 14mer and
Single-Stranded 16mer Substrates ..........................................................
194
Maxam-Gilbert Sequencing of 16mer and 36mer DNAs ........................................ 194
Synthesis of Site-Specifically Platinated 195mer Substrates ........................................
196
Nucleotide Excision Repair Assay of 195mer DNAs .......................................................197
16
Gel Electrophoresis Mobility Shift Assay of HMGB1 DomA with ds-16mers ..........197
Results
..........................................................
197
Synthesis and Purification of Platinated Oligonucleotides
for 195mer Repair Substrates. .........................................................
Sequencing of 16mer and 36mer Substrates ..........................................................
197
198
Time Course of Platination of 14mer and 16mer Substrates ..........................................
199
EMSA Assay of dsDNA with HMGB1 Dom A..........................................................
199
Effect of Methylation on NER of 195mer Substrates ......................................................
200
Discussion .................................................................................................................................201
DNA Methylation Modulates the Formation of Platinum-DNA Adducts ..................201
DNA Methylation Modulates the Interaction between HMGB1 Dom A
and Cisplatin-DNAAdducts.
.........................................................
203
DNA Methylation Modulates NER of Cisplatin-DNA Adducts ..................................
205
Implications in Cancer and Processing of Cisplatin.
........................................ 207
Acknowledgment
..........................................................
209
References .........................................................
209
Appendix 6.1 Oligonucleotides sequences.
233
.........................................................
Chapter 7. Transcription-Coupledand DNA Damage-Dependent
Ubiquitination of RNA Polymerase II In Vitro ...........................................
234
Introduction
..........................................................
235
Materials and Methods ..........................................................
236
Chemical Reagents................................................................................................................236
17
Preparation of Nuclear Extracts .
Platination of Plasmid DNA .
.............................................................
.............................................................
237
237
In Vitro Transcription Assay ..............................................................
238
In Vitro Ubiquitination Assay ..............................................................
239
Results ........................................................................................................................................ 240
Alpha-amanitin
Stimulates Ubiquitination of RNA Pol II ........................................
Dependence of Ubiquitination on DNA ..............................................................
240
243
Ubiquitination is Induced by Arrest of Transcription
on Cisplatin-Damaged DNA ..............................................................
Dependence of Ubiquitination on Transcription .
.......................................
244
245
D iscussion .................................................................................................................................246
Implications for Transcription-Coupled DNA Repair .
....................................... 246
The Cellular Signals Triggering Ubiquitination ....................................................... 248
Implications for the Cisplatin Mechanism of Action .......................................................
250
Acknowledgments
..............................................................
251
References .............................................................
252
Part II. Synthesis of Site-SpecificallyCisplatin-Modified DNA
Templates and In Vitro Transcription Study .............................................................
262
Introduction..............................................................................................................263
Materials and Methods .............................................................
Synthesis and Purification of DNA Fragments .
.......................................
265
266
Platination, Purification, and Characterization of Platinated DNA Primer 20GG......266
18
267
Synthesis of DNA 296-Pt by the Enzymatic Ligation Method .....................................
268
Site-Directed-Mutagenesis (SDM) .....................................................................................
269
Synthesis of DNA 296-Pt by the Primer Extension Method ..........................................
271
In Vitro Transcription with a Reconstituted Transcription System ............................
272
In Vitro Transcription Assay with Nuclear Extracts.......................................................
Results and Discussion .
273
.............................................................
..............
273
Platination Purification and Characterization of DNA Primer 20GG.........
273
Synthesis of DNA 296-Pt by the Enzymatic Ligation Method ......................................
..............................................................
Site-Directed-Mutagenesis
273
Synthesis of DNA 296-Pt by the Primer Extension Method....................................
274
In Vitro Transcription Assay .............................................................
276
.................................
..............................................................
Acknowledgments
279
References .............................................................
279
Appendix 7.1. Sequence of 296 mer ..............................................................
290
Chapter 8. Synthesis and Characterizationof Platinated Dumbbell DNA for
In Vitro Transcription and Repair Studies ..............................................................292
..............................................................
Introduction
293
Experimental Section ..............................................................
294
294
Synthesis and Characterization of the Dumbbell DNA probe .......................................
Excision Assay of Dumbbell DNA .............................................................
295
.......................
Reconstitution of Site-Specifically Cisplatin-Modified Nucleosome ...........................295
In Vitro Transcription Assay with DNA Template Containing a G-less Cassette ..... 296
19
Assessment of Transcription from Dumbbell DNA using RNase
Protection Assay...........................................................
296
Results and Discussion...........................................................
297
Synthesis and Characterization of Dumbbell DNA Probes ............................................
297
Nucleotide Excision Repair from Dumbbell Probes .......................................................
299
Nucleosome Assembly of Dumbbell DNA ......................................................................
300
Cisplatin Blocks the Transcription from Dumbbell Probes ..........................................
300
TFIIB Dependent Transcription from Dumbbell Probes ...............................................
300
Acknowledgment
...........................................................
301
References ..........................................................
301
Appendix 8.1. Dumbbell GG and GTG sequences...........................................................314
Appendix Al. The Interaction of Human SWI/SNFwith Platinated
Nucleosomes and Its Effect on NER...........................................................
315
Introduction ................................................................................................................. 316
Materials and Methods ...........................................................
319
M aterials ................................................................................................................
319
Synthesis and Purification of Site-SpecificallyPlatinated DNA Probes ......................319
Proteins Preparation ...........................................................
319
Nucleosome Assembly of Site-SpecificallyCisplatin-Modified Nucleosomes............320
Glycerol Gradient Purification ...........................................................
DNase I Footprinting Assay ..........................................................
320
.....................................
320
Excision Assay for Nucleosome Pre-incubated with SWI/SNF ...................................
321
__
20
Restriction Enzyme Characterization ......................................
Resultsand Discussion.
322
.....................................
Characterization of Nucleosomes by Footprinting .........
321
...................................
Restriction Enzyme Characterization ......................................
322
322
The Stimulatory Effect of hSWI/SNF on NER ......................................................... 323
SWI/SNF and Histone Modifications......................................
325
The Role of SWI/SNF in Cancer.........................................................................................
325
Acknowledgment
. ..................................... 326
R eferences .................................................................................................................................326
333
Biographical N ote ....................................................................................................................
Curriculum Vitae ......................................................................................................................
334
21
List of Tables
Table 5.1 Stimuli that Activate a MAPK Pathway to Induce Histone
Phosphorylation .....................................................................................................
185
Table 6.1 The Ion Exchange HPLC Gradient for Isolation of Platinated 16mer
Products
. ......................................................... 215
Table 6.2 Buffer Conditions for the Time Course of Platination of
Single-Stranded 16mer Substrates by Ion Exchange HPLC..............................
215
Table 6.3 Buffer Conditions for the Time Course of Platination of DoubleStranded 14mer Substrates by Reverse Phase Ion Exchange HPLC ...............216
Table 6.4 Electrospray Mass Spectrometry Results for 16mer Substrates .......................
216
Table 6.5 UV/Vis and AA Spectroscopy Results for 16mer Substrates
......................216
Table 6.6 Extent of Platination for the Single-Stranded 16mer Substrates ....................217
Table 6.7 Extent of Platination for the Double-Stranded 14mer Substrates ...................217
I
22
List of Figures
Figure 1.1 Cisplatin-DNA adducts cause a wide variety of cellular responses ..............95
96
Figure 1.2 Mechanisms of cisplatin uptake and efflux ........................................................
Figure 1.3 HMGB1 protein has multiple roles ..........................................................
97
98
Figure 1.4 The p53 pathway can partially mediate cisplatin cytotoxicity ........................
Figure 1.5 Cisplatin activates MAPK pathways .......................................................... 99
Figure 1.6 Cisplatin induces necrosis and apoptosis, two different modes
100
of cell death ..........................................................
Figure 2.1 Structures of cisplatin and carboplatin .............
............................. 122
Figure 2.2 Strategy for synthesizing site-specifically platinated DNA probes ..............123
Figure 2.3 Denaturing and non-denaturing PAGE gel of ligation products of
124
site-specifically platinated DNA probes...........................................................
Figure 2.4 SDS-PAGEgel of recombinant histones and histone octamer.......................125
126
Figure 2.5 Non-denaturing PAGE gel of nucleosome and free DNA .............................
Figure 2.6 Excision assay with nucleosomal and free DNA ........................................ 127
Figure 2.7 Analysis of repair of native and recombinant nucleosomal
platinated DNA by the excision assay .
.......................................
128
Figure 3.1 Strategy for synthesizing the blunt-ended site-specifically
platinated DNA probe ..................................................................................144
Figure 3.2 4-20%SDS-PAGEgel of recombinant histones, histone (H3/H4)2
tetramer and histone octamer .........................................................
Figure 3.3 Non-denaturing PAGE gel of purified double-stranded DNA
145
23
146-mer GTG-Pt ......................................................
146
Figure 3.4 Analysis of site-specifically cisplatin-modified tetrasome,
{Pt(DACH))2+-modified tetrasome, and cisplatin-modified nucleosome .....147
Figure 3.5 Nucleosome and tetrasome inhibition of nucleotide excision
repair of cisplatin-DNA adduct ..........................................................................
148
Figure 3.6 Tetrasome inhibition of the excision repair of cisplatinand {Pt(DACH))}2-DNAadduct ...........................................................................
149
Figure 4.1 Strategy for synthesizing site-specifically platinated-DNA probe ................163
Figure 4.2 Non-denaturing PAGE gels of nucleosomal and free DNA ...........................
164
Figure 4.3 Non-denaturing
gel of 5'-32 P-labeled nucleosomal DNA of
199GTG (unmodified) and 199GTG-Pt (cisplatin-modified)
..........................165
Figure 4.4 Restriction enzyme digestion of nucleosome 199GTGand
199GTG-Pt
......................................................
166
Figure 4. 5 DNase I footprinting of cisplatin-modified and unmodified
nucleosomes..........................................................................................................
167
Figure 4.(6 Hydroxyl footprinting of the 146GTGand 146GTG-Ptnucleosomes ...........168
Figure 4.57 Model of the n146GTG nucleosome ......................................................
169
Figure 4.E38Hydroxyl footprinting of the n146GTGand n146GTG-Ptnucleosomes .......170
Figure 5.11 Cisplatin-induced
phosphorylation
Figure 5.22 Inhibition of cisplatin-induced
of histone H3 at Ser 10 ..........................186
phosphorylation
of H3
Ser 10 by SKF86002 ......................................................
Figure 5.'3 Cisplatin induction of phosphorylation of histone H3 in
187
24
MCF-7 (top) and Ntera II (bottom) cell lines ......................................................188
Figure 5.4 Cisplatin induction of phosphorylation of H3 Ser28 and
acetylation of H4 .........................................................
189
Figure 6.1 Strategy for synthesizing site-specifically platinated 195mer
DNA probes .........................................................
218
Figure 6.2 HPLC trace of platination products of 16mer DNAs
after 18 h incubation .........................................................
219
Figure 6.3 Denaturing PAGE analysis of Maxam-Gilbert sequencing of
16GG and 16GGMe substrates .............................................................................220
Figure 6.4 Maxam-Gilbert sequencing results for the 16GG and 16GGMe ....................221
Figure 6.5 Maxam-Gilbert sequencing of 16GTGand 16GTGMe substrates ................222
Figure 6.6 Maxam-Gilbert sequencing results for the 16GTGand 16GTGMe...............223
Figure 6.7 PAGE analysis of Maxam-Gilbert sequencing of 36mer substrates ..............224
Figure 6.8 Maxam-Gilbert sequencing results for the 36CC and 36 MM ......................225
Figure 6.9 The Maxam-Gilbert sequencing results for the 36mer CAC
226
and 36mer MAM ...................................................................................................
Figure 6.10 Comparison between platinated and non-platinated substrate
of Maxam-Gilbert sequencing .
........................................................ 227
Figure 6.11 Time course of platination of 16GG and 16 GGMe .........................................228
Figure 6.12 Time course of platination kinetics of 16GTG and 16GTGMe .....................229
Figure 6.13 Time course of platination kinetics of ds14 CC and ds14MM......................230
Figure 6.14 EMSA of dsl6mers with HMGB1-dom A .
....................................... 231
25
Figure 6.15.A Non-denaturing PAGE gel of dsl95mers .
....................................... 232
Figure 6.15.B Excision assay with methylated and non-methylated DNA probes........232
Figure 6.15.C The relative excision efficiencies of ds 195mers .........................................
232
Figure 7.1 Ubiquitination of Pol II is induced by a-amanitin in an in vitro assay.........257
Figure 7.2 Comparison of ubiquitination and transcription activities
of various drug-induced cell cycle stage nuclear extracts ...............................
258
Figure 7.3 Alpha-amanitin induction of Pol II ubiquitination is DNA-dependent ........259
Figure 7.4 Ubiquitination of Pol II is induced to a similar extent
by cisplatin-modified DNA and alpha-amanitin ........................................ 260
Figure 7.5 Stimulation of polymerase ubiquitination by alpha-amanitin
or cisplatin-modified DNA is suppressed by inhibiting transcription ..........261
Figure 7.6 Two-phase enzymatic ligation strategy for synthesizing
a platinated 296mer .............................................................
282
Figure 7.7 Procedure for synthesizing a platinated DNA 296mer
by primer extension .............................................................
283
Figure 7.8 Maxam-Gilbert sequencing results for 20GG-Pt ........................................ 284
Figure 7.9 Denaturing PAGE of the two-phase ligation product
and purified 296-Pt .............................................................
285
Figure 7.10 Concentration of 296mer is estimated by using
the low DNA mass ladder .............................................................
286
Figure 7.11 Agarose gel electrophoresis of purified single-stranded DNA ..................286
Figure 7.12 Agarose gel electrophoresis of DNA primer extension products ...............
287
26
Figure 7.13 In vitro transcription assay with cisplatin-modified template ..................288
Figure 7.14 In vitro transcription assay with cisplatin-modified template ..................289
Figure 8.1 Strategies for the synthesis site-specifically platinated dumbbell
DNA Probes ...........................................................
305
306
Figure 8.2 Denaturing PAGE gel analysis of dumbbell ligation products ......................
Figure 8.3 Denaturing PAGE gel analysis of dumbbell ligation products (sixfragment strategy) ...........................................................
307
308
Figure 8.4 Digestion sites of restriction enzymes on the dumbbell DNA ......................
Figure 8.5 Non-denaturing gel of restriction enzyme digestion of
dumbbell DNA ...........................................................
309
Figure 8.6 Denaturing gel of T7 exonuclease digestion of dumbbell DNA ....................310
311
Figure 8.7 Denaturing PAGE gel of repair assays for dumbbell DNA ...........................
312
Figure 8.8 Reconstitution of dumbbell into nucleosome....................................................
313
Figure 8.9 Effect of anti-TFIIBon transcription from dumbbell DNA ..........................
Figure A1.1 Analysis of site-specifically cisplatin-modified nucleosome
330
on a 5% non-denaturing PAGE gel electrophoresis .......................................
Figure A1.2 The effect of hSWI/SNF on nucleosome structure
characterized by Nla III digestion ........................................................... 331
Figure A1.3 The stimulatory effect of hSWI/SNF on nucleotide excision repair ...........332
27
Chapter 1 Cellular Processing of Platinum Anti-Cancer
Drugs - Identifying Pathways for Chemogenotherapeutic
Drug Design*
* Submitted as an invited paper in Nature Reviews Drug Discovery.
28
Introduction
Cisplatin and carboplatin have been widely for many years to treat
several kinds of cancer, including as testicular, ovarian, head and neck, and nonsmall cell lung.' Over this period, thousands of platinum analogues have been
synthesized and screened for anti-cancer activity. Among them, the only one that
has been approved by the FDA is oxaliplatin, for treatment of colorectal cancer.2 3
Several interesting platinum compounds, including those with a trans geometry
at the platinum center, platinum(IV) molecules with tethered substituents, and
multinuclear species, have been reported to be as active as cisplatin in one or
another assay, as has been reviewed elsewhere.4-7
Although many cellular components interact with cisplatin, DNA is the
primary biological target of the drug.' The platinum atom of cisplatin forms
covalent bonds to the N7 positions of purine bases to form primarily 1, 2- or 1, 3intrastrand cross-links and a fewer number of interstrand cross-links. These
linkages to DNA occur at the positions where the chloride, CBDCA, or oxalate
ligands reside in the original platinum drug. Structural studies of cisplatin and
oxaliplatin DNA adducts have been reviewed elsewhere.'
I The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II);trans-DDP,transdiamminedichloroplatinum(II); carboplatin, cis-diammine(cyclobutane-1,1-dicarboxylato)platinum(II); CBDCA, cyclobutane-1,1-dicarboxylate; ER, estrogen receptor; ATM, ataxiatelangiectasia mutated; tsHMG, testes-specific HMG box protein; ATR, ataxia-telangiectasia and
Rad3-related; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH 2-terminal kinase; ERK,
extracellular signal-regulated kinase; NER, nucleotide excision repair; TCR, transcription-coupled
repair; GGR, global genomic repair; MMR. mismatch repair; XIAP, X-linked inhibitor of
apoptosis; MDM2,murine double minute-2; MEF,mouse embryo fibroblasts; PARP, poly(ADPribose)polymerase; SCC, serum squamous cell; Cdc25C, cell division cycle 25C; PCNA,
proliferating cell nuclear antigen; GADD, growth arrest and DNA damage; ES, embryonic stem.
29
Cisplatin-DNA adducts cause a wide variety of cellular responses (Figure
1.1).8 Understanding the mechanisms of how cells process cisplatin provide
important insights for designing more efficient platinum-based drugs. For
example, the discovery that treatment of ER(+) cells with estradiol sensitizes
them to cisplatin inspired the design and synthesis of a series of active estrogentethered Pt(IV) compounds.9 Information of this kind facilitates the introduction
of new families of molecules in which the fundamental genotoxicity of a
platinum-DNA adduct can be made selective for cancer cells by targeting the
compound selectively to tumors or by interrupting a pathway used by the cancer
cell to survive the chemical assault. The current trend among cancer biologists to
downplay the importance of chemotherapy in favor of pathway-directed
modalities is, in our opinion, failing to take advantage of a time-proven weapon
against the disease. Combining the best of chemotherapeutic action with specific
targets in cellular pathways offers a powerful new approach to cancer treatment
that perhaps may impede the many clever ways that human cells have in
becoming drug-resistant.
This purpose of the present review is to highlight recent discoveries in
those pathways involved in regulating platinum drug transport, DNA damage
signaling, cell cycle checkpoints and arrest, DNA repair and mechanisms of cell
death. From the understanding of these cellular pathways it has become possible
to form a biological basis for the chemical synthesis of new platinum drugs for
cancer treatment, a process that we term chemogenotherapy.
30
Mechanisms of Cisplatin Transportinto Cells
The mechanisms of cisplatin cellular uptake and efflux are still not fully
understood. Early studies suggested that cisplatin enters the cell mainly by
passive diffusion, since its uptake proceeded linearly with time up to 60 min and
could not be saturated up to a concentration of 1.0 mM. In addition, cisplatin
absorption was not inhibited by structural analogues and there did not appear to
be an optimum pH for its entry into the cell.1 -'2
More recently, a growing body of evidence reveals a link between Ctrlp, a
high-affinity copper transporter, and cisplatin uptake (Figure 1.2). Mutation or
deletion of the CTR1 gene results in increased cisplatin resistance and reduction
of platinum levels in both yeast and mouse cells.'3 Moreover, CTR1-deficient
cells exhibit impaired
accumulation of cisplatin analogues
carboplatin,
oxaliplatin, and ZD0473[cis-amminedichloro(2-methylpyridine)platinum(II)].14,15
Another direct link between the cellular management of copper and
platinum concentrations comes from studies of cisplatin efflux. Coppertransporting P-type adenosine triphosphate (ATP7B),which plays an important
role in regulating copper levels in the cell, is associated with cisplatin resistance
in vitro.'6 Moreover, ATP7Blevels correlate with cisplatin resistance in a variety
of cancers. For example, in the case of primary oral serum squamous cell (SCC)
carcinoma, patients with ATP7B-positive tumors have reduced overall survival
following cisplatin treatment than those who are ATP7B-negative.2 0 The
31
expression level of the ATP7B gene was significantly increased in patients with
undifferentiated ovarian carcinoma and gastric carcinoma, which is less sensitive
to cisplatin treatment.17 -'9 Although ATP7A is expressed in a considerable
fraction of many common tumor types, its role is less clear; however, enrichment
of human ovarian cancer cells that express ATP7A is associated with poor
response to platinum-based therapy. 21 In addition, MRP2 (cMOAT), an exporter
protein, also plays a role in cisplatin resistance, presumably by promoting drug
efflux. Overexpression
resistance.22-
of MRP2 is associated with increased cisplatin
24
The observation that both cisplatin uptake and efflux are seemingly linked
to the copper metabolic pathway leads to the hypothesis that copper and
cisplatin might interfere with their mutual transport.2 s In fact, both copper and
cisplatin have the ability to reduce the uptake of the other and can trigger the
degradation and delocalization of Ctrlp.l 3 Moreover, copper and cisplatin also
exhibit bidirectional cross-resistance.2 6 ,27 Expression of ATP7B in human
carcinoma cells modulates sensitivity to both cisplatin and copper by causing
more rapid efflux of the two agents.'6
These observations offer a potential new mechanism for targeting
platinum to tumor cells by taking advantage of an emerging receptor for
delivering the drug into the cancer cell. Any compound having a substituent that
can direct the platinum complex to a tumor or organ tissue population in a
specific manner is of potential interest for chemogenotherapy. Included in this
32
category would be molecules that target the tumor vasculature28-31 or deliver the
molecule specifically to cells having a receptor for the appended moiety.93 2
Cisplatin-DNA Damage Recognition Proteins - A Postulated Key
Role for HMGB1
Cisplatin modifications distort the structure of the DNA duplex, bending
it significantly toward the major groove and exposing a wide, shallow minor
groove surface to which several classes of proteins bind. Included are high
mobility group box proteins, repair proteins, transcription factors, and other
proteins such as histone H1, which preferentially recognize 1,2-intrastrand crosslinked platinum-DNA adducts, as summarized elsewhere.' X35Here we focus on
recent progress in understanding the functions of the HMG box family of
proteins and their putative role in facilitating the anticancer activity of cisplatin.
The abundant HMG box protein, HMGB1, also named Amphoterin, has
been connected to several DNA-dependent pathways (Figure 1.3). HMGB1
stimulates RAG1/2 cleavage in V(D)J recombination and promotes binding of
sequence-specific transcription factors.- 38 In addition, HMGB1 physically
interacts with MutSa and plays a role in mismatch repair.3 9 Moreover, HMGB1
enhances p53 DNA binding activity and directly interacts with p53 in vitro.40
Both the C-terminal and A box domains of HMGB1 regulate p53-mediated
transcription activation and its downstream effects.41 HMGB1 affinity for
33
cisplatin-damaged DNA is significantly enhanced by p53. Interaction between
p53 and HMGB1 after platination may provide a molecular link between DNA
damage and p53-mediated DNA repair.4 2 In vitro nucleotide excision repair
studies indicate that HMG box proteins such as HMGB1 and tsHMG shield the
Pt-1,2-d(GpG) cross-link from repair proteins by stably binding to this DNA
adduct.43-4 5 Until recently, the postulated in vivo relationship between HMG box
protein levels and cisplatin toxicity has been somewhat controversial. Several
studies report that HMG box protein levels correlate with cisplatin sensitivity.
For example, overexpression of HMGB1 induced by addition of the steroid
hormone estradiol sensitizes breast and ovarian cancer cells to cisplatin
treatment. 46 Deletion of yeast Ixrl, a gene that encodes a transcriptional repressor
containing two HMG boxes, results in 2-6-fold increased resistance to cisplatin,
4 7 48
depending on the strain investigated.
, On the other hand, cancer cell lines that
have developed resistance to cisplatin often have elevated levels of HMGB1.49 In
addition, no significant difference in sensitivity to cisplatin was observed
between and Hmgbl knockout and wild type mouse embryonic fibroblast cell
lines.50 Moreover, S. pombemutant deficient in the HMGB protein Cmbl is more
sensitive to cisplatin treatment.5 l These discrepancies most likely reflect the
importance of cell type and history in determining the ability HMGB1 and
related proteins to mediate cisplatin cytotoxicity. Recent work from our
laboratory using RNAi clearly demonstrate the strong correlation with HMGB1
levels and the sensitivity of cells to cisplatin.5 2
34
HMGB1 interacts with the nucleosome at the entry and exit points of
DNA and facilitates binding of the remodeling complex ACF to nucleosomal
DNA, thereby accelerating the ability of ACF to induce structural changes in the
nucleosome.535 4 Of greater interest is the recent discovery that the dynamic
behavior of HMGB1 is completely altered in apoptotic cells, where it binds
irreversibly to chromatin, most likely due to the change of the nature of
chromatin.55 In addition, HMGB1 plays an important role as a cytokine in the
inflammatory response.5 6-58 In monocytes activated by inflammatory stimuli,
HMGB1 relocalizes from the nucleus to the cytoplasm, and eventually to
secretory organelles.5 5 HMGB1 thus appears to act as a signal of tissue damage,
inducing mesoangioblast migration and proliferation.5 9
The activity
of
HMGB1/2
is modulated
by
post-translational
modifications. Histone acetyltransferase CBP acetylates HMGB1/2 proteins in
vitro.6 0 The acetylated HMGB1/2 full-length protein is 3-fold more effective in
inducing ligase-mediated circularization of a 111-bp DNA fragment than
unmodified full length protein. In addition, acetylation of HMGB1 plays an
important role in regulating relocalization of HMGB1 from nucleus to cytoplasm,
and subsequent secretion when monocytes receive an appropriate second
signal.6 l The export of HMGB1 from necrotic cells does not appear to require
63 If viable cancer cells surrounding the
such post-translational modification.5562
necrotic core of a solid tumor took up HMGB1,it could potentiate the ability of
35
cisplatin to kill such cells and account for the specificity of the drug for cancer
cells.
The structure-specific recognition protein, SSRP1, contains a single HMG
domain that was initially isolated from expression screening of a human B-cell
cDNA library for proteins that bind to cisplatin-modified DNA.64Human SSRP1
and Sptl6 form a complex named FACT, which facilitates RNA polymerase II
66 The isolated HMG
transcription elongation through a nucleosome block.65,
domain of SSRP1 and FACT, but not SSRP1 alone, can bind to the major 1,2d(GpG) intrastrand cisplatin adduct, which suggests that Spt16 primes SSRP1for
cisplatin-damaged DNA recognition by unveiling its HMG domain.6 7 Also,
SSRP1functions as a co-activator of the transcriptional activator p63.68
Yeast Nhp6, an abundant HMG box protein, is a component of yeast
FACT complex in S. cerevisiae.Nhp6 is required for proper regulated expression
of a number of genes that are transcribed by RNA polymerase II or III. Either
Nhp6 alone or the yFACT complex were able to bind nucleosomes and
significantly reorganize their structure.6 9 In addition, Nhp6Ap binds to cisplatinmodified intrastrand cross-links on duplex DNA with a 40-fold greater affinity
than to unmodified DNA with the same sequence. Nhp6A/Bp appears to
function directly or indirectly in yeast to enhance cellular resistance to cisplatin.7 0
36
Cell Cycle Checkpoints and Arrest
Cell cycle checkpoints monitor the proper order of events in the cell.
When DNA is damaged, the cell cycle is arrested to provide time for repair.
Failures to do so leads to the acquisition and accumulation of genetic alterations,
ultimately causing tumorigenesis. 71
The G1/S checkpoint ensures that damaged DNA is not replicated.
Following DNA damage, ATM (ataxia-telangiectasia mutated) cells may control
the fate of a G1/S checkpoint through direct phosphorylation of p53 or through
phosphorylation
of c-Abl, which in turn upregulates p21 through p73
activation.7Z73 ATM-dependent phosphorylation of p53 contributes to p53
stabilization by reducing its interaction with MDM2 (murine double minute2).74,75 In addition, p21, a downstream target of p53 and a universal inhibitor of
cyclin-dependent kinases, plays a major role in mediating p53-dependent cell
cycle G1 arrest. p21 inhibits G1 cyclin/cdks and blocks cell cycle progression. 7 6 ,77
Exogenous expression of p21 exerts cell growth inhibition and enhances
sensitivity to cisplatin in hepatoma cells.78
Cisplatin does not cause G1 arrest in all cases, however. In fact, the drug
fails to induce a G1 arrest response in synchronized wild-type MEF cells, even
though it can activate p53 and cause an S phase arrest.79
The G2/M checkpoint allows for repair of DNA that was damaged in late
S or G2 phase of the cell cycle prior to mitosis to prevent damaged DNA from
37
being segregated into daughter cells. It has been proposed that such G2 arrest is
essential to the process of engaging cell death following cisplatin treatment.80 8, l
Upon the occurrence of DNA damage, ATM and its related ATR (ataxiatelangiectasia and Rad3-related) kinase activate checkpoint kinases Chkl and 2
through
phosphorylation,
which
in turn
phosphorylate
Cdc25C. The
phosphorylated Cdc25C promotes its binding to 14-3-3adaptor proteins and
thereby is separated from Cdc2 by translocation of Cdc25c to the cytoplasm. As a
result, Cdc2 phosphorylation is elevated and causes cells to arrest in G2.82-88
In addition, p21 plays a regulatory role in maintaining cell cycle arrest at
G2 by blocking the interaction of Cdc25C with PCNA.8 9-9 1 More recently, SHP-2
extra-cellular signal-regulated kinases (ERK) and p38 mitogen-activated protein
kinases (p38 MAPK) have also been implicated in the G2/M cell cycle
checkpoint.9 2- 95 Therefore, it appears that multiple pathways contribute to the
regulation of the G 2/M checkpoint following genotoxic stress.
p53 and p73 Pathways
p53 is an important tumor suppressor protein which is mutated in greater
than 50% of all human tumors.9 The p53 protein plays a crucial role in many
cellular processes; in particular, it inhibits cell proliferation by inducing either
cell cycle arrest or apoptosis in response to cellular stress. p53 functions as a
transcription factor and upregulates a number of genes involved in these
38
7- 99 In the absence of cellular stress, p53 is maintained at
processes (Figure 1.4).9
low steady-state levels and exerts very little effect on the cell. Upon cellular stress,
p53 is overexpressed and undergoes post-translational modification.
p53 has also been connected with DNA repair, specifically nucleotide
00-102 One of the activities of
excision repair (NER) and base excision repair (BER).1
p53 involves its transcriptional activation of the Gadd45gene. GADD45 interacts
with
PCNA and
stimulates
nucleotide excision repair
activity when
overexpressed.'0 3 p53 is also involved in repair through direct interactions with
critical components of the NER machinery. XPC, an important damage
recognition protein in NER, is induced after DNA damage in a p53-dependent
manner.104XPC also can help initiated cisplatin damage-mediated cell cycle
regulation.l0 5 In addition, p53 binds to three of the subunits of the TFIIH
transcription/repair
complex, XPB, XPD, and the p62 subunit, suggesting an
additional role in NER.10 6 Moreover, p53 physically interacts with RPA.107Such
interaction is disrupted following UV or cisplatin damage in p53-wild type cells,
but not in p53-mutant repair-deficient cells.08 These results suggest that release
of RPA and p53 plays a role in facilitating NER.109The repair defect in p53mutant ovarian carcinoma cells IGROV-1/Pt may be attributed to a reduced
removal/recycling of PCNA at repair sites.l09
Sensitivity to cisplatin correlates positively with the presence of wild-type
p53 function in a National Cancer Institute (NCI) panel of 60 human tumor cell
lines."1° In addition, tumor cell lines that lack functional p53 are more resistant to
39
cisplatin than cells that contain functional p53, but can be sensitized when
3 Other studies, however, show no or even
reconstituted with wild-type p53.111-11
negative correlation between p53 status and response to cisplatin, For example,
p53 mutation promotes increased sensitivity to cisplatin, with loss of G1/S
checkpoint control and decreased cisplatin-DNA adduct repair.l 4 Furthermore,
one study demonstrated that, in SaOS-2 osteosarcoma cells, p53 function was
associated with increased cisplatin sensitivity under high serum growth
conditions but with decreased sensitivity under low serum conditions.l5
Previous work from our own laboratory using murine testicular teratocarcinoma
cells also revealed that, although rapid apoptosis is induced in p53-wild-type but
not in p53-/- teratocarcinoma cells upon cisplatin treatment, the absence of p53
does not alter cellular sensitivity to cisplatin as measured in clonogenic assays.l 6
The relationship between p53 status and cisplatin cytotoxicity depends upon
several factors, including tumor cell type, activation of specific signaling
pathways, and the presence of other genetic alterations.
Besides p53, there are two other members of the p53 family, p63 and p73,
which are homologous to p53 in terms of overall domain structure and
conformation. Both p63 and p73 are expressed as multiple isoforms, truncated at
the N- or C-terminus through alternative splicing and use of several
promoters.
7
,"'8 p 7 3 can cooperate with DNA damaging agents or p53 to induce
some p53 target genes and activate their promoters. Signaling pathways for p53
and p73 in inducing cell cycle arrest and apoptosis are similar but also have
40
important differences. p73 differentially regulates some p53 target genes, either
several fold lower than p53, such as p21, or several fold higher than p53, such as
14-3-3a.119 DNA damage can stabilize p73 and enhance p73-mediated apoptosis
in a c-Abl dependent manner. 73 p73 upregulates MDM2 expression, but unlike
p53, it is not targeted by MDM2 for ubiquitin-dependent degradation. Instead,
MDM2 downregulates p73 transactivator function by disrupting its interaction
with p300/CBP, a component of the eukaryotic transcription complex.2 0
Proteins involved in p53 related pathways modulate p53 activity and
cisplatin cytotoxicity. Aurora kinase A is a key regulatory component of the p53
pathway and phosphorylates p53 at Ser315, leading to its ubiquitination by
Mdm2 and proteolysis. Aurora kinase A is overexpressed in many human
cancers, which leads to increased degradation of p53, and down-regulation of
checkpoint-response pathways. Silencing of aurora kinase A results in less
phosphorylation of p53 at Ser315, greater stability of p53 and cell-cycle arrest at
G2-M, and ultimately increased sensitivity to cisplatin treament.'2 1
MDM2 interacts with the N-terminus of p53 and inhibits p53
transcriptional activity.2 2 In addition, MDM2 acts as the E3 ligase for p53 and
thus promotes
p53 ubiquitination
and degradation. 12 3
Post-translational
modifications of p53 and MDM2, including phosphorylation, acetylation and
sumoylation, modulate their interactions and thus influence p53 stability and
activity.'2 4 In addition, STAT-1 interacts directly with p53 acts as a co-activator
for p53 and a negative regulator of Mdm2 expression.' 2
41
Cyclin G is another transcriptional target gene of tumor suppressor p53.
Cyclin G may play a central role in the p53-MDM2 autoregulated module.
Inhibition of cyclin G by small interfering RNA (siRNA) causes the accumulation
of p53 levels in response to DNA damage.'2 6 This cyclin G-mediated p53
regulation is dependent upon the status of ATM protein, which activates p53 in
response to DNA damage. 2 7
The human BRCA1gene contains one RING finger domain and two BRCT
domains. BRCA1 interacts directly or indirectly with numerous proteins
involved in transcriptional regulation, cell cycle/checkpoint control, protein
ubiquitination, chromatin remodeling, and DNA repair. BRCA1 functions as a
molecular determinant of response to a range of cytotoxic chemotherapeutic
agents.l28 BRCA1 is a substrate of ATM kinase in vitro and in vivo, and is
regulated by an ATM-dependent mechanism as a part of the cellular response to
DNA damage.l2 9 The p53 and BRCA1 tumor suppressors are involved in repair
processes and may cooperate to transactivate certain genes, including p21WAF/cm
and GADD45. BRCA1 also enhances p53 binding to the DDB2 promoter in vivo.
Antisense of BRCA1 abrogates upregulation of DDB2 after cisplatin exposure.
DNA repair activity is significantly restored by introduction of BRCA1into wildtype as compared to DDB2-deficientcells, suggesting that BRCA1plays a role in
DNA damage repair-mediated cisplatin resistance.3 0,13 '
These studies demonstrate that many cellular functions controlled directly
or indirectly by p53 respond to cisplatin treatment. Since p53 is not essential for
42
cisplatin-mediated cell-killing,l1 6 however, it is unlikely that any one of them is
critical to the selectivity and curative properties of the drug for certain cancers
such as testicular.
MAPK and Other Related Pathways
Akt Pathway
Akt, protein
kinase B (PKB), is a member
of the family of
phosphatidylinositol 3-OH-kinase-regulated serine/threonine
kinases. 3 2 Akt
promotes cell survival and down-regulates apoptotic pathways through
phosphorylation and modulation of several downstream target proteins.' 33
Therefore, Akt protects cells from apoptotic death induced by a variety of stimuli,
including cisplatin.3 4
Akt phosphorylates X-linked inhibitor of apoptosis (XIAP) and thus
stabilizes XIAP by inhibiting both its auto-ubiquitination and cisplatin-induced
ubiquitination activities. Increased levels of XIAP are associated with decreased
cisplatin-stimulated caspase 3 activity and apoptosis. Inhibition of XIAP and/or
Akt expression/function
may be an effective means
of overcoming
chemoresistance in ovarian cancer cells expressing either endogenous or
37 Akt also phosphorylates the MDM2 protein
reconstituted wild-type p53.13-1
promoting its translocation to the nucleus and destabilization of p53, therefore
impairing the cellular stress response and increasing the survival of tumor cells.
43
Akt also phosphorylates and inactivates several other apoptotic pathway
proteins. For example, Akt phosphorylates BAD, promoting its association with
14-3-3 proteins in the cytosol and inactivating its proapoptotic function.lM
Moreover, Akt phosphorylates and inactivates apoptosis signal-regulating
kinase-1 and mixed lineage kinase 3 (MLK3), the mitogen-activated protein
kinase kinase kinases (MAPKKK), leading to cell survival.'3,
active Akt might be due to down-regulation
39
Constitutively
of FADD-protein or the
upregulation of the caspase 8 inhibitor c-FLIP to increase cisplatin resistance.l40
Expression of adenovirus Ela gene is able to block selectively Akt activation
mediated by cisplatin and increases sensitivity to cisplatin.l4 0
In addition, Akt also phosphorylates IKBkinase, subsequently activating
nuclear factor of KB (NFKB).NFKBplays an important role in controlling cell
survival.l3 3 Increase of NFKB activity correlates with decreased apoptosis,
whereas inhibition of NFKBincreases the efficacy of cisplatin both in vitro and in
vivo. 13 3 , 41
c-Abl and Bcr-Abl
c-Abl is a member of the Src family of non-receptor tyrosine kinases,
which contain Src- homologue regions 2 and 3 (SH2 and SH3) at N-terminus and
a large C-terminus region that contains nuclear localization motifs and nuclear
export sequences. In addition, c-Abl also carries a DNA binding motif and an
actin-binding domain at its extreme C-terminus. Nuclear c-Abl tyrosine kinase
44
activity can be stimulated by cisplatin and other DNA damaging agents and acts
to transmit DNA damage signals from the nucleus to the cytoplasm.14 2 The
activation of c-Abl tyrosine kinase might be a downstream event in ATMdependent apoptosis. ATM may directly phosphorylate c-Abl at Ser465 or
indirectly phosphorylate c-Abl through DNA-PK or other unidentified protein
kinases.l43 c-Abl tyrosine kinase plays a role in the activation of apoptosis
induced by cisplatin. Overexpression of c-Abl tyrosine kinase in Saos-2 cells and
NIH3T3 cells can activate apoptosis,44 14 5 whereas c-Abl deficient mouse
fibroblasts and chicken DT-40 cells are resistant to cisplatin-induced apoptosis.
Re-introduction of a functional c-Abl in these Abl-deficient cells restore their
sensitivity to the drug.73 In addition, knocking down c-Abl expression by RNA
interference confers resistance to cisplatin-induced apoptosis.46
Retinoblastoma tumor suppressor protein (RB) plays a negative role in
DNA damage-induced activation of c-Abl kinase.73 RB binds to the tyrosine
kinase domain of c-Abl, thereby blocking c-Abl tyrosine kinase activity and
preventing c-Abl from being activated by DNA damage signals. Phosphorylation
of RB at G1/S disrupts the RB/c-Abl interaction, leading to the release of c-Abl
in the nucleus of S phase cells.14 7
The transcription factors p73 and p53 are possible downstream effectors of
c-Abl. Cisplatin can induce the accumulation of p73 protein in wild type and
p53-deficient, but not in c-Abl-deficient, primary mouse embryo fibroblasts.7 3
Furthermore, c-Abl phosphorylates p73 both in vitro and in vivo, and the SH3
45
domain of c-Abl can directly bind to and phosphorylate the p73 protein.14 8s149 cAbl is not essential to the induction and activation of p53 by DNA damage,
although c-Abl can stabilize and activate p53 in transient co-transfection
experiments.
15 0
c-Abl is also an upstream effector of MEKK-1 and the stress-activated
kinases, c-Jun N-terminal kinases
NK) and p38 MAPK.5 - l53 The treatment of
wild-type fibroblasts, but not Abl-/- cells, with cisplatin is associated with an
increased level of tyrosine phosphorylation of MEKK-1.154In addition, cells
deficient in c-Abl fail to activate p38 MAP kinase and JNK after treatment with
cisplatin, but not after exposure to UV and methyl methanesulfonate.
Reconstitution of c-Abl in the Abl-/- cells restores the activation of p38 MAPK
and JNK.1 55 These findings indicate that c-Abl dependent activation of p38 MAP
and JNK protein kinases is specific to cisplatin. p38 MAPK and JNK are
differentially regulated in response to different classes of DNA-damaging agents.
Another interesting feature of c-Abl is its ability to associate with
chromatin. c-Abl contains three HMG-like boxes that preferentially bind to A/T
duplexes and distorted DNA structures, such as four-way junctions. Whether cAbl could recognize and interact with cisplatin-DNA adducts like HMGB1 is a
very interesting question, the answer to which might provide more insight into cAbl activation by the drug.l56,15 7
Different subcellular localizations might contribute to the opposing effects
of c-Abl and Bcr-Abl kinase on apoptosis.l5 8 Cytoplasmic Bcr-Abl can inhibit
46
apoptosis through the activation of Akt.147 Mutagenesis assays demonstrated that
cells expressing a deletion mutant of BCR/ABL that lacked the SH2 and SH3
domains of ABL (BCR/ABLAA)were sensitive to cisplatin. Moreover, Bcr-Abl
tyrosine kinase can be converted into a potent inducer of apoptosis by allowing it
to function in the nucleus, which is consistent with the role of the nuclear c-Abl
tyrosine kinase in DNA damage-induced cell death.l4 7
MAPK/JNK/ERKPathways
The MAPK family of proteins play important roles in signal transduction
processes through specific phosphorylation cascades in response to a variety of
extracellular stimuli.l59 ,160 There are three major mammalian MAPK subfamilies,
ERK, JNK (also called stress-activated protein kinase), and p38 MAPK. The ERK
pathway is highly induced in response to growth factors and cytokines, and it is
also activated by some conditions of stress, particularly oxidative stress. In
contrast, JNK and p38 are highly activated in response to a variety of stress
signals including ionizing and short wavelength ultraviolet irradiation (UVC),
tumor necrosis factor, and hyperosmotic stress, and they are only weakly
activated by growth factors.5 9 ,160 Cisplatin triggers the activation of the ERK,
JNK and p38 MAPK cascades in tumor cells or transformed cell lines (Figure 1.5),
as discussed in following section.
47
p38 MAPK Path7way
A growing body of evidence suggests that activation of the p38 MAPK
pathway plays an important role in the cellular response to cisplatin.l55 16-1 63
Cisplatin, but not trans-diamminedichloroplatinum(II) (trans-DDP) or PtCl4,
induces sustained activation of p38 MAPK.161Cisplatin activates p38 MAPK for
8-12 hr in sensitive cells and for 1-3 hr in resistant cells.l64 This difference in the
kinetics of activation could account for their differential cytotoxicity. Moreover,
lack of p38 MAPK activation/function induces a resistant phenotype in human
cells.l6 1 "64 MAP kinases may also play a role in modifying the chromatin
environment of target genes.16 5 This kinase regulates immediate-early (IE) gene
expression and other cellular responses through phosphorylation of various
substrates, including transcription factors, chromatin protein constituents, and
downstream Ser/Thr effector kinases.16616 7 Furthermore, p38 MAP kinase and
downstream kinase MSK-1 phosphorylate histone H3 Ser in vitro.l6 7 The p38
MAPK pathway is also involved in cisplatin-induced phosphorylation of histone
H3 at Ser 10.168
ERK Pathluay
Cisplatin treatment results in dose- and time- dependent activation of
69- 71 However, the extent of ERK activation might reflect the nature or
ERK1/2.1
context of specific cell lines, since some cells show no or only weak activation of
48
ERK following cisplatin treatment.72 There is also conflicting evidence for the
role of ERK in influencing cell survival of cisplatin-treated cells. Some studies
have suggested that ERK activation is associated with enhanced survival of
74 Such is not always the case, however. Although
cisplatin-treated cells.73,1l
inhibition of cisplatin-induced ERK activity by treatment with the MEK1
inhibitor PD98059 sensitizes the human melanoma cell line C8161 to cisplatininduced apoptosis, PD98059 does not potentiate cisplatin-induced apoptosis in
three other human melanoma cell lines. Instead, PD98059 protects against
cisplatin-induced cytotoxicity in one of these cell lines, partly via an
enhancement of cisplatin-induced of NFKBactivation.l75-77Moreover, inhibition
of cisplatin-induced ERK activity by PD98059results in decreased levels of p53,
p21WAF1, GADD45 and Mdm2.'7 8 In addition, elevated expression of Ras, an
upstream component of the ERK signaling pathway, has been connected with
enhanced sensitivity to cisplatin.7 9 HeLa cell variants selected for cisplatin
resistance show reduced activation of ERKfollowing platinum treatment.l6 9 This
discrepancy suggests that the relationship between the activity of ERKs and the
cellular response to cisplatin may depend upon individual cellular context and
levels of stress.
INK Path7ay
The mechanisms of JNK1 activation in response to UV irradiation and
cisplatin treatment have important differences. UV light is able to induce
49
translocation of JNK1 from the cytoplasm to the nuclear compartment, whereas
cisplatin does not.72 Although a number of studies have established that JNK is
activated in response to cisplatin treatment, a role for this signaling pathway in
determining survival is far from clear. Several studies have provided evidence
64 ,1728 0-1
84
that the JNK pathway contributes to cisplatin-induced apoptosis,1
whereas others have suggested that JNK activation in response to cisplatin
8 7 Such
signals a protective response and is important for cell survival.174,182z1s3,l5-
differential effects observed from one study to another could reflect the nature of
different cell types and specificity or transient vs persistent activation patterns,
respectively. Interestingly, one study showed that the duration ofJNK induction
may be the crucial factor in mediating the signaling decision resulting in either
cell proliferation or apoptosis.l 88
MKP-1/CL100
Expression of phosphatase MKP-1/CL100 inhibits JNK and p38 activation
after cisplatin treatment and correlates with an increase in survival after drug
treatment. JNK1 induction by cisplatin is delayed and persistent, whereas transDDP induces JNK in a transient manner. This difference in duration of JNK
induction may be due to the differential ability of inducing of MKP-1/CL-100.
trans-DDP efficiently induces MKP-1/CL100, and its expression correlates with
JNK inactivation, whereas cisplatin is only a very weak inducer of MKP1/CL100.1 8 9 Moreover, ectopic expression of a catalytically inactive mutant of
50
MKP-1/CL-100 in 293 cells increases the duration of JNK activation by trans18 9o190Overexpression
DDP and increases toxicity of both cisplatin and trans-DDP.
of MKP-1 induces resistance to Fas-ligand triggered apoptosis in human prostate
cancer cells. 91 These results suggest that the persistent activation of JNK in
response to cisplatin may be due to its inability to induce the expression of MKP1/CL-100 and inhibition of MKP-1/CL100 activity in tumor cells might improve
the therapeutic response to cisplatin in human cancer.
From the foregoing brief summary it is clear that cisplatin treatment of
cells triggers or alters numerous defined pathways. Deciding which to apply in
designing new cancer treatment strategies is critical in any chemogenotherapeutic approach. Our recommendation is to attack those processes that
occur closest in time to the formation of DNA adducts and lead most rapidly to
irreversible mechanistic steps. Interruption of NER and potentiation of
transcription inhibition are two such activities and, consequently, a focus of
much attention in our own laboratory.
DNA Repair Pathways
DNA repair plays a significant role in modulating cisplatin cytotoxicity.
Nucleotide excision repair, the major pathway responsible for removal of
cisplatin-DNA adducts, is composed of several steps including recognition of
DNA lesion, excision and removal of - 30-bp single-stranded DNA fragments
containing the lesion, and re-synthesis and ligation of the newly-synthesized
51
repair patch to the preexisting strand.19 2 There are two distinct sub-pathways of
nucleotide excision repair, transcription-coupled
repair (TCR) and global
genomic repair (GGR). TCR refers to the preferential repair of transcribed
strands of RNA polymerase II-transcribed active genes, whereas GGR refers to
repair throughout the genome.19 31, 94 In human cells, the lesions are recognized by
95
different sets of proteins for GGR and TCR; XPC/hHR23B is involved in GGR,1
whereas the Cockayne syndrome genes, CSA and CSB, are involved in TCR.16
After the different initial recognition, the two sub-pathways share common
subsequent repair events; XPA and RPA are recruited and act as another
damage-recognition complex that may participate in lesion verification. TFIIH is
then recruited. This protein complex is involved both in transcription and in
DNA repair. XPB and XPD, the component helicases of TFIIH, unwind the DNA
duplex that surrounds the DNA adduct, prior to the incision step. Subsequently,
XPF-ERCC1and XPG, two structure-specific endonucleases, incise on the 5' and
3' sides of the DNA adduct, respectively, to remove a 24- to 32-base
oligonucleotides containing the lesion.97 These steps are irreversible.
Cisplatin-DNA adducts are removed primarily by the NER pathway in
vitro and in vivo.43 1 98-200 It has been suggested that the favorable clinical
response of testicular cancer to cisplatin reflects its intrinsically low capacity for
removal of cisplatin-DNA adducts and low levels of nucleotide repair proteins
XPA, ERCC1 and XPF.2mThese observations are consistent with the notion that
52
modification of DNA by cisplatin is the major mechanism of its cytotoxicity and
that the dominant cisplatin lesions are primarily corrected by the NER pathway.
Human cells lacking functional p53 exhibit partial deficiency in GGR.202
The ability of p53 to modulate GGR is mediated through its ability to control the
expression of p48 (also called DDB2). BRCA1 upregulates p48 expression in a
p53-depedent manner following cisplatin treatment.2 0 5206 p48 is involved in
DNA damage recognition that is missing in most XPE cell lines and in CHO cells.
In addition, GADD45, a protein downstream of p53, plays a role in GGR,
coupling chromatin assembly and DNA repair. Overexpression of GADD45
protects cells from cisplatin, whereas inhibition of GADD45 expression by
antisense DNA results in GGR deficiency and increased cell sensitivity to
20 7 2 08 On the other hand, the role of p53 in TCR is somewhat
cisplatin.
controversial. Some laboratories reported that p53 mutant cells retained the
normal TCR activity,2 0 2 whereas others showed that p53 also functions in
204 This discrepancy may reflect differences in damage type and the
TCR.23,
method used to monitor repair.
The mismatch repair (MMR) process involves the recognition of base pair
mismatches or other DNA damage and assembles a multimeric complex that
coordinates subsequent repair events. The MMR pathway also activates other
cell-cycle regulators such as p53, p7 3 or c-Abl, mediating the induction of cell
cycle arrest and apoptosis.20 9 Cisplatin-induced activation of c-Abl depends upon
functional
MSH2 and MLH1. MLH1-defective
HCT-116 cells and MSH2-
53
defective HEC-59 cells fail to activate c-Abl following cisplatin treatment; and,
cisplatin activation of c-Abl kinase activity is re-established after restoration of a
functional MMR in HCT-116 or HEC-59 cells.97 Moreover, p73 induction and
apoptosis by cisplatin are also MMR and c-Abl dependent. The induction of p73
by cisplatin is abrogated in Mlhl-null and Abl-null mouse embryo fibroblasts
(MEFs)but not in wild-type or Tp53-nullMEFs.Both Mlhl-null and Abl-null
20
MEFs show reduced sensitivity to cisplatin killing.73,
Two models have been proposed for how MMR induces cell death. One
model envisions futile cycles of repair whereas the other invokes direct signaling
of apoptosis. In the first model, MMR of mismatches containing cisplatin-DNA
guanine (G) adducts leads to the removal and resynthesis of the paired C base in
the newly synthesized strand instead of removing the cisplatin-DNA lesion,
ultimately inducing cell death. In the second model, recognition of DNA damage
by MMR proteins serves as a lesion-sensor and initiates a signal transduction
cascade through signal transducer ATM/ATR, activating the cell cycle
2 09
checkpoint and apoptosis.l1,
However, a direct connection between the MMR pathway and cisplatin
cytotoxicity has not been established. Several observations suggest that MMR
deficiency is associated with low-level cisplatin resistance.2 -24 In human
ovarian tumor cell lines selected for cisplatin resistance, the resistance phenotype
was often found to be correlated with a reduction or loss of expression of MSH2
212 -2 14 There are notable exceptions, however. Although Msh2-/or MLH1.
54
deficient cells treated with cisplatin in vivo exhibit a reduction in cell death, no
difference was occurs in the mutation rates of MMR-proficient and -deficient
2 51 216 Moreover, another in vivo study using
cells after cisplatin exposure.
immuno-histochemical staining for hMLH1 and hMSH2 revealed no association
between MMR protein expression and overall survival of ovarian cancer patients
treated with cisplatin.2 17 The most convincing evidence comes from a recent
study in which the Cre-lox system was applied to create a cell line in which the
Msh2 gene can be inactivated de novo and then reactivated to test the contribution
of MMR to the cytotoxicity of cisplatin. With this system, no changes in the
sensitivity to cisplatin were observed upon de novo inactivation of Msh2 cell lines.
In addition, the cisplatin-resistant subclones from the freshly generated MMRdeficient cell line could not be derived. Therefore, no evidence for direct
involvement of MMR deficiency in cisplatin resistance was found in ES cells.2 18
The discrepancy may result from the lack of specific genetic changes that are
needed, in addition to MMR deficiency, to affect cisplatin cytotoxicity. For
example, one report suggested that defective MMR was only a minor contributor
to cisplatin resistance, accounting for less than 1.3-fold of observed cellular
resistance, and that loss of p53 response was the main determinant of
resistance.219 Alternatively, alterations related to DNA protection and cell cycle
progression after drug damage have been also proposed as resistance
modulators instead of MMRfailure in one study using human ovarian carcinoma
cells.220
55
Cell Death Pathways: Apoptosis and Necrosis
Cisplatin induces necrosis and apoptosis, two different modes of cell
death (Figure 1.6). Necrosis is characterized by a cytosolic swelling and early loss
of plasma membrane integrity. In contrast, early features of cells undergoing
apoptosis include cell shrinkage, loss of attachment to the monolayers, chromatin
condensation, and DNA fragmentation. The mode of cell death induced by
cisplatin is concentration dependent.221 Primary cultures of mouse proximal
tubular cells undergo necrotic cell death over a few hours after treatment with
high concentrations of cisplatin (800 pM), whereas cells undergo apoptosis
following exposure to only low concentrations (8 M) of the drug over several
days.2 22 PARP has been very recently identified as a novel target for cancer
2 23 - 225 Excessive DNA damage induces hyper-activation of PARP. PARP
therapy.
cleaves the NAD+ and transfers ADP-ribose moieties (ADPR) to carboxyl groups
of nuclear proteins, thereby causing NAD+/ATP depletion and resulting in
necrotic cell death if ATP depletion reaches lethal-inducing levels.22 6
Apoptosis, or programmed cell death, is the main response of cells to
chemotherapeutic agents. Apoptosis results from activation of members of the
caspase family of aspartate-specific proteases. Upstream initiator caspases such
as caspase-8 and caspase-9 are activated early in the apoptotic process and they
then activate other downstream caspases, caspase-3 and caspase-7. The latter are
56
largely responsible for cleavage of many other cellular proteins, leading to
apoptosis.221
Two major distinct apoptotic pathways have been described for
mammalian cells. One involves caspase-8, which is recruited by the adapter
molecule Fas/APO-l-associated death domain protein to death receptors upon
extracellular ligand binding. 227 The other involves cytochrome c releasedependent activation of caspase-9 through Apaf-1.22 8 Increased levels of
cytochrome c are observed in the cytoplasm of cisplatin-treated cells, suggesting
that cytochrome c release is required for cisplatin-induced apoptosis.2 2 9
Caspase-3 plays an essential role for procaspase-9 processing and
cisplatin-induced apoptosis. Cytochrome c- and caspase-8-mediated procaspase2 3 0 -232 The apoptosis of caspase-39 processing are highly dependent on caspase-3.
deficient MCF-7 breast cancer cells is defective in response to cisplatin treatment.
Stable transfection of CASP-3 cDNA into MCF-7 cells results in increased
apoptosis after cisplatin treatment. 232 Some studies also reported that cisplatininduced apoptosis has caspase-3 independent pathways in cisplatin-resistant and
sensitive human ovarian cancer cell lines.233 Another study showed that at least
50% of cisplatin-induced
RPTC apoptosis is independent of p53 and caspases 3, 8,
and 9.23 4 Moreover, cisplatin can also up-regulate Fas and Fas ligand (FasL)
proteins. 22 7
In addition, cell density could also be a factor that modulates cisplatin
cytotoxicity. Recently it was reported that, at high cell density, there is a separate
57
cell-interdependent pathway of cisplatin killing in which damaged cells can
transmit a death signal to neighboring cells. This signal is produced within the
damaged cell by Ku/DNA-dependent
through
gap
junctions.23 5
However,
protein kinase signaling transduced
another
study reported
that
p53
transcriptional activation by DNA damage and subsequent p53-dependent
apoptosis is attenuated at high cell density, possibly through modulation of cellcell and cell-matrix interaction.2 3 6 Such molecular mechanisms remain to be
elucidated. Nevertheless, the modulation by high cell density of cisplatin
cytoxicity and cellular processing pathways is a subject of significant interest that
clearly warrants further investigation.
Cisplatin and Nucleosomes
In the eukaryotic cell, DNA is packaged in a chromatin context. The basic
unit is the nucleosome, in which the DNA is wrapped around a histone octamer.
The nucleosome modulates a variety of biological processes, such as replication,
transcription, and repair. Incorporation of DNA into chromatin alters the
dynamic and structural properties of the DNA. Therefore, nucleosome context
must be taken into consideration in order to understand fully the mechanism of
cisplatin cytotoxicity in vivo.
The influence of chromatin structure on cisplatin-DNA adduct formation
has been investigated both in vitro and in vivo. Studies showed DNA in the
linker region of polynucleosomes to be a preferred site of cisplatin DNA
58
2 37,23 8 but this apparent preference is no longer apparent at higher drug
binding,
The preference for linker DNA might be attributed to histonelevels.238,239
induced constraints on the core DNA. In addition, some studies also reveal an
influence of chromatin proteins on the extent and sequence specificity of
cisplatin-DNA binding. For example, enhanced damage in intact cells occurs at
CACC sequences where a member of the Spl family of proteins is thought to
bind, presumably by bending the DNA double-helix such that enhanced cisplatin
binding occurs.240 Moreover, cisplatin and trans-DDPshow different nucleosome
binding
preferences.
Cisplatin
targets
the
DNA, whereas
trans-DDP
23 8,2 41,242
preferentially targets the histones.
Cisplatin
modification
influences
inter-nucleosomal
DNA-protein
interactions. The inhibition of micrococcal nuclease digestion rate and the change
of digestion profile of cisplatin-modified chromatin suggest that cisplatin-DNA
adducts alter DNA-protein interactions associated with the higher order
structure of chromatin.2 43 On the other hand, micrococcal nuclease digestion
indicates that cisplatin binding does not significantly disrupt the structure within
the core particle, since for cisplatin modified nucleosome cores there is little
effect on the digestion rate and the relative distribution of DNA fragments
2 4 3 DNase I digestion assays also reveal no
produced by enzymatic cleavage.
detectable change in the DNA-protein interactions upon DNA modification.244
Nucleosome structure and DNA accessibility can be altered by several
multiprotein
remodeling
complexes or
by
covalent
post-translational
59
modification of histones. Pretreatment with TSA or SAHA, two histone
deacetylase inhibitors, increased, the killing efficiency of cisplatin, which
indicates that relaxation of the chromatin structure by histone acetylation
increases the cytotoxicity of the drug.245 Similarly, the combination of arginine
butyrate and cisplatin resulted in a concentration-responsive increase in
cisplatin-DNA adduct formation in PC-3 cells and an overall increase in
cisplatin-DNA adduct formation in three other human cancer cell lines and a
significant increase in cytotoxicity of cisplatin.2 4 6 These results suggest that
chromatin configuration can affect cisplatin adduct formation and modulate
cytotoxicity.
With histones present, both trans-DDP and cisplatin inhibit the
reconstitution of nucleosomes. Platinated DNA, however, can be successfully
incorporated into nucleosome core particles.2 38 These results suggest that
platination of histones impedes reconstitution of free DNA, and that cisplatin
may have an inhibitory role on new chromatin assembly in vivo. In addition, one
study reported that cisplatin, not trans-DDP, significantly inhibits chromatin
remodeling and transcription factor binding, as well as transcription from mouse
mammary tumor virus promoter in vivo.247 In addition, recent work from our
laboratory using DNA alone, nucleosomes prepared with native histone, and
nucleosomes prepared with recombinant, unmodified histones, each containing
the same site-specific platinum-DNA adduct, reveals that the nucleosome
60
significantly inhibits NER. Moreover, post-translational modification of histones
can modulate NER from damaged chromatin in vitro.19 8
Future Directions
An impressive body of knowledge of the cellular processing of cisplatin
has emerged in recent years, which has given us a much more detailed
understanding of how cisplatin-DNA damage is recognized, how the damage
signals are transduced, how the cell cycle arrests, and how DNA repair and
apoptosis are activated. This knowledge provides us with an important basis and
new insights to develop improved combined therapeutic strategies through
chemogenotherapy and/or rational design of new platinum-based compounds.
Disruption of certain pathways by inhibitors, activators or in combination with
drugs that modulate cellular sensitivity to cisplatin are likely to lead to clinical
benefit. DNA microarray, RNA interference, pharmaceutical intervention as well
as other combined biochemistry and genetic methods will provide us with
powerful tools for discovery of new proteins or pathways involved in the
signaling transduction network of cellular response to cisplatin. We can
anticipate more exciting discoveries in this field in coming future and, with the
commitment and backing of the pharmaceutical industry, a new generation of
platinum-based anticancer drugs should emerge.
61
Acknowledgments
This work was supported by National Cancer Institute Grant CA34992 (S.J.L).
We thank Ms. Katie R. Barnes for a critical reading of the manuscript.
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95
NH 3
/CI
/PN
NH:
Cl
Cisplatin
Guanine N7 position
H
Replication Inhibition
Transcription Inhibition
Cell Cycle Arrest
DNA Repair
Cell Death
Figure 1.1. Cisplatin-DNA adducts cause a wide variety of cellular responses.
The platinum atom of cisplatin binds covalently to the N7 position of purines
to form 1,2- or 1,3-intrastrand crosslinks, and interstrand crosslinks.
Cisplatin-DNA adducts cause a variety of cellular responses such as
replication arrest, transcription inhibition, cell cycle arrest, DNA repair,
apoptosis.
96
Copper
%W
onj
ricnlitin
Wla
PO I 64611
I
rtY4
d-W
0
Pi
*15
kV,
Ir~j
\
)
I/
/
Figure 1.2. Mechanisms of cisplatin uptake and efflux. In addition to passive
diffusion, cisplatin is also actively imported by the copper transporter Ctrlp.
Copper-transporting P-type adenosine triphosphate (ATP7B) plays a role in
cisplatin efflux. Both copper and cisplatin have the ability to reduce each
other's uptake and can trigger the degradation and delocalization of Ctrlp.
Moreover, copper and cisplatin also exhibit directional cross-resistance.
97
Nucleosome
Remodeling
ACF
~.C
F .. -Apoptosis
.... ~Aft
,
,
Irrevesible
Binding
d
ecrotic Cell
I1II~
S
' _.
Secretion
MAPK
Inflammation
MAPK
Proliferati ion
NF B
Migration
MMR
NER
recombination
Figure 1.3. HMGB1 protein has multiple roles. HMGB1 recognizes cisplatindamaged DNA and modulates NER efficiency in vitro. Moreover, HMGB1
has been connected with RAG1/2, p53, MutSa. HMGB1 has been connected
to MMR, V(D)J recommendation, p53 and MAPK pathways. It also facilitates
nucleosome remodeling and serves as a cytokine, being secreted by necrotic
and immune cells.
98
cisplatin
ATM/ATR
\K!
t
X PC
R'PA
T FIIH
P CNA
N
active
p53
C
.... ,
_
P ro-a p c)ptotic genes
BRCA1
CSB
..
Figure 1.4. The p53 pathway can partially mediate cisplatin cytotoxicity. p53
is linked to DNA repair, cell cycle arrest and apoptosis. Upon DNA damage,
p53 is activated
and
subsequently
trans-activates different sets of
downstream target genes, which in turn induce a variety of cellular
responses.
99
/
MEK1,2
ERK1/2
MKK4,7
JNK
MKK3,6
p38 MAPK
IC
Proliferation
Apoptosis
Figure 1.5. Cisplatin activates MAPK pathways. There are three
major
mammalian MAPK subfamilies: ERK,JNK and p38 MAPK. Cisplatin triggers
the activation of the ERK, JNK and p38 MAPK cascades in
tumor cells or
transformed cell lines.
100
Cisplatin
DNA damage
Transcription
Inhibition
. .--I
Cell surv
rIVal
C------------
~-f"
~
~
Cell cycle arrest
TCR
I
IhiTR I
Signal Transduction
Repairfailure
DNA repair
I
ATP/NAD*
Futile repair
Apoptosis
Depletion
Necrosis
Figure 1.6. Cisplatin induces necrosis and apoptosis, two different modes of
cell death. DNA damage arrests the cell cycle, inhibits transcription and
initiates apoptosis. Excessive DNA damage induces hyper-activation of
PARP. PARP cleaves NAD* and transfers ADP-ribose moieties (ADPR) to
carboxyl groups of nuclear proteins. It thereby causes NAD+/ATP depletion,
resulting in necrotic cell death if ATP depletion reaches lethal-inducing levels.
101
Chapter 2 Nucleotide Excision Repair from a Site-Specifically
Platinum-Modified Nucleosome*
* The work in this chapter has been published in Biochemistry,42(22), 6747-6753
(2003).
102
ABBREVIATIONS
NER, nucleotide excision repair; CHO, Chinese hamster ovary; XPA,
xeroderma
pigmentosum
pigmentosum
complementation
complementation
group
group
A;
XPF, xeroderma
F; ERCC, excision repair
cross
complementing; GTG-Pt, DNA probe containing a site-specific intrastrand
cisplatin cross-link at a single d(GpTpG) site; GG-Pt, DNA probe containing a
site-specific intrastrand cisplatin cross-link at a single d(GpG) site; PMSF,
phenylmethylsulfonyl fluoride; DTT, dithiothreitol; bp, base pair(s); IE HPLC,
ion exchange high performance liquid chromatography; AAS, atomic absorption
spectroscopy; PAGE, polyacrylamide gel electrophoresis; TBE, Tris-borateEDTA; BSA, bovine serum albumin.
103
Introduction
Cisplatin, cis-diamminedichloroplatinum(II), and other platinum-based
drugs such as carboplatin, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (Figure 2.1), are used to treat testicular tumors as well as a variety of
other cancers (1, 2). DNA is the principal cellular target of these compounds (3),
the platinum atom forming covalent bonds to the N7 positions of the purine
bases. The major adducts are 1,2-intrastrand and 1,3-intrastrand cross-links (4-6).
Although cisplatin and carboplatin both form these identical bifunctional
adducts, the relative frequency of individual platinum-DNA cross-links is quite
different. For cisplatin, 1,2-intrastrand adducts comprise 50-90%, and 1,3intrastrand 10-25%,of all DNA lesions in cultured Chinese hamster ovary (CHO)
cells treated with the drug. The percentage of 1,2- and 1,3-intranstrand crosslinks for carboplatin treatment of the same CHO cells is 35-50% and 30-40%,
respectively, however
(6). Since carboplatin is widely used in cancer
chemotherapy, it is important to study both 1,2- and 1,3-intrastrand cisdiammineplatinum(II) cross-links formed by the two compounds.
The cellular events that are triggered by platinum-DNA damage are the
subject of considerable interest, and detailed knowledge of these processes could
facilitate the rational design of better platinum-based drugs. For example, the
platinum compounds with different ligands which block DNA repair and
transcription more efficiently, will be considered as a strategy for designing new
drugs. Cells deficient in DNA repair are hypersensitive to cisplatin, suggesting
104
that repair plays an important role in the molecular mechanism of the drug (7, 8).
To date, most studies of platinum-DNA adducts have used platinated DNA
substrates, with little work having been done with nucleosomal DNA (9, 10). In
eukaryotic cells, however, DNA is packaged into chromatin. The fundamental
unit of chromatin structure is the nucleosome, comprising a core particle and
linker DNA (11). X-ray crystal structure analysis of the nucleosome core particle
has revealed how the histone octamer (H2A, H2B, H3, H4)2 is assembled and the
wrapping of a 146 bp DNA duplex around the outside of this core as a shallow
superhelix (12). Nucleosomes modulate many cellular processes, including DNA
recombination, replication, transcription and repair. (11, 13-15). It is therefore of
interest to investigate the effects of platinum-DNA adducts at the nucleosome
level, which is more physiologically relevant, in order to achieve a full
understanding of the mechanism of action of cisplatin and its analogs.
Two major classes of chromatin-modification factors that increase DNA
accessibility have been recently identified. The first class, chromatin-remodeling
complexes such as SWI2/SNF2, ISWI and Mi, alter histone-DNA interactions by
utilizing energy released from ATP hydrolysis (16). The second class of factors
alters DNA-histone interactions through covalent modification of histones,
including acetylation, phosphorylation, methylation and ubiquitination (17-19).
In the present study, we address how nucleosome structure modulates
nucleotide excision repair of platinum-DNA adducts. Since we wished to learn
the extent to which nucleosome structure affects the excision of different types of
105
platinum-I)NA
adducts, it was necessary to prepare substrates containing
specific adducts. Accordingly, two site-specifically platinated nucleosomes were
prepared, each containing a single 1,2-d(GpG-Pt) or 1,3-d(GpTpG-Pt) intrastrand
cis-diammineplatinum(II) cross-link. In vitro repair assays using these substrates
were carried out to compare the effect of the nucleosome core on the efficiency of
platinum removal from DNA. In addition, we studied platinum excision from
core particles reconstituted with either native or recombinant histones in order to
determine their relative activities as substrates. The results, which are described
herein, reveal the first direct evidence linking nucleotide excision repair of
platinated mononucleosomes with post-translational modification of histones.
Materials and Methods
Materials. Cisplatin was obtained as a gift from Johnson-Matthey. T4
polynucleotide kinase and T4 DNA ligase were purchased from New England
Biolabs (Beverly, MA). Phosphoramidites and chemicals for DNA synthesis were
obtained from Glen Research. y-32P-ATPwas purchased from ICN Biomedicals.
Synthesis of Platinated Oligonucleotides. All oligonucleotides
were
synthesized on a 1 unol scale using an Applied Biosystems DNA synthesizer
(Model 392) and
purified
by conventional
methods.
The platinated
oligonucleotide 20-mers, 20GG-Pt or 20GTG-Pt, were prepared as described
previously (20). Briefly, a 179-p.L aliquot of cisplatin (0.84 mM) was mixed with a
106
100-pL portion of the oligonucleotide (1.50 mM) in a 1:1 ratio in 0.01 M sodium
phosphate buffer (pH 6.75). The mixture was incubated in the dark at 37 °C for
12 to 14 h. The platinated oligonucleotides were purified by ion exchange HPLC
and characterized by mass spectrometry, UV spectroscopy, and atomic
absorption spectroscopy (AAS).
Characterizationof Oligonucleotidesby UV-vis Spectroscopy,AAS, and Mass
Spec. DNA concentrations were determined by UV absorption at 260 nm using a
Hewlett Packard 8453 UV-vis instrument. The concentrations of bound platinum
were measured on a Perkin Elmer Analyst 300 atomic absorption spectrometer
equipped with a Perkin Elmer Pt lumina lamp. The ratio of bound platinum to
DNA was calculated by Pt concentration divided by DNA concentration. The
molecular masses of platinated oligonucleotides were conformed by MIT
biopolymers lab using mass spectrometry.
Preparationof Site-SpecificallyModifiedPlatinum-DNA Probes.The synthesis
of DNA duplexes containing a unique, site-specific platinum-DNA adduct was
performed by enzymatic ligation of three sets of complementary oligonucleotides
(Figure 2.2), as previously described (20, 21). Briefly, six fragments were
annealed and ligated. The ligation products were then separated by denaturing
PAGE, re-annealed, and purified by non-denaturing PAGE. The resulting DNA
probes contain internal labels with
32p
at the 10th phosphodiester bond 5' to the
d(GpG) cross-link and at the 9th phosphodiester bond 5' to the d(GpTpG) crosslink (Figure 2.2).
107
Cell-FreeExtract Preparation.Cell-free extracts were prepared from CHO
AA8 cells by the method described previously (22).
Native Histone OctamerPreparation.Native histones H2A, H2B, H3, and H4
from HeLa S3 cells were isolated and purified as reported (23, 24). HeLa cells
were grown in suspension with Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum at 37 C. Thereafter, the cells were
harvested and used for whole cell extract preparation according to a published
procedure (22). The remaining nuclear pellets were lysed with a glass
homogenizer and washed with 0.4 M and 0.6 M NaCI solutions. The washed
nuclear pellets were adsorbed onto a dry hydroxylapatite resin. Subsequently,
the core histone proteins were eluted with 2.5 M NaCl.
Expression and Purification of Recombinant Histone Proteins. Expression
plasmids encoding each of the four histone proteins were transformed into E. coli
BL21(DE3)cells and expressed under the control of a T7 promoter. The histones
were then purified by following literature procedures (25). The histone octamer
was refolded and the solution was separated by gel filtration on a 16/60
Superdex 200 (Pharmacia) column. The purified histone octamer was analyzed
by 18% SDS-PAGE.
NucleosomeReconstitution and Purification. The radioactively labeled 199bp DNA probes were assembled into nucleosomes with the histone octamer as
described previously (26). A 1 pmol quantity of DNA substrate was incubated
with the HeLa core histones in a 1:1 molar ratio, 1 g of BSA, and 2 M NaCI in a
108
final volume of 10 A.Lfor 15 min at 37 C. The reaction mixture was serially
diluted by adding 3.3, 6.7, 5.0, 3.6, 4.7, 6.7,10, 30, and 20
L portions of 50 mM
HEPES (pH 7.5), 1 mM EDTA, 5mM dithiothreitol (DTT), and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) at 15 min intervals over a period of 2.5 h
incubating at 30 °C. The resulting solution was reduced to 0.1 M in NaCI by
adding 100 gL of 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 0.1% Nonidet P-40,
5 mM DTT, 0.5 mM PMSF, 20% glycerol, and 100 pg/mL of BSA, and incubated
for 15 min at 30 C. After reconstitution, the mononucleosomes were purified
from free DNA by centrifugation through an 11-mL, 5-25% sucrose gradient in
10 mM HEPES-KOH (pH 7.9)-1 mM EDTA-0.1% Nonidet P-40 using a SW41
rotor (25,000rpm,
18 h, 4
C) according to published
methods
(27).
Reconstitution products and fractions separated by sucrose gradient were
analyzed
by
polyacrylamide;
non-denaturing
polyacrylamide
1X Tris-borate-EDTA
gel
electrophoresis
[TBE]) (28). Fractions
(6%
containing
mononucleosomes were used for the excision assay, which measures the release
of 24 to 32 nt-long oligomers carrying the damage site (29, 30). The recombinant
nucleosomes were prepared in the same manner.
ExcisionRepairAssay. Cell-free extracts from CHO AA8 cells were used to
measure excision with the 199-bp DNA substrates in the form of a
mononucleosome or free DNA as described elsewhere (28). Briefly, in a typical
excision assay, 1.5 to 3 fmol of substrate DNA (either free DNA, native
nucleosome or recombinant nucleosome) were incubated with 50 pg of cell-free
109
extracts at 30 C in 25 pL of excision repair buffer (32 mM HEPES-KOH, pH 7.9,
64 mM KCI, 6.4 mM MgC12, 0.24 mM EDTA, 0.8 mM DTT, 2 mM ATP, 0.2
mg/mL BSA, 5.5% glycerol, 4.8% sucrose). The reaction mixtures were incubated
at 30 C for 2 h to 4 h. The reaction products were purified by phenol-chloroform
extraction and analyzed on a denaturing PAGE gel (8%polyacrylamide; 1XTBE).
The extent of excision was determined by measuring the levels of radioactivity in
the bands of excised products (20-30 nt range) and unexcised substrate with a
phosphorimager and ImageQuant system (Molecular Dynamics).
Results
Synthesis and Purificationof DNA 199GG-Pt and 99GTG-Pt Probes.The
oligonucleotides were prepared and purified by PAGE and the purity was
confirmed by HPLC and mass spectrometry. The platinated oligonucleotides
(GG-Pt and GTG-Pt) were characterized by UV-vis spectroscopy, AAS (31), and
mass spectrometry. The 199-mer was synthesized by modification of a previously
described strategy (Figure 2.2) (21). The ligation products were then purified by
PAGE (Figure 2.3), and the bands corresponding to the 199-mer (Figure 2.3A)
were excised and eluted. The double stranded 199-mer (ds-199-mer) was purified
by non-denaturing PAGE (Figure 2.3B). The total yield of ds-199-mer ranged
from 1-5% based on starting material.
Reconstitution and Purifcation of Nucleosomes.The recombinant histone
octamer was refolded and purified. The SDS-PAGEgel analysis revealed that the
110
individual recombinant histones and recombinant histone octamer are > 95%
pure (Figure 2.4). The internally
3 2P-labeled DNA
probes and octamer were
reconstituted into nucleosomes with native or recombinant histones. The
reconstituted nucleosomes were separated from free DNA by sucrose gradient
centrifugation. Analysis on a non-denaturing gel (Figure 2.5) revealed that the
nucleosome band migrates more slowly than its DNA component, in agreement
with previous work (28). The GTG-Pt and GG-Pt modifications did not
significantly alter the migration of either free or nucleosomal DNA in the gel.
The presence of a sharp band in lanes 2 and 4 (Figure 2.5) indicates good
substrate positioning of the nucleosomal DNA wrapped around the histone
octamer.
ExcisionAssay on NucleosomalDNA. The nucleosomal DNAs were used as
substrates for in vitro nucleotide excision repair under the same conditions as for
free DNA. The reaction products were analyzed by 8% denaturing PAGE (Figure
2.6A). Comparison of repair signals for free and native nucleosomal DNA
containing the same cisplatin adduct clearly indicates that the nucleosome
inhibits excision under our assay conditions. For the free GTG-Pt DNA
substrates, the excision is about 10% + 2% (averaged results from 4 experiments)
of total DNA, whereas for the native nucleosomal GTG-Pt DNA, the excision is
only 1.2% + 0.4% (averaged results from 3 experiments). Upon changing from
free GG-Pt DNA to native nucleosomal GG-Pt DNA, the excision signal
decreases from 1.1% + 0.3% (averaged results from 4 experiments) to 0.35% +
111
0.14% (averaged results from 3 experiments) of total DNA. Thus, the efficiency
of excision repair of nucleosomal DNA GG-Pt is about 33% of free DNA GG-Pt,
whereas the efficiency of excision of nucleosomal DNA GTG-Pt is about 12% that
of free DNA GTG-Pt (Figure 2.6B).
To investigate the role of histone post-translational modification in
modulating NER of platinum-DNA adduct at the mononucleosome level, native
histone octamers composed of post-translationally modified histones and
recombinant histone octamer composed of non-modified histones were prepared
and purified. In vitro nucleotide excision repair of the native and recombinant
nucleosomal DNA probes were carried out under the same conditions. The
reaction products were analyzed by 8% denaturing PAGE (Figure 2.7).
Comparison of excision signals for native and recombinant nucleosomal DNA
containing the same cisplatin adduct clearly indicates that the efficiency of NER
of native nucleosomal DNA GTG-Pt is 2.5 + 0.4 fold higher than that of
recombinant nucleosomal DNA GTG-Pt. Similar stimulation of NER (2.2 + 0.4
fold) was also observed from native nucleosomal GG-Pt, compared to that of
recombinant nucleosome GG-Pt (data not shown).
Discussion
In this work we prepared site-specifically platinated mononucleosomes
and compared the relative excision of platinum damage from free versus
112
nucleosomal DNA, and from nucleosomes reconstituted with native versus
recombinant histone core proteins.
Comparisonof NER from Free vs NucleosomalDNA. This study reports the
first synthesis and characterization of mononucleosomes containing site-specific
2 + d(GpG) or d(GpTpG) intrastrand cross-links. With the use of
cis-(Pt(NH3)2}
these substrates, we found the excision of damage from nucleosomal DNA to be
significantly less than that from free DNA. This observation reveals that
nucleosomes protect platinum-DNA adducts from being repaired. There are
several possible reasons for this result. First, core histone-DNA interactions
reduce the association constants for damaged DNA-binding proteins of the
repair complex by tenfold (24, 28). Second, the site of damage might be shielded
by the histone octamer surface or histone protein tails, thus limiting access of the
repair machinery. Third, the nucleosome significantly alters DNA structure.
Nucleosomal DNA is overwound by about 0.3 bp per turn compared to free
DNA (12).Finally, the distortion caused by the cisplatin adducts on nucleosomal
DNA might differ from that produced by the same adduct on free DNA, thus
lowering the overall recognition efficiency of damage sensor proteins. Taken
together, these effects can limit the ability of NER proteins to access or recognize
the platinum cross-link owing to a modulation of nucleosome structure. This
result is consistent with the previous studies of UV- or AAF-damaged
nucleosomes (24, 28, 32, 33).
113
Our results are also in accord with the observation that repair in actively
transcribed genes is more rapid and efficient than in non-transcribed regions of
the genome (34). We suggest that one possible factor contributing to this
phenomenon of transcription-coupled repair is that, in the transcribed gene,
when damage is in the template strand, DNA is more exposed compared to its
tight packaging in nucleosomes comprising chromatin of inactive genes (35).
The degree of nucleosome protection differs for the GG-Pt and GTG-Pt
cross-links. This difference probably reflects the degree of distortion of GG-Pt
and GTG-Pt adducts. Previous structural studies reveal that DNAs containing
GG-Pt and GTG-Pt lesions are strikingly different (2, 36). Gel electrophoresis
experiments indicate that the 1,2-d(GpG) cisplatin adduct bends the DNA helix
by 32-34° towards the major groove of DNA and unwinds the DNA helix by 13°,
whereas the 1,3-d(GpTpG) adduct bends 35° and unwinds the DNA helix by 230
(37, 38). Further confirmation is provided by X-ray and NMR structural studies
(39, 40). The NMR structure of a single 1,3-d(GpTpG) cisplatin adduct shows it to
be more distorted than the structure of DNA containing a single 1,2-d(GpG)
cross-link (41-44). The central T is extruded into the minor groove and base
pairing is lost at the 5' G and central T/A base pair (44). These significantly
different structural distortions between 1,2- and 1,3-intrastrand cross-links are
already known to produce differential recognition and processing by cellular
proteins (45). The nucleosome modulation adds another level of complexity, the
114
detailed nature of which must await the availability of structural information on
site-specifically platinated nucleosomes.
Comparison of NER from Nucleosomes zwith Native vs Recombinant Histones.
Repair synthesis of UV-damaged DNA is enhanced within nucleosome cores of
hyperacetylated chromatin in butyrate-treated human cells (46). The origin of
enhanced repair synthesis was not identified, however, and could have arisen
from a greater population of minor UV photoproducts, such as pyrimidinepyrimidone (64) dimers, in the highly acetylated nucleosomes. The substrates
employed in this study were globally modified and then digested to
mononucleosomes. Since the DNA sequence differs for each mononucleosome,
the types, the amount, and the locations of UV damage are also variable. Thus it
is difficult to evaluate the effects of post-translational modification on a specific
type of DNA damage or location within the nucleosome.
The present study, which reports the first reconstituted, site-specifically
platinated mononucleosome containing either recombinant or native, and
therefore post-translationally modified histones, provides a powerful tool to
investigate these above questions. In our biochemically well-defined system, the
excision efficiency of the native nucleosomes is significantly greater than that of
recombinant nucleosomes, revealing that overall post-translational modification
of histones stimulates NER in this context.
There are at least three possible reasons for this effect. First, histone
modification changes the nucleosome structure and surface environment, and
_·
115
thus can improve the accessibility of nucleosomal DNA to the repair apparatus.
Acetylation occurs on lysine residues at the basic, N-terminal tail domains of the
core histones. One consequence of acetylated N-terminal tails is a reduced
affinity for DNA owing to charge neutralization, thus destabilizing chromatin
structure (for reviews, see (47, 48)).
Second, the histone modification can recruit remodeling factors. Transient
histone hyperacetylation acts as a signal for ATP-dependent remodeling
complexes at the PHO8 promoter in vivo (49). Moreover, acetylation of histone
H4 K8 mediates recruitment of the SWI/SNF complex to DNA (50), and it was
recently reported that SWI/SNF can stimulate excision repair of human excision
nuclease in the mononucleosome core particle, made with post-translationally
modified histones, by increasing DNA accessibility (24). Given these findings, we
conclude that histone covalent modifications, nucleosome remodeling and
nucleotide excision repair are linked processes.
Third, histone modification also enlists proteins involved in NER. Access
of DNA repair machinery to UV lesions within chromatin is facilitated by TBPfree-TAFii complex (TFTC) via covalent modification of chromatin. A subunit of
TFTC (SAP130)shares homology with the large subunits of UV-damaged DNA
binding factor (DDB). TFTC is recruited to UV damaged DNA in parallel with
the nucleotide excision repair protein XP-A. The fact that TFTC can recognize
UV-damaged DNA and preferentially acetylate nucleosomes assembled on UVirradiated DNA suggests a possible role for TFTC in making the DNA damage
116
accessible for repair in the context of chromatin (51). In addition, CREBbinding
protein (CBP) and p300 histone acetyl-transferases can interact with the small
subunit of XP-E damage-specific DNA binding protein (DDB) (52). Taken
together, these findings may indicate that selective acetylation of histones on
damaged nucleosomes may provide a general strategy for recruiting NER factors
more efficiently to overcome the nucleosome barrier to excision repair. These
three possibilities are not mutually exclusive. Our results are consistent with the
previous studies of the function of histone post-translational modification and
ability to stimulate transcription (53-55). It is likely that cells utilize posttranslational modifications as a signal or factor, through some common
mechanism, to regulate NER and transcription on chromatin.
The relative stimulation of NER by post-translationally modified histones
is similar for both GG-Pt and GTG-Pt cross-links. This result indicates that
histone modification is likely to affect overall nucleosome structure in a general
rather than adduct-specific manner.
The present experiments were carried out at the mononucleosome level in
vitro. Histone modifications can significantly alter higher order nucleosome
packing, however, and thus change the accessibility of nucleosome to NER (for a
review, see (17, 18)). We suggest that post-translational modification of histones
will have an important effect on NER in vivo, as has already been demonstrated
for transcription regulation (53-55).
117
Conclusion. With
the use of site-specifically platinated
DNA in
mononucleosomes, we determined that nucleotide excision repair in mammalian
cell extracts is substantially diminished compared with free DNA containing the
same adducts. A comparison of the extent of repair of native and recombinant
nucleosome substrates revealed excision from native nucleosomal DNA to be
about 2-fold higher than the level observed with recombinant, unmodified
nucleosomal DNA. This result indicates that post-translational modification of
histones can play a key role in modulating nucleotide excision repair of
platinum-DNA adducts in chromatin. The relative effect of the nucleosome does
not depend on the nature of the adduct. The in vitro system we established in
this study will facilitate investigation of other cellular processing of platinumDNA damage and help to elucidate the role of specific post-translational
modifications in NER of platinum-DNA adducts at the nucleosome level.
Acknowledgment
We thank J. T. Reardon for providing AA8 cell extracts and A. Danford for
assistance in purifying some of the oligonucleotide fragments. We thank Dr. K.
Luger for providing histone plasmids. We also acknowledge members of our
laboratories for helpful discussion and comments on the manuscript.
118
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A.
H3N\
Cl
H3 N
Cl
B.
H3N\
H3N
/
Figure 2.1. Structures of cisplatin and carboplatin. A: cisplatin, cis-diamminedichloroplatinum(II). B: carboplatin, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II).
123
A
B
C
D
E
P
DNA
-I
p Lization
199mer:
A:83rmer; B20GG-Pt or 20GTO-Pt; C: 96er,
D:72rter, E:4OCC or40CAC; F: 87mer.
FRAGMENT A:
GCTrGACAACAAAAAGATTGTCTmTCTGACCAGATGGACGCGGCCACCTCAAAG
TCACCGCGGGCCAGGTGAATATCA
GCA
FRAGMENT B:
2000GG-PT:AATCCTCCTGGTTTTCCAC
20GTG-PT:AATCCTCCGTGTrT'CCAC
FRAGMENT
C:
GTATTATGAATrCAGCTGCTCGAGCTCAATTAGTC
A ACCCCAACAGCTGGAGAAAA
GCGACCCGCGGGCCCCGGCAGGCOGCTCAAGCAGGAG
FRAGMENT D:
C TGGCC CGCGO TGATGCCTTTAGGGTGGCCGCG
TCCATCTGG TC AAAAAOACAATCTT
rTTTTGTC AAG
FRAGMENT
E:
40CC: TCATAATACGTGGAAAAACCAGGAGGArTGATATCAC
40CAC: TrCATAATACGTGGAAAAACACGGAGGATTITGATAITCAC
FRAGMENT
G
F:
CTGCGCCTGCCCCGCGGGTCGCTG'TCTCTCCAGCTGTTGGGGTC
TGACTAATTGAGCTCGAGCAGCTGAA
Figure 2.2. Strategy for synthesizing site-specifically platinated DNA probes. Six
synthetic oligonucleotides, one bearing the platinum intrastrand cross-link and a
5'-32pradiolabel denoted by an asterisk, were annealed and ligated as shown.
Sequences of the individual strands are depicted.
124
1
A.
2
' 04[
199 nt
B. 12
3
4
2
3
4
1
B.
aj
400 bp
300 bp
· 200 bp
0,
_
Figure 2.3. (A) Denaturing
_
100bp
PAGE gel of ligation products of site-specifically
platinated DNA probes. Lane 1: ligation reaction of 199-mers DNA GG-Pt; lane 2:
ligation reaction of 199-mer DNA GTG-Pt. The 199-mers are shown by the arrow.
(B) Non-denaturing
PAGE gel of purified site-specifically platinated
ds-DNA
probes. Lane 1: ds-199-mer DNA GG-Pt; lane 2, ds-199-mer DNA GTG-Pt; lane 3,
25 bp DNA ladder; lane 4, 100 bp DNA ladder.
125
1
2
3
4
5
ISOM.WOMM-~
Figure 2.4. SDS-PAGE gel of recombinant histones and histone octamer. Lane 1,
H2A; lane 2, H2B; lane 3, H3; lane 4, H4; lane 5, octamer.
126
1
2
3
4
Nucleosomal
DNA
I
Free
DNA
Figure 2.5. Non-denaturing PAGE gel of nucleosome and free DNA. Lane 1: free
199GG-Pt DNA; lane 2: nucleosomal
199GG-Pt DNA; lane 3: free 199GTG-Pt
DNA; lane 4: nucleosomal 199GTG-Pt DNA.
127
t
A
D
4
'
A.
¢'I'
_l,_ .
FV pi
IA
be
1Zi
...
(C-!'
-
.....i.4 ... --
._..
e
a
;:w I,
4OW 0II0%
040,16 -
e( E.I]a
ID1 t
r
. I.0%.
a.
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0,2^
:*
;
in
dfe
Figure 2.6. (A) Excision assay with nucleosomal and free DNA. Lane 1:
nucleosomal
199GG-Pt DNA; lane 2: free 199GG-Pt DNA; lane 3: NER
nucleosomal 199GTG-Pt DNA; lane 4: free 199GTG-Pt DNA. The numbers the
bottom of each lane are the excision percentages of the repair reactions. (B)
Efficiency of excision of cisplatin lesions from nucleosomal DNA. The efficiency
of excision of nucleosomal DNA GTG-Pt is about 12% of free DNA GTG-Pt,
whereas the efficiency of nucleosomal GG-Pt is about 33% of free DNA GG-Pt.
128
1
2
Excision
..,.
.·
.....-.
,.~
Figure 2.7. Analysis of repair of native and recombinant nucleosomal platinated
DNA by the excision assay. Lane 1: native nucleosomal 199GTG-PtDNA; lane 2:
recombinant nucleosomal 199GTG-Pt DNA. The efficiency of excision from
native nucleosomal DNA was 5.3% (lane 1) and from nucleosomes reconstituted
from recombinant histones was 2.2% (lane 2). The absolute per cent efficiency in
lane 1 cannot be compared with that in lane 3 of Figure 2.6 because of different
incubation times, buffer conditions, and batch of cell extract.
____
____
129
Chapter 3 Nucleotide Excision Repair of Site-Specifically
Platinum-Modified Tetrasomes
130
Introduction
The nucleosome, the basic structural unit of chromatin, is composed of a
histone (H3/H4)2 tetramer flanked by two H2A/H2B dimers, around which is
wrapped 146 base pairs (bp) of DNA.(1) The histone octamer is maintained by
hydrogen bonds between the (H3/H4) 2 tetramer and the two H2A/H2B
dimers.(2) The interaction of the H2A/H2B dimer with the (H3/H4) 2 tetramer is
significantly weaker than the interaction between histones H3 and H4 within the
tetramer.(2,3) An H2A/H2B dimer exhibits different dynamic behavior
compared to the (H3/H4) 2 tetramer, being more easily released and exchanged
from the octamer in vivo and in vitro.(4-6) In addition, the turnover rate of H2A
and H2B is also faster than that of H3 and H4.(7-9)
The (H3/H4) 2 tetramer plays a central role in maintaining the structural
integrity and positioning of the nucleosome core particle.(10-12) The tetrasome
particle, formed by a histone (H3/H4) 2 tetramer and DNA, is similar in overall
dimensions to the entire nucleosome core particle. The full 1.75 superhelical
turns of DNA can be maintained. The shielding effects of the (H3/H4) 2 tetramer
are sufficient to allow for the generation of many nuclease resistant fragments
that are characteristic of complete nucleosomes.(13) Nuclease digestion results
reveal that the (H3/H4) 2 tetramer is bound to, and protects the central 70-80 bp
region, of nucleosomal DNA.(14,15)
131
During DNA replication, the H3 and H4 histones together behave as an
entity that is distinct from histones H2A and H2B.(16-18) Newly synthesized H3
and H4 deposit as a tetramer and associate with old H2A and H2B.(18) The
(H3/H4) 2 histone tetramer interacts specifically with chromatin assembly factors
and chaperones, which are required for nucleosome assembly at DNA
replication forks.(19,20)Moreover, histones H3 and H4 together with ASF1 (antisilencing function 1 protein) assemble into the RACF complex (replicationcoupling assembly factor), which mediates the assembly of newly synthesized
DNA into chromatin during DNA replication and the repair of UV damaged
DNA.(19,21)
The effects of the (H3/H4) 2 tetrasome on transcription depend upon the
nature of the RNA polymerase. Both tetrasomes and nuclesomes can serve as
absolute blocks of transcription elongation by RNA polymerase II, even in the
presence of elongation factors such as TFIIS in vitro.(22) On the other hand, the
tetrasome and nucleosome core particles exhibit different effects on T7 RNA
polymerase, a much simpler transcription system. The histone (H3/H4) 2
tetramer has little effect on transcription initiation, thus allowing efficient
transcription in vitro by T7 RNA polymerase, whereas the histone octamer
causes transcriptional inhibition mainly by blocking initiation.(15)
The presence of the nucleosome also serves as a barrier for DNA repair.
Previously we reported that nucleosomes significantly inhibit nucleotide excision
repair (NER) of cisplatin-modified DNA in vitro,(23) however, little is known
132
about the effect of the tetrasome on such repair. It is of interest to learn whether
histone tetramers are also sufficient to block the repair of cisplatin-DNA adducts.
In the present chapter, we describe the results of our studies of this process. In
addition, the {Pt(DACH))2 +-modified tetrasomes were also prepared to evaluate
how changes in the spectator ligands on platinum might affect excision repair at
the tetrasome level.
Method and Materials
Materials.Cisplatin was obtained as a gift from Engelhard. Reagents for
DNA synthesis were purchased from Glen Research. SequaGel diluent and
concentrate solutions for preparing denaturing polyacrylamide gels were
purchased from National Diagnostics. T4 polynucleotide kinase, T4 DNA ligase,
and NEB buffer 3 were obtained from New England Biolabs. y-32P-ATP was
purchased from Perkin-Elmer.
Synthesis of Platinated Oligonucleotides. All oligonucleotides
were
synthesized on a 1 pLmolscale with an Applied Biosystems DNA synthesizer
(Model 392) and
purified
by
oligonucleotides were prepared,
conventional
methods.
The platinated
purified and characterized as described
previously.(23)
Preparation of Site-Specifically Modified Platinum-DNA Probes. The synthesis
of blunt-ended DNA duplexes containing a unique, site-specific platinum-DNA
adduct was performed by enzymatic ligation of complementary oligonucleotides
133
(Figure 3.1), as previously described.(24,25) Briefly, five oligonucleotides
fragments were phosphorylated using T4 polynucleotide kinase and ATP (y32p_
ATP, where appropriate). All the oligonucleotides were annealed and then
incubated with T4 DNA ligase at 16 C for 16 h. The ligation products were
separated by denaturing PAGE. The band corresponding to the desired product
was excised from the gels. The DNA was eluted, re-annealed, and separated by
non-denaturing PAGE. The resulting DNA probes contain internal labels with
32p
at the 7th phosphodiester bond 5' to the 1,3-d(GpTpG) cross-link (Figure 3.1).
Cell-FreeExtract Preparation.Cell-free extracts were prepared from CHO
AA8 cells by the method described previously.(26)
Expressionand Purificationof RecombinantHistone Proteins.The four histone
proteins were expressed under the control of a T7 promoter in E. coli BL21(DE3)
cells and purified following published procedures.(27,28) Purified histones were
mixed
with
appropriate
molar
quantities
(equimolar
amounts
of
H2A:H2B:H3:H4 for the octamer; for the tetramer, H3:H4 = 1:1) in 7 M
guanidinium HCI, 20 mM Tris-HCl (pH = 7.5), and 10 mM DTT. The histone
protein solution was dialyzed against refolding buffer (2 M NaCl, 10 mM
Tris-HCl (pH = 7.5), 1 mM Na-EDTA, 2 mM DTT) at 4 °C through three changes
over a period of 48 h. The dialyzed histone octamer and/or tetramer solution
were then separated by gel filtration on a 16/60 Hi-Load Superdex 200 column
(Pharmacia). The purified histone octamer and tetramer were analyzed on a 420% Tris.HCl SDS-PAGE.
134
Reconstitution and Purfication of the Tetrasomeand Nucleosome.The
32p
radioactively labeled DNA probes were assembled into either nucleosome or
tetrasome with the histone octamer or tetramer, respectively, in the manner
described previously.(23) The nucleosome and tetrasome products were then
purified from free DNA by centrifugation through an 11-mL, 5-25% sucrose
gradient in 10 mM HEPES-KOH (pH = 7.9), 1 mM EDTA, 0.1% Nonidet P-40
using a SW41 rotor (35,000rpm, 18h, 4 C). The purified nucleosome and
tetrasome products were used as substrates in an in vitro nucleotide excision
repair assay.
ExcisionRepairAssay. Cell-free extracts from CHO AA8 cells were used to
measure excision with the DNA substrates in the form of a nucleosome,
tetrasome, or free DNA as described previously.(23) Briefly, in a typical excision
assay, 1.5 to 3 fmol of substrate DNA (either free, tetrasomal, or nucleosomal
DNA) were incubated with 50 gg of cell-free extracts at 30 C in 25 gL of excision
repair buffer (32 mM HEPES-KOH (pH = 7.9), 64 mM KCI, 6.4 mM MgC12,0.24
mM EDTA, 0.8 mM DTT, 2 mM ATP, 0.2 mg/mL
BSA, 5.5% glycerol, 4.8%
sucrose). The reaction mixtures were incubated at 30 C for 3 h. The reaction
products were purified by phenol-chloroform extraction and analyzed on a
denaturing PAGE gel (8% polyacrylamide; 1X TBE). The extent of excision was
determined by measuring the levels of radioactivity in the bands of excised
products (24-32 nt range) and unexcised substrate with a phosphorimager and
ImageQuant system (Molecular Dynamics).
135
Results and Discussion
The histone octamer and (H3/H4) 2 tetramer was prepared, purified and
analyzed on a 4-20% Tris-HCl SDS-PAGE (Figure 3.2). The two bands in lane 6
(histone tetramer) correspond to histone H3 and H4 with a 1:1 ratio.
The site-specifically cisplatin-modified DNA was synthesized
and
purified by 5% denaturing gel electrophoresis and subsequently on a 5% nondenaturing gel (Figure 3.3). The purified dsDNA probes were then assembled
into nucleosome and tetrasome substrates and purified by sucrose gradient
centrifugation (Figure 3.4). The resulting purified tetrasomes and nucleosomes
were used as substrates for the in vitro nucleotide excision repair assay under the
same conditions as for free platinated DNA. The reaction products were
analyzed on an 8% denaturing PAGE (Figure 3.5). For the naked 1,3-d(GpTpG)Pt DNA templates, the amount of excised product is about 16.6% + 0.3%,
determined by the ratio of the counts of the rapidly moving bands (24-32nt) to
the total counts in the lane. The excision efficiency is only 1.7% + 0.3% and 1.6%
+ 0.1%, however, for the nucleosomal and tetrasomal 1,3-d(GpTpG)-Pt DNA,
respectively. The nucleosome inhibits excision to about 10% of the level observed
with free DNA, which is consistent with our previous report with a 199mer
DNA.(23) The presence of linker DNA does not appear to be an important factor
in the inhibitory effect of the nucleosome on NER.
136
Comparison of the repair signals of free and tetrasomal DNA indicates
that the tetrasome can also significantly inhibit excision repair of DNA, the level
of inhibition being similar to that of the nucleosome. The 1,3-d(GpTpG)
platinum-DNA adduct is located at the 70-72nd nucleotides from 5' terminus of
the 146mer DNA, presumably interacting with the adjacent H3/H4 tetramer in
the nucleosome, as revealed in the previously reported nucleosome structure.(29)
The similarity in repair inhibition is therefore likely a result of the similar local
environment around the damage site within the tetrasome and nucleosome.
Similar to cisplatin-modified DNA, the site-specifically 1,3-d(GpTpG)
{Pt(DACH))2+-modified DNA and tetrasome were prepared as substrates for in
vitro nucleotide excision repair. Comparison of the excision efficiency of
2 +{Pt(DACH)}2+-modified tetrasomal and free DNA indicates that {Pt(DACH)}
modified tetrasome also significantly inhibits the excision repair (Figure 3.6). The
extent of excision from 1,3-d(GpTpG) {Pt(DACH))2 +-modified DNA is 19%,
whereas for the {Pt(DACH)}2+-modifiedtetrasomal DNA, the excision signal
decreases to 3%, or about 6-fold inhibition. In agreement with earlier reports,
2 + or cisplatin modification was excised at a similar
DNA with either {Pt(DACH)}
level in naked DNA.(30) However, comparison of the excision efficiencies of the
modified substrates reveals that of the cisplatin-modified tetrasomal DNA to be
about half (53%) that of {Pt(DACH)}2+-modified probe. The influence of 1,2-
diammineplatinum(II) spectator ligand is therefore much more significant in
tetrasomes than in free DNA. The presence of cyclohexane ring has only a minor
137
effect on the adduct geometry, even considering formation of a H-bond with a
2 +nearby guanosine, the overall structures of the cisplatin-DNA and {Pt(DACH)}
DNA complexes being very similar.(31,32) Therefore, the differential repair we
observe here is most likely a consequence of the interaction of the DACH
cyclohexane ring and the core histones. Alternatively incorporation into the
2+ structure differently,
tetrasome may modulate the cisplatin and {Pt(DACH)}
thus changing the repair efficiency.
Previous work revealed that the histone tetramer can protect from
nuclease digestion only about 70 bp of DNA, whereas the full nucleosome blocks
146 bp. The histone tetramer thus provides similar protection as the nucleosome
when the cisplatin lesion is located within the central 70 bp range. On the other
hand, the tetrasome may be less able to protect the damage site when it is located
beyond the central 70 bp range. Therefore, globally platinated DNA may be
better repaired in tetrasomes compared with nucleosomes in vivo. Moreover, the
absence of H2A/H2B dimer decreases the folding of nucleosome arrays into
higher-order structures.(33) Therefore, the change in higher order chromatin
structure that occurs by releasing H2A/H2B may also stimulate repair in vivo.
In addition to disassembling nucleosomes, the cell has other means by
which to increase DNA accessibility hence maintain efficient transcription and
repair. Two major mechanisms, covalent histone post-translational modifications
and nucleosome remodeling complexes, increase the efficiency of transcription
and repair from nucleosomes in vivo.(34,35) In particular, the (H3/H4) 2 histone
138
tetramer is important in both mechanisms. The presence of the tail domains of
core histones, especially those in the (H3/H4)2 tetramer, inhibits transcriptional
initiation.(36,37) Removal of the histone H3 and H4 tails is sufficient to restore
the access of nucleosomal DNA to TFIIIA and increases the transcriptional
activity of the 5S nucleosomal templates in vitro.(38,39) In addition, acetylation
of the (H3/H4) 2 histone tetramer is accompanied by substantial stimulation of
transcription.(39-41) Histone post-translational modifications also stimulate
repair of damage from the nucleosome.(23,42,43)In the case of the remodeling
complex SWI/SNF, which has been linked to the stimulation of transcription and
DNA repair from nucleosomes, interactions of the global domain of histone H3
are crucial for SWI/SNF association with nucleosomes.(44) SWI-SNF can also
remodel
(H3/H4) 2
tetrasomes,
although
less efficiently compared
to
nucleosomes.(45) Therefore, releasing H2A/H2B, modifying histone tails, and
remodeling by remodeling complex may act synergistically to relieve the
nucleosome barrier for repair the cisplatin-DNA adducts in vivo.
In conclusion, We demonstrate that the (H3/H4)2 tetramer is sufficient to
2+-DNA adducts in vitro;
block the excision of both cisplatin- and {Pt(DACH)}
therefore, releasing H2A/H2B alone is not sufficient to relieve the inhibition at
mononucleosome level. Cells may adopt several ways to relieve such a barrier.
2 +- and cisplatinIn addition, the tetrasome structure inhibits {Pt(DACH)}
modified differentially, which may be important for their different profiles of
anticancer activity.
139
Acknowledgment
I thank Dr. J. T. Reardon and Dr. A. Sancar for providing AA8 cell extracts
and A. Danford for assistance in purifying some of the oligonucleotide fragments.
We thank Dr. K. Luger for providing histone expression plasmids and G. Singh
for preparation of histone H3 and H4. I also acknowledge members of our
laboratories for helpful discussion and comments on the manuscript.
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144
A
B
C
_~~~Ns=I
t.,,WF^~f.9
Bf
M W
agR%%M,,
E
D
DNA
Ligation
146mer:
A: 63mer; B:14GTG-Pt; C: 69mer; D: 86mer; E: 60mer.
Fragment 63mer:
5'-ATCAATATCCACCTGCAGATTCTACCAAAAGTGTATTTGGAAACTGCTCCATCAAAAGGCA
TG-3'
Fragment 14merGTG: 5'-TTCACCGTGATTCC-3'
Fragment 69mer:
5'-CCTCAACATCGGAAAACTACCTCGTCAAAGGTTATGTGAAAACCATCTTAGACGTCCACC
TATAACTA-3'
Fragment 86mer:
CAC: 5'-ATGTTGAGGGGAATCACGGTGAACATGCCTTrGATGGAGCAGTTTCCAAATACACT
TTTGGTAGAATCTGCAGGTGGATATTGAT-3'
Fragment 60mer:
5'-TAGTTATAGGTGGACGTCTAAGATGGTTTTCACATAAACCTTGACGAGGTAGTTrTCCG-3'
Figure 3.1. Strategy for synthesizing the blunt-ended site-specifically platinated
DNA probe. Five synthetic oligonucleotides, one bearing a 1,3-d(GpTpG)
intrastrand platinum-DNA cross-link and a 5'-32P radiolabel denoted by an
asterisk, were annealed and ligated as shown. Sequences of the individual
strands are depicted.
145
1
2
3
4
5
__
__rrr~-,AAI
_
e~~~MWU
6
MIIM
Mosibw
Figure 3.2. 4-20% SDS-PAGE gel of recombinant histones, histone (H3/H4) 2
tetramer and histone octamer. Lane 1, histone H2A; lane 2, histone H2B; lane 3,
histone H3; lane 4, histone H4; lane 5; histone octamer; lane 6, histone (H3/H4) 2
tetramer.
146
1
2
Figure 3.3. Non-denaturing PAGE gel of purified double-stranded DNA 146-mer
GTG-Pt. The site-specifically platinated DNA probe was prepared by the
enzymatic ligation described in the text. The resulting products were purified by
5% denaturing PAGE gel electrophoresis and subsequently by 5% nondenaturing
PAGE gel electrophoresis.
purified ds DNA 146-mer GTG-Pt.
Lane 1, 100-bp DNA ladder; lane 2,
147
A.
2 3
1
4
5 6
7
8
9 10 1112
4
5
.·-·
.rr-
Tetrasome
Free DNA
12 3456
B.
Tetrasome
00a
I
Free DNA
1
C.
Nucleosome
Free
DNA
am
-
A
2
_"
3
-
.
6
r
MOM.wof
Figure 3.4. Non-denaturing PAGE gel electrophoresis analysis of site-specifically
cisplatin-modified tetrasome (A), Pt(DACH)2 +)-modified tetrasome (B), and
cisplatin-modified nucleosome (C). Different fractions collected from sucrose
gradient centrifugation for each sample were loaded onto the non-denaturing
PAGE gel and visualized by autoradiography.
148
1
2 3
,
.: ...
. - -.. .
Figure 3.5. Nucleosome and tetrasome inhibition of nucleotide excision repair of
cisplatin-DNA adduct. The cisplatin-modified substrate, either free (lane 1),
tetrasomal (lane 2), or nucleosomal DNA (lane 3), was incubated with a cell-free
extract at 30 C for 3h and products were analyzed on an 8% denaturing gel and
visualized by autoradiography.
149
r
fr~
'1 2"3
H3/-14 Tetranmer
-+
4
m.e sm,*
as
+
h·-i
Figure 3.6. Tetrasome
inhibition of excision repair
of cisplatin- and
{Pt(DACH)}2+-DNAadduct. The substrates Pt(DACH))2+-modifiedDNA (lane 1),
its tetrasome (lane 2), cisplatin-modified DNA (lane 4) and its tetrasome (lane 3)
were incubated with a cell-free extract at 30 C for 3h and the products were
analyzed on an 8% denaturing gel and visualized by autoradiography.
150
Chapter 4 Cisplatin Adducts Change the Rotational
Positioning of DNA on the Nucleosome
151
Introduction
In eukaryotic cells, DNA is packaged into chromatin. The nucleosome, the
basic structural unit of chromatin, consists of a histone octamer, which is composed
of a (H3/H4)2tetramer and two H2A/H2B dimers, around which is wrapped 146 bp
(base pairs) of DNA.(1, 2) Incorporation of DNA into chromatin alters the structural
and functional properties of the DNA. Nucleosomes modulate a variety of biological
processes, such as replication, transcription, and repair.(3-5) The rotational setting of
the DNA on the nucleosome core provides an asymmetric environment for the
double helix, which limits transcription factors to the bases facing away from the
histone core surface.(6-8)
Cisplatin, cis-diamminedichloroplatinum (II), is used to treat testicular,
ovarian, cervical, head and neck, esophageal, or non-small cell lung cancer.(9)
Cisplatin enters the cell through either passive diffusion or transporter-mediated
uptake.
The lower intracellular chloride ion concentration (-20 mM ) facilitates
displacement of chloride ligand by a water molecule, transforming cisplatin into an
activated, aquated form, cis-[Pt(NH
3 )2 Cl(OH2 )1+. This activated form of cisplatin
reacts readily with DNA to form a covalent bond with the N(7) atom of a purine
base.(9) Cisplatin-DNA adducts significantly distort the DNA structure by
unwinding the helix and bending it in the direction of the major groove. (9)
The influence of chromatin structure on cisplatin-DNA adduct formation has
been investigated both in vitro and in vivo. Cisplatin and trans-DDP show different
152
nucleosome binding preferences. Cisplatin predominantly targets the DNA and
forms intrastrand cross-links, whereas trans-DDPpreferentially forms histone-DNA
cross-links.(10-12)In polynucleosomes, the DNA in the linker region is the preferred
binding sites for cisplatin,(13, 14) but higher drug levels abolish this apparent
preference.(12, 13, 15) In addition, the nucleosome structure has a significant
inhibitory effect on the repair of cisplatin-DNA adducts.(16)
Understanding the interaction of cisplatin-DNA adducts with nucleosomes
and their effects on nucleosome structure and functions can contribute to the
rational improvement of the anticancer properties of the drug. Here we use
hydroxyl radical and DNAse I footprinting to characterize site-specifically cisplatinmodified nucleosomes. Preformed cisplatin-DNA adducts change the rotational
setting of DNA with respect to the histone surface during nucleosome formation.
Materials and Method
Materials.Cisplatin was obtained as a gift from Engelhard. Reagents for DNA
synthesis were purchased from Glen Research. SequaGel diluent and concentrate
solutions for preparing denaturing polyacrylamide gels were obtained from
National Diagnostics. T4 polynucleotide kinase, T4 DNA ligase, and DNase I were
purchased from New England Biolabs. Phosphoramidites and chemicals for DNA
synthesis were from Glen Research. w32 P-ATP was received from Perkin-Elmer. All
other reagents were obtained from Sigma or Mallinckrodt.
153
Synthesis of Platinated Oligonucleotides.Oligonucleotides were synthesized
using an Applied Biosystems DNA synthesizer (Model 392) and purified by
conventional methods. The platinated oligonucleotides 14GTGand 21GTG, in which
the Pt atom forms 1,3-cross-link between two guanine bases, were prepared,
purified and characterized as described previously.(16)
Preparationof 5'-End RadiolabeledSite-SpecificallyPlatinum-ModifiedDNA Probes.
The synthesis of DNA duplexes containing a unique, site-specifically platinummodified DNA adduct was performed by enzymatic ligation of complementary
oligonucleotides as previously described.(17, 18) In brief, an equimolar amount of 5'3 2 P-labeled
63-mer was combined with the site-specifically cisplatin-modified
oligonucleotide and three other oligonucleotide fragments. All fragments were
annealed and then incubated with T4 DNA ligase at 16 C for 16 h. The resulting
ligation products were separated by denaturing PAGE and subsequently isolated by
non-denaturing PAGE. Unmodified DNA probes were synthesized and purified in
the same manner as their platinated counterparts.
Nucleosome Reconstitution. The 5'-3 2 P-radiolabeled DNA probes (146GTG,
146GTG-Pt, n146GTG and nl46GTG-Pt) were assembled into nucleosomes in a
manner similar to that in a previous report, except the glycerol was excluded from
the nucleosome assembly buffer.(16) The nucleosomes were used as substrates for
footprinting assays. The 5'-3 2 P-labeled 199GTG-Pt or 199GTG mononucleosomes
were assembled into nucleosomes and purified in the same manner as described.(16)
154
Restriction Enzyme Digestion. About 1 fmol of 5'- 32 P-labeled 199GTG-Pt or
199GTG mononucleosomes or the corresponding free DNAs were treated with a
restriction enzyme (20 U EcoR I, 5 U BsaA I, or 8 U PflM I) at 37 °C for 60 min. The
reaction products were then treated with proteinase K and SDS at 30 °C for 15 min,
and then phenol extracted and ethanol precipitated. The reactions were analyzed on
an 8% denaturing PAGE gel.
DNase I FootprintingAssay. About 10 fmol of 5'-32 P-labeled 146merGTG or
146GTG-Pt mononucleosome or the corresponding the free DNA was treated with
0.2 U DNase I at 25 C for 1 min or 5 min in TE buffer (10 mM Tris 'HC1 (pH = 8.0), 1
mM EDTA). The reactions were quenched by the treatment with proteinase K and
SDS at 30 C for 15 min, followed by phenol extraction and ethanol precipitation
steps. The reaction products were analyzed on a 6% denaturing PAGE gel.
Hydroxyl RadicalFootprintingAssay. A hydroxyl radical footprinting assay was
carried out according to published methods(9, 20) with reconstituted nucleosome
substrates but omitting the step involving sucrose gradient centrifugation. About 10
fmol of 5'-32Plabeled mononucleosome or free DNA were treated with 0.6% H20 2, 1
mM Fe(II)-2 mM EDTA, and 2 mM sodium ascorbate. Reactions were quenched
with 5% glycerol. The mixtures were loaded onto a 5% native PAGE gel to separate
the nucleosome and free DNA bands. The labeled DNA substrates were excised,
purified separately and analyzed on a 6% denaturing PAGE gel.
155
Results and Discussion
Synthesis of 5'-End RadiolabeledSite-SpecificallyPlatinum-ModifiedDNA Probes.
To test the effect of cisplatin modification on nucleosome structure, two similar sitespecifically platinum-modified DNA probes and their unmodified counterparts
were synthesized by enzymatic ligation (for sequences see Figures 3.1 and 4.1). The
site of cisplatin cross-link was shifted by about half a turn of a double helix toward
the 3'-end in the nl46GTG-Pt probe compared to the 146GTG-Pt substrate, in order
to provide two different rotational configurations with respect to the nucleosome
octamer core. All four DNA probes were assembled into nucleosomes and
characterized by gel electrophoresis (Figure 4.2).
Band corresponding to the
nucleosomes were excised from the gel to provide material for the footprinting
experiments.
RestrictionEnzyme Assay. BsaA I, EcoR I and PflM I were used to probe DNA
accessibility near the center of the nucleosome core particle and of the linker region
of the 199 mer-containing nucleosome (sequence see Figure 2.1). The purified 5'-end
radiolabeled site-specifically platinum-modified nucleosome and the unmodified
nucleosome were prepared and analyzed on a 5% non-denaturing PAGE gel (Figure
4.3). The cut bands were quantified and are indicated in Figure 4.4. Both unmodified
and modified nucleosomes are cleaved with significantly lower efficiency than their
corresponding free DNAs. The platinum adducts on the free DNA did not affect the
cleavage efficiency of any enzyme, whereas the platinated nucleosome has relatively
low cleavage efficiency by PflM I and EcoR I, compared to the unmodified
156
nucleosome. This result indicates that the local distortion induced by the cisplatin
damage changes the access ibility and recognition efficiency of the nucleases for
DNA in the nucleosome. In particular, our data suggest that these sites are facing the
histone core as a consequence directing the site of platinum cross-link toward the
outside of the nucleosome particle (see below).
DNAse I Footprinting Characterizationof the Nucleosomes. The DNase I
footprinting results for nucleosome substrates 146GTG and 146GTG-Ptrevealed -10
bp periodicity, whereas a greater number of bands were observed for the DNA
substrates alone (data not shown).(21, 22) The cleavage rate of nucleosomal DNA is
much slower than that of free DNA, indicating that the core histones protect the
DNA from DNase I digestion. Comparison of the cleavage pattern from platinated
versus unmodified nucleosomes revealed differences over the region of 30 bp to 120
bp (see Figure 4.5).
Characterizationof Nucleosomesby Hydroxyl Radical Footprinting.In contrast to
DNase I, the small size of hydroxyl radicals makes them relatively non-specific
cleavage agents. The hydroxyl radical footprinting method has been used to map the
location of DNA-binding drugs, such as distamycin, actinomycin, netropsin, and
mithramycin.(23-26) Hydroxyl radical footprinting was applied here to examine the
rotational settings of the nucleosomal DNAs 146GTG-Pt and 146GTGby measuring
the accessibility of the minor groove and DNA phosphate backbone to the
solvent.(19) The cleavage pattern of nucleosomal DNA exhibited -10 bp periodicity,
a characteristic feature of the nucleosome structure,(2, 27) which is absent in naked
157
DNA. Cisplatin-modification does not disrupt the overall nucleosome structure,
since the 10 bp pattern is also observed in modified nucleosome. More strikingly, the
146GTG-Pt nucleosome exhibits three strong bands corresponding to the site of GTG
platinum cross-link ( Figure 4.6). This result indicates the DNA backbone of the
cisplatin damage site faces outward and is more exposed to hydroxyl radicals than
other positions in the modified nucleosome. Another characteristic feature is that the
peaks in the cleavage pattern are shifted about 2-3 bp towards to 3'-end of the
146GTG-Pt nucleosome for two to three helical turns near the cisplatin-DNA adduct
compared to the cleavage pattern of the unmodified nucleosome. This shift indicates
that the change in rotational setting most likely propagates to two or three adjacent
turns. In addition, the most dramatic change in the cleavage pattern occurs + 1
helical turns around the damage site indicating that the cisplatin-DNA adduct
induces a significant local distortion.
To check the effect of the cisplatin-DNA d(GpTpG) cross-link on rotational
position, we synthesized a new platinated probe n146GTG-Pt,in which the platinum
atom was placed half of a helical turn towards to the 3'-end of the 146mer; its
unmodified counterpart n146GTG was also prepared.
A nucleosome model was built up based on the solved nucleosome crystal
structure on a similar DNA sequence,(2) using MOLMOL software. As shown in
Figure 4.7, if incorporation of the cisplatin-cross-link did not alter the rotational
setting of the DNA, the sugar-phosphate backbone in the vicinity of the cisplatinDNA adduct should faced toward the inside of the nucleosome and be shielded by
158
the histone core. However, three strong bands corresponding to the GTG platinum
damage site occur in the hydroxyl radical footprinting from this new probe (Figure
4.8). As for the previous probe, the cleavage pattern peaks also shift by 1-2 bp over
two-three helical turns surrounding the site of the cisplatin-DNA adduct. Thus,
formation of cisplatin-DNA adducts changes the rotational setting by about 180°.
That DNA lesions affect the rotational setting of nucleosomal DNA seems to
be a general phenomenon. Other DNA lesions, such as UV photo-products, also
affect nucleosome rotational setting,(28, 29) as does the bisintercalating antitumor
antibiotic echinomycin and several non-intercalating groove binding ligands. DNA
rotation by 180° on the protein core are not unprecedented.(30-33)
The rotational setting of DNA in the nucleosome is determined by the
anisotropic flexibility of DNA and the helical periodicity of defined structural
properties.(34, 35) The resulting bending in a preferred direction determines which
sequences will face in or out, relative to the histone octamer.(36) Therefore, the
rotational settings surrounding a DNA lesion must be the consequence of the
combined forces of reduced DNA flexibility and the DNA bending by both the
nucleosome and the lesion. Changes of the rotational setting may be necessary in
order to lower the total energy of modified nucleosome. Some regulatory factors
can bind to nucleosomal DNA only if the target sequence is presented in a particular
rotational orientation on the histone surface.(37) It is conceivable that the
accessibility of DNA damage is modulated by changing the rotational setting for the
specific purpose of attracting transcription factors or other DNA-binding proteins.
159
The formation of DNA lesions modulates the stability of nucleosomes
containing the damaged DNA. Benzo[a]pyrene diol epoxide (BPDE) adducts
significantly enhance nucleosome formation, whereas UV photoproducts inhibit the
process for the same sequence.(38) The effects of cisplatin may depend on both
location and sequence. We observed less stable nucleosomes for 146GTG-Pt than for
the unmodified 146GTG nucleosome (shorter storage time at 4 C). However, we
observed the nucleosome containing the cisplatin-modified DNA n146GTG -Pt is
more stable than that of n146GTG (data not shown). The detailed mechanisms by
which cisplatin-adducts alter nucleosome formation remain to be elucidated.
In conclusion, we have characterized site-specifically cisplatin-modified
nucleosomes and DNA by restriction enzyme and DNase I digestion analysis, along
with hydroxyl radical footprinting. Cisplatin-DNA adducts change the rotational
settings of, and enzyme accessibility to, the DNA. These changes may contribute to
the biological properties of cisplatin.
Acknowledgment
I thank A. Danford for assistance in purifying some of the oligonucleotide
fragments and some experiments. I thank Qun Wang and Dr. T. D. Tullius for the
technical help of hydroxyl radical footprinting experiment. I also acknowledge
members of our laboratories for helpful discussion and comments on the manuscript.
160
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R. B., Solomon,
M. J., Varshavsky,
A., and Lippard,
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Wang, D., Hara, R., Singh, G., Sancar, A., and Lippard, S. J. (2003) Biochemistry
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Zamble, D. B., Mu, D., Reardon, J. T., Sancar, A., and Lippard, S. J. (1996)
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Tullius, T. D., Dombroski, B. A., Churchill, M. E., and Kam, L. (1987) Methods.
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Hayes, J. J., Clark, D. J., and Wolffe, A. P. (1991) Proc. Natl. Acad. Sci. USA 88,
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21.
Cousins,
D. J., Islam, S. A., Sanderson,
M. R., Proykova,
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Robinson, C., and Staynov, D. Z. (2004) J. Mol. Biol. 335, 1199-1211.
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S A 94, 2215-2220.
163
B
A
C
*
I
I
D
DNA
Ligation
-q
*A
nl46mer
ul
I
·
-
-
I
~~~~~s~s,~~
~i~~iu~i~~n
B i~~ta~~i~:
1·~~i~~'1A~~i:~:
A: 63mer; B:21merGTG-Pt or 21mer GTG; C: 62mer; D: 90mer; E: 56mer.
Fragment A (63mer):
5'-ATCAATATCCACCTGCAGATTCTACCAAAAGTGTATTTGGAAACTGCTCCATCAAAAGGCA
TG-3'
Fragment B (21merGTG):
5'-TTCACCCTCATTGTGCTCAAC-3'
Fragment C (62mer):
5'-ATCGGAAAACTACCTCGTCAAAGGTTTATGTGAAAACCATCTTAGACGTC
CACCTATAACTA-3'
Fragment D (90merGTG):
5'-TAGTTATAGGTGGACGTCTAAGATGGTTTTCACATAAACCTTTGACGAGG
TAGTTTCCGCATGTTGAGCACAATGAGGGTGAACATGCCT-3'
Fragment E (56mer):
5'-TTTGATGGAGCAGTTTCCAAATCAACTTTGGTAGAATCTGCAGGTGGAT
ATTGAT-3'
Figure 4.1. Strategy for synthesizing site-specifically platinated-DNA probe. Five
synthetic oligonucleotides, one bearing a 1,3-d(GpTpG)intrastrandplatinum-DNA
cross-link, were annealed and ligated as shown. Sequences of the individual strands
are depicted.
164
A.
1
Nucleosome
4-
2
3
5
...
400bp
','--- 300bp
m
m~
Free DNA
4
m
.l
-'-
200bp
r"
100bp
B.
Nucleosome
Free DNA
3
2
1
is
U
S
dm
4
so
Figure 4.2. Non-denaturing PAGE gels of nucleosomal and free DNA. A. n146GTG
(unmodified) and n146GTG-Pt (cisplatin-modified) are loaded onto a 5% nondenaturing PAGE gel. Lane 1, cisplatin-modified
nucleosome of n146 GTG-Pt; lane 2,
cisplatin-modified free DNA of nl46GTG-Pt; lane 3, unmodified nucleosome of
n146GTG; lane 4, unmodified free DNA of n146GTG. B. 146GTG (unmodified) and
146GTG-Pt (cisplatin-modified) are analyzed on a 5% non-denaturing PAGE gel.
Lane 1, unmodified nucleosome of 146GTG; lane 2, cisplatin-modified nucleosome
of 146GTG-Pt;lane 3, cisplatin-modified free DNA of 146GTG-Pt;lane 4, unmodified
free DNA of 146GTG.
165
1
Nucleosome
-
ZA
-
Free DNA
Figure 4.3. Non-denaturing
2
gel of 5'- 32 P-labeled
3
uI
nucleosomal
DNA of 199GTG
(unmodified) and 199GTG-Pt (cisplatin-modified). Lane 1, cisplatin-modified
nucleosome
199 GTG-Pt;
lane 2, unmodified
unpurified nucleosome 199GTG.
nucleosome
199 GTG; lane 3,
166
BsaAl
A
C
I
nc.M
Uncut
DNA
wlabomm
Pt
_P.
-
DNA
EcoRi
Pt
BsfaAl
Pr
Cut
4
Cut/o:
*M1O
I ,1
25 27 89 91 29 40 81 79
m
1WO
S~
B
1wo~
Position:
m
¶99r~e 1UP~I
Pt: 92
BsaAI:101
EcoRJ:l
111
PfiI :144
cOLM-
l
go
4g
28
Figure 4.4. Restriction enzyme digestion of nucleosomes 199GTGand 199GTG-Pt. A.
BsaA I and PflM I digestions of 199GTG and 199GTG-Pt nucleosomes.
B. EcoR I
digestion of nucleosome 199GTG and 199GTG-Pt. C. Locations of BsaA I, PflM I, and
EcoR I cutting sites.
167
1
I_
2
3
4
;
a.f
100bp
-am
- "y
Wx
0
50bp -.
p
'i I
A
_i Iw
II
Figure 4.5. DNase I footprinting of cisplatin-modified and unmodified nucleosomes.
Lane 1, 100-bp DNA ladder; lane 2, 10-bp DNA ladder; lane 3, nucleosome 146GTG;
lane 4, cisplatin-modified nucleosome 146GTG-Pt.
168
---
E
ti
1u
I
2s
---------------"-----
800
700
600
500
400
300
200
100
0
0
200
400
600
800
1000
1200
Migration/Pixel
(1 Pixel = 0.2 mm)
Figure 4.6. Hydroxyl footprinting of the 146GTG and 146GTG-Ptnucleosomes. Both
nucleosome substrates showed 10-bp periodicity in their cleavage pattern. The
modification sites of 1,3-d(GpTpG) (70-72) are indicated. The lengths of bands are
also indicated.
169
Figure 4.7. Model of the n146GTG nucleosome. The nucleosomal DNA is shown in
gray and the histone core particle is omitted for the clarity. The platinum-bound
guanines of 1,3-d(GpTpG) (76-78) are colored in green. The sugar-phosphate
backbone of 1,3-d(GpTpG) is buried inside.
170
r.
I
l
120
1-1
Ls-
.2
100
80
60
40
20
0
0
200
400
600
800
1000
1200
Migration/Pixel
(1 Pixel = 0.2 mm)
Figure 4.8. Hydroxyl footprinting of the n146GTG and n146GTG-Pt nucleosomes.
Both nucleosome substrates showed 10-bp periodicity in their cleavage pattern. The
modification sites of 1,3-d(GpTpG) (76-78)and the lengths of bands are indicated.
171
Chapter 5 Cisplatin-Induced Post-TranslationalModification of
Histones H3 and H4*
* This chapter was published as a paper in J. Biol. Chem. 279 (20),20622-5 (2004).
172
Introduction
Cisplatin [cis-diamminedichloroplatinum(II)],1 widely used to treat of a variety of
human cancers, binds to DNA to form covalent platinum-DNA adducts.(1,2) Platination
of the genome triggers cellular responses involving several pathways, including DNA
repair, transcription inhibition, cell cycle arrest and apoptosis, all of which require
remodeling of the structural and dynamic properties of chromatin.(3-6)
The p38 mitogen-activated protein kinase (MAPK) pathway has been implicated
in osmotic stress, cell cycle regulation, differentiation, inflammation, development and
many other biological processes.(7-12)In particular, the p38 MAPK pathway is involved
in the cellular response to various DNA damaging agents, including UV irradiation and
cisplatin.(13-15) Recently, a growing body of evidence suggests that activation of the
p38 MAPK pathway plays an important role in the therapeutic response to
cisplatin.(16,17) Cisplatin, but not the inactive stereoisomer trans-DDPand not the Pt(IV)
compound PtCl4, induces long-term activation of p38 MAPK.(16) This kinase regulates
immediate-early (IE) gene expression and other cellular responses by phosphorylating
various substrates, including transcription factors, chromatin proteins, and downstream
Ser/Thr
effector kinases.(18) Understanding
the detailed mechanism of signal
transduction following of cisplatin-stimulated cellular response could lead to the
rational design of better drugs and more efficient therapy.
IThe abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); MAPK, mitogen-activated
protein kinase; EGF, epidermal growth factor; PBS, phosphate-buffered saline; IE gene, immediate-early
gene; HAT, histone acetyltransferase; HDAC, histone deacetylases; JNK, c-Jun NH 2 -terminal kinase; ERK,
extracellular signal-regulated kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate.
173
Histone post-translational modification regulates chromatin structure and cell
division.(19-23) Phosphorylation of histone H3 at Ser 10 has dual, and opposing, roles in
interphase and metaphase. During interphase, phosphorylation of H3 at Ser 10 can
facilitate transcription
of the IE genes,(24-28) whereas
during
mitosis, such
phosphorylation facilitates chromosome remodeling and condensation.(29-31) Several
stimuli including UV irradiation, arsenite treatment, and EGF (epidermal growth factor)
bring about phosphorylation of histone H3, but until now, it was unknown whether
cisplatin could induce such phosphorylation. In present study, we investigated the
phosphorylation state of histone H3 in cells treated with cisplatin as well as the signal
transduction pathway mediating such an event. We discovered that the drug triggers
specific phosphorylation of histone H3, mediated by p38 MAPK pathway. We further
report that hyperacetylation of histone H4 also occurs. These findings reveal a direct
link between cisplatin treatment and chromosome modifications that can lead to
structural changes with attendant modulation of function.
METHODS AND MATERIALS
Reagents. Cisplatin was obtained as a gift from Johnson-Matthey. Antibodies
were purchased from Upstate Biotechnology. Sodium butyrate was purchased from
Sigma. DMEM, RPMI-1640 medium and L-glutamine were obtained from Invitrogen.
SKF86002was bought from Calbiochem.
174
Cell Culture. HeLa and MCF-7 cells were cultured in DMEM supplemented with
10% (v/v) fetal bovine serum on tissue culture plates at 37 C in a humidified
atmosphere of 5% CO2 . Ntera II cells were cultured in RPMI-1640 supplemented with
10% (v/v) fetal bovine serum and 2 mM glutamine on tissue culture plates at 37 °C in a
humidified atmosphere of 5% CO2 . Cells were grown in medium to near 90%
confluence, then pretreated with various concentrations of cisplatin for fixed times
before the preparation of nuclear extracts. For studies with the p38 MAPK inhibitor
SKF86002,cells were pre-incubated with 10 pM of the inhibitor for 1 hr before cisplatin
treatment.
Preparation of Nuclear Extracts. Nuclear extracts were prepared from cells as
described in the Clontech TransFactor Extraction Kit (PT3612-2) with a slight
modification. Briefly, HeLa cells were washed twice with 5 packed cell volumes of cold
phosphate-buffered saline (PBS). The cells were collected and transferred to clean
centrifuge tubes, then centrifuged for 5 min at 450 X g, after which the pellets were
rinsed twice with an equal volume cold PBS. The cell pellets were resuspended with 5
packed cell volumes of ice-cold lysis buffer (10 mM Hepes (pH = 7.9), 10 mM KCl, 1.5
mM MgC12,1 mM DTT, 2 mM PMSF) and incubated on ice for 15 min. The cells were
spun down and the pellets were resuspended with 2 packed cell volumes of ice-cold
lysis buffer. The cells were lysed with 10 strokes of a narrow-gauge syringe (No. 263/4).
The suspension was spun at 11,000X g for 20 min. The pellet was resuspended in a twothirds volume extraction buffer comprising 20 mM Hepes (pH = 7.9),25% glycerol, 0.42
M NaCl, 1 mM DTT, 2 mM PMSF, and 1.5 mM MgC12.The solution was homogenized
175
with the narrow-gauge syringe. The suspension was shaken for 30 min at 4 C. The
nuclear extract was spun at 20,000 X g for 5 min at 4 C. The supernatant was
transferred in small aliquots and stored at -80 °C.
Immunoblotting.Proteins were analyzed on a 4-20%SDS-PAGEminigel (Bio-Rad).
Membranes containing proteins were incubated with specific histone antibodies as
indicated in the figures and horseradish peroxidase-conjugated goat anti-rabbit or antimouse antibody separately, and were detected with the chemiluminescence ECL plus
detection system (Amersham Pharmacia Biotech). Actin was used as the loading control.
RESULTS
CisplatinStrongly Induces Histone H3 Ser 10 Phosphorylationin HeLa Cells.To study
whether cisplatin activates a pathway leading to phosphorylation of histone H3 at Ser
10, HeLa cells were pretreated with 1 gM of the drug for 16 h. HeLa nuclear extracts
were prepared and analyzed by western blotting using a phospho-specific antibody
against phosphorylated
Ser 10 of histone H3. Cisplatin strongly induces the
phosphorylation of histone H3 at Ser 10 (Figure 5.1A). A time course study revealed
that
platinum-stimulated
phosphorylation
of histone H3 at Ser 10 increases
monotonically with incubation time from 1 to 24 hr (Figure 5.1B).The phosphorylation
level of histone H3 at Ser 10 also rises with increasing concentrations of cisplatin (Figure
5.1C). trans-DDP, a stereoisomer of cisplatin that is not active against cancer, has no
significant effect on the degree of H3 Ser 10 phosphorylation compared with cisplatin
under the same experimental conditions (Figure 5.2, lane 1).
176
Cisplatin Induces Histone H3 Phosphorylationin Other Cell Lines. To address
whether this cisplatin-induced phosphorylation of histone H3 at Ser 10 is cell specific,
we also tested MCF-7 breast cancer cells and the Ntera II testicular cancer cells.
Cisplatin induces phosphorylation of histone H3 at Ser 10 in both of these cell lines.
Interestingly, the induction of histone H3 phosphorylation was significantly greater in
the Ntera II than the MCF-7 cells (Figure 5.3).
SKF86002Inhibits Phosphorylationof H3 at Ser 10 Activated by Cisplatin. Cisplatin
causes activation of p38 MAP kinase. To explore whether p38 MAPK has a role in
mediating cisplatin-induced phosphorylation of histone H3, we tested the effect of a
specific p38 MAPK inhibitor, SKF 86002, on the cisplatin-induced phosphorylation of
histone H3 Ser 10. We prepared nuclear extracts from HeLa cells pretreated or
untreated with SKF86002.The western blot results revealed that the phosphorylation
level of H3 Ser 10 induced by cisplatin is significantly reduced when cells were
pretreated with SKF86002 (Figure 5.2). This result suggests that cisplatin-induced
phosphorylation of histone H3 at Ser 10 is mediated by the p38 MAPK pathway.
Cisplatin Induces Other Histone Modifications.To explore other possible histone
modification pathways activated by cisplatin, we analyzed HeLa nuclear extracts by
western blot using several modification-specific antibodies. The results demonstrate
that cisplatin also induces phosphorylation of H3 at Ser 28 (Figure 5.4A) and acetylation
of histone H4 (Figure 5.4B).
177
DISCUSSION
In the present study, we discovered that cisplatin treatment strongly induces
phosphorylation of histone H3 at Ser 10 in HeLa cells. Phosphorylation of this amino
acid is associated with mitotic and meitotic chromosome condensation,(22,29,31) helps
to regulate transcription,(24) and is brought about by various stimuli, including TPA
(12-O-tetradecanoylphorbol-13-acetate),
EGF, UV irradiation
and arsenite.(27,32,33)
Cisplatin, a potent anti-cancer drug, forms platinum-DNA adducts and causes a variety
of cellular responses. However, a relationship between cisplatin treatment and histone
H3 modification had not previously been established.
Cisplatin activates p38 MAPK for 8-12 h in sensitive cells and transiently for 1-3
h in resistant cells.(16,17)Moreover, lack of p38 MAPK activation/function induces a
resistant phenotype in human cells.(16,17) MAP kinases may also play a role in
modifying the chromatin environment of target genes. Here we show that SKF86002,a
potent p38 inhibitor, inhibits cisplatin-induced phosphorylation of histone H3 at Ser 10.
Furthermore, the p38 MAP kinase and downstream kinase MSK-1 have been reported
to phosphorylate histone H3 Ser in vitro.(13,27) Taken together, this evidence indicates
that p38 MAPK mediates phosphorylation of histone H3 at Ser 10 in response to
cisplatin.
Our results also indicate that the inactive isomer of cisplatin, trans-DDP,fails to
induce phosphorylation of histone H3 at Ser 10. Previous work revealed that both
cisplatin and trans-DDP activate p38 MAPK differently. Only the active drug cisplatin
178
can bring about long-term activation.(16) This difference in the kinetics of activation
could account for their differential cytotoxicity.
Cisplatin also modestly induces phosphorylation of histone H3 at Ser 28.
Phosphorylation at this site can be also triggered by UV irradiation, TSA and other
stimuli.(34,35) The kinetics are very similar to those for Ser 10. Both Ser 10 and Ser 28 on
histone H3 N-terminal tails are highly conserved and occur in the same consensus
sequence, ARKS.(22)
There are two distinct modes of histone H3 phosphorylation, mitotic and
stimulus-inducible. Both Ser 10 and Ser 28 on histone H3 are highly phosphorylated on
condensed chromosomes during mitosis. The exact role of histone phosphorylation is
still not fully understood, although several models have been proposed to explain such
a process during mitosis.(30) Diverse stimuli induce rapid phosphorylation of H3 at Ser
10, which has been correlated with transcription activation of certain IE genes, by
changing the nucleosome environment or serving as a binding motif for recruiting coactivators or chromatin remodeling complexes.
Cisplatin-induced phosphorylation of histone H3 also occurs in other cell types
than HeLa. In testicular cancer cell lines, a high level of H3 phosphorylation is observed.
Since the Ntera II cell line is at least twice as sensitive to cisplatin than MCF-7 cells (data
not shown), there may be a link between H3 phosphorylation and cisplatin cytoxicity.
Acetylation of histones correlates well with transcription activation of many
genes,(36) presumably by increasing the accessibility of particular genomic regions for
transcription activator or remodeling protein complexes. The acetylation of histones is
179
under the control of histone acetyltransferases (HATs) and histone deacetylases
(HDACs). We find that cisplatin treatment induces hyperacetylation of histone H4.
Although this process could occur by a mechanism independent from that by which
histone H3 is phosphorylated, it might be that MAP kinase-dependent recruitment of
co-activators with HAT activity to sequence-specific regulatory elements contributes to
acetylation of histones, as has been reported.(37,38) Acetylation and phosphorylation of
histones are linked with transcriptional activation and facilitate DNA repair.(39,40)
Phosphorylation of histone H3 Ser 10 is also induced by UV, arsenite, EGF and
other stimuli through activation of the MAPK pathway (Table 1).(18) In general, the
extracellular
signal-regulated
kinases (ERKs) are activated by mitogenic and
proliferative stimuli, such as EGF and TPA. The c-Jun NH2-terminal kinases UNKs) and
p38 MAPKs respond to environmental stress and chemotherapeutic drugs, such as UV,
arsenite, and cisplatin. The different patterns of MAPK pathway activation caused by
these various stimuli may represent a regulatory signal by which cells response to stress
in a stimuli-specific manner.
Besides p38 MAPK, both JNK and ERK1/2 have been implicated in the cellular
response to cisplatin.(41-43) ERK was weakly activated (2-3 fold) by a 33 pM cisplatin
treatment for 24 h, whereas JNK was more significantly activated (10-fold).(41)Another
study showed no activation of ERK 1/2 in HaCaT cells following a 33-132 pM cisplatin
treatment for 4 h, whereas JNK and p38 MAPK could be activated by 33 ,uM cisDDP.(16) In addition, a third group reported no activation of ERK1/2 in HeLa cell lines
cells after a 10 M cisplatin treatment for 12 h, even though ERK activation was
180
observed at higher cisplatin concentrations.(43) Taken together, these results make it
unlikely that ERK1/2 was activated by cisplatin under the experimental conditions (1
giM)whereby H3 was phosphorylated. Although we do not exclude other kinases for
the cisplatin-induced phosphorylation of histone H3, the MAPK pathway seems to be a
prominent one.
Cisplatin-DNA adducts arrest the cell cycle.(44-46)Nucleotide excision repair
complexes are then recruited to repair the DNA damage. p38 MAPK-mediated
phosphorylation and acetylation of histones causes nucleosomal structural changes,
increasing accessibility of other proteins. These events facilitate the binding of the
remodeling complex and repair proteins to the nucleosome, thereby stimulating DNA
repair (39,40,47) and helping cells to survive cisplatin-induced stress. When DNA
damage is extensive, cells will undergo apoptosis. Phosphorylation and acetylation of
histonesleading to-the-activation-and
ranseiption -o
f I-gene will-facilitate-apeptsis-under these conditions.
ACKNOWLEDGMENT
We thank Dr. K. B. Lee and Ms. K. R. Barnes for the technical help with the tissue
culture experiments, and Prof. A. Y. Ting and Mr. C.-W. Lin for helpful discussions.
-
181
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185
Table 5.1. Stimuli that Activate a MAPK Pathway to Induce Histone Phosphorylation
Stimulus
Activation of MAPK pathway
Histone modification
cisplatin
p38
H3 Ser 10/28 phosphorylation
JNK, ERK
Unknown
UV
p38, ERK, JNK,
H3 Ser 10/28 phosphorylation
arsenite
p38, ERK2
H3 Ser 10 phosphorylation
(33,49)
EGF
ERK
H3 Ser 10 phosphorylation
(32,38)
TPA
ERK
H3 Ser 10/28 phosphorylation (25)
(48)
186
A
1
2
B
t(hr)
C
Cisplatin:
1
2
3
4
5
0
1
4
8
12 24
1
0
2
3
4
6
5
0.2 1.0 10 40
(jM)
Figure 5.1. Cisplatin-induced phosphorylation of histone H3 at Ser 10. HeLa nuclear
extracts were analyzed by western blot using an antibody against phospho-Ser 10 of
histone H3. A, lane 1: HeLa nuclear extract (control); lane 2: HeLa nuclear extract
pretreated
with 1
M cisplatin overnight. B, time-course of cisplatin-induced
phosphorylation of histone H3 Ser 10. HeLa nuclear extracts were prepared from cells
pretreated
with 10
M cisplatin for 0, 1, 4, 8, 12, or 24 h. C, dose-dependence
of
cisplatin-induction of phosphorylation of histone H3 Ser 10. HeLa cells were treated
with 0, 0.2,1.0,10, or 40 M cis-DDP for 8 hr before harvesting.
187
1
2
3
4
5
Figure 5.2. Inhibition of cisplatin-induced phosphorylation of H3 Ser 10 by SKF86002.
HeLa nuclear extracts were pretreated with either by 1 plM trans-DDP (lane 1); 1 JIM
cisplatin and 10 gIM p38 MAPK inhibitor (SKF86002) (lane 2); 10 LM p38 MAPK
inhibitor (SKF86002)(lane 3); or 1 jiM cisplatin (lane 4) overnight. Lane 5 shows a HeLa
nuclear extract control. HeLa nuclear extracts were analyzed as described in the caption
to Fig. 5.1.
188
2
1
MCF-7
1
2
Ntera II
Figure 5.3. Cisplatin induction of phosphorylation of histone H3 in MCF-7 (top) and
Ntera II (bottom) cell lines. Lane 1: nuclear extracts (control). Lane 2: nuclear extracts
prepared from cells pretreated with 1 !M cisplatin overnight. Nuclear extracts were
analyzed as described in the caption to Fig. 5.1.
189
A
1
2
B Anti-acetylated H4
1
2
3
Anti-acetylated Lysine
1
2
3
Figure 5.4. Cisplatin induction of phosphorylation of H3 Ser28 and acetylation of H4. A,
HeLa nuclear extracts were analyzed by western blot using antibody against phosphoSer 28 of histone H3. Lane 1: HeLa nuclear extract (control). Lane 2: HeLa nuclear
extract pretreated with 1 pM cisplatin overnight. B, nuclear extracts prepared from
HeLa cells were analyzed by western blot using anti-acetyl-lysine antibody or antiacetylated histone H4 as indicated. Lane 1: HeLa nuclear extract (control). Lane 2:
HeLa nuclear extract pretreated with 1 M cisplatin overnight. Lane 3: HeLa nuclear
extract pretreated overnight with 4 mM sodium butyrate, an inhibitor of histone
deacetylases (positive control).
190
Chapter 6 The Modulating Effect of DNA Methylation on the
Cellular Processing of Cisplatin-Damaged DNA
191
Introduction
Cisplatin, cis-diamminedichloroplatinum(II),
has been used to treat
various cancers for over 25 years. The drug interacts covalently with DNA
forming mostly 1,2- and 1,3-intrastrand cross-links. These platinum-DNA
adducts trigger several cellular responses, including transcription inhibition,
DNA repair, cell cycle arrest, and apoptosis.(1) Understanding the detailed
mechanisms by which cells process such cisplatin damage, identifying the factors
that manipulate the processing and unraveling the cytotoxic mechanism of the
drug hold promise of providing rationales to improve the efficacy of the drug
and to develop new anticancer agents.
Epigenomic regulation of genes is essential for proper cellular function.
One major form of epigenetic information in mammalian cells is DNA
methylation, mostly 5-methylcytosine within a d(pCpG) dinucleotide. DNA
methylation
has
profound
effects on the
mammalian
genome, with
approximately 3-5% of the cytosine residues in genomic DNA existing as 5methylcytosine.(2) Hypermethylation of the promoter region of a gene silences
its expression. In addition, DNA methylation also modulates chromatin structure,
inactivates the X chromosome, and affects DNA repair and genomic
imprinting.(3-8) The pattern of genomic methylation is conserved even following
DNA replication.(9)
192
Cancer cells frequently exhibit altered genomic methylation patterns,
mostly showing global hypomethylation with region-specific hypermethylation
within the promoter region of certain genes.(9-12) These aberrant patterns of
DNA methylation have been linked to the onset of cancer presumably by
silencing the expression of tumor suppressor genes. It is therefore of interest to
investigate how such different methylation profiles might affect the processing of
cisplatin-DNA lesions in cancer and normal cells.
In the present study we have examined the role of DNA methylation in
the formation of platinum-DNA adducts, on protein-DNA interactions, and on
the repair of platinum damage. The rates and extents of platination of DNA
probes containing 5-methylcytosine residues adjacent to the platinum binding
site were compared to those for non-methylated DNA. The effect of DNA
methylation on the interaction of a protein with platinated DNA was studied for
domA of HMGB1 binding to methylated and non-methylated DNA probes.
Finally, the effect of DNA-methylation on the NER of cisplatin-modified DNA
was investigated using 195mer substrates containing site-specifically cisplatinmodified DNA with adjacent 5-methylcytosine residues.
Materials and Methods
Materials.Cisplatin was obtained as a gift from Engelhard. Reagents for
DNA synthesis were purchased from Glen Research. SequaGel diluent and
concentrate solutions for preparing denaturing polyacrylamide gels were
193
purchased from National Diagnostics. Hydrazine (98%, anhydrous), dimethyl
sulfate, and other reagents for DNA sequencing were purchased from Aldrich.
T4 polynucleotide kinase, T4 DNA ligase, and NEB buffer 3 were obtained from
New England Biolabs. y-3 2P-ATP was purchased from PerkinElmer. The
analytical
and
preparative
ion
exchange
high-performance
liquid
chromatography (HPLC) columns (Dionex DNApac PA100) and analytical C-18
reverse phase HPLC columns were obtained from Dionex.
Synthesis of OligonucleotideFragments. All oligonucleotides (sequences are
supplied in Appendix 6.1) were synthesized by using an Applied Biosystems
DNA Synthesizer (Model 392) and purified by denaturing polyacrylamide gel
electrophoresis. The gel slices containing the desired oligonucleotides were
excised and eluted with 300 ptL of elution buffer (0.5 M ammonium acetate, 10
mM magnesium acetate, 1 mM EDTA, 0.1% SDS).
Preparative-ScalePlatination of 16mer Fragments. Four different singlestranded
platinated
16mer fragments
(16mer GG-Pt, 16mer GTG-Pt, 16mer
GGMe-Pt, and 16mer GTGMe-Pt) were prepared in the same manner as
described (13). Briefly, a portion of 16mer (100 nmol) was incubated in the dark
with one equivalent of cisplatin in 0.01 M sodium phosphate buffer (pH = 6.75) at
37 C for 18 h. Platinated oligonucleotides were then purified by ion exchange
HPLC utilizing the gradient in Table 6.1. Following HPLC purification, the
16mer samples were analyzed by mass spectrometry and UV-vis (HewlettPackard 8453) and AA spectroscopy (Perkin-Elmer HGA 800).
194
Time Courseof the Platinationof Double-Stranded14mer and Single-Stranded
16mer Substrates. The time-course for the formation of Pt adducts on four
different single-stranded 16mer fragments was investigated.
An aliquot of
oligonucleotide (20 nmol) was incubated in the dark with 20 nmol of cisplatin in
a total volume of 180 !rL containing 0.01 M sodium phosphate (pH = 6.75) at
37 °C. A 10 jiLaliquot was removed from the reaction mixture after 30 min, 1 h,
2 h, 4 h, 6 h, 8 h, and 24 h. The extent of platination at different time points was
evaluated by ion exchange HPLC utilizing the gradient in Table 6.2. The area
under the peak for the platinated product was compared with that for the peak
corresponding to the starting material. In a similar manner, 20 nmol of 14mer
duplex DNA was incubated with 20 nmol of cisplatin in a total volume of 180 pL
of 0.01 M sodium phosphate (pH = 6.75) at 37 °C. A 10 JIL aliquot was removed
from the reaction mixture after 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. The extent
of platination was evaluated by reverse phase HPLC using a C-18 analytical
column and utilizing the gradient in Table 6.3. The area under the peak
corresponding to platinated DNA product was compared with that of the
starting material peak.
Maxam-GilbertSequencingof 16merand 36mer DNAs. The 16mer and 36mer
fragments were sequenced to verify the presence and location of the 5methylcytosine residues as well as to characterize the platinum-DNA crosslink.
Maxam-Gilbert reactions were performed as described.(14)
195
Briefly, the guanine-specific reaction (G-reaction) was conducted by
adding 4
L of 1 g/L salmon sperm DNA and 5
L of 5'-32 P-labeled DNA
substrate (approximately 10,000 cpm) to 190 [L of dimethyl sulfate buffer (50
mM sodium cacodylate, 1 mM EDTA). A portion of 5 pL of 10% dimethyl sulfate
was added to the solution, and the reaction was then incubated for 5 min at
ambient temperature. The reaction was stopped by adding 50 IL of dimethyl
sulfate stop solution (1.5 M sodium acetate, 1 M P-mercaptoethanol, 250 Pg/mL
yeast tRNA).
The purine-specific reaction (G + A reaction) was performed by
incubating with a mixture of 4 gL of 1 g/L salmon sperm DNA, 10 pL of 5'-32p
labeled DNA substrate (approximately 20,000 cpm), 10 tL of doubly distilled
water, and 4 pL of 1 M piperidine formate (pH 2.0) at 37 C for 15 min. The
reaction was stopped by adding 240 IlL of stop solution A (0.3 M sodium acetate,
0.1 mM EDTA, 100 plg/mL yeast tRNA).
The pyrimidine-specific reaction (C + T reaction) was performed by
combining 4
L of 1 g/L salmon sperm DNA, 10 [IL of 5'-3 2 P-labeled DNA
substrate (approximately 20,000cpm), 10 CiLof doubly distilled water, and 30 PL
of hydrazine
(anhydrous, 98%). The reaction was incubated at ambient
temperature for 7 min. The reaction was then stopped by adding 200 L of stop
solution A.
The cytosine-specific reaction (C-reaction) was conducted by combining
15 tL of 5 M sodium chloride, 4 iL of 1 g/L salmon sperm DNA, 10 plL of 5'-32p-
196
labeled DNA substrate (approximately 20,000 cpm), and 30 plL of hydrazine
(anhydrous, 98%). The reaction was first incubated at ambient temperature for 5
min, and was then stopped by adding 200 pL of stop solution A.
For each of the sequencing reactions, the DNA was cleaved by piperidine.
Briefly, the DNA was precipitated from solution with ethanol and then incubated
in 100 tIL of freshly prepared 1 M piperidine in water at 90 C for 30 min. The
piperidine was removed under vacuum. Substrates were treated with 50 L of
0.2 M sodium cyanide overnight at 37 C in order to remove the platinum from
the DNA. Following the piperidine treatment, the samples were then analyzed
by denaturing polyacrylamide gel electrophoresis. The intensity of the bands in
each lane was quantified by ImageQuant Software (Molecular Dynamics).
Synthesis of Site-Specifically Platinated 195mer Substrates. The 195mer probes
for the repair assay were synthesized from six oligonucleotides in the same
manner as described.(13) Briefly, the substrates were constructed as indicated in
Figure 6.1. For each 195mer, the 36mer, 72mer, and 96mer oligonucleotides were
phosphorylated by using T4 polynucleotide kinase and ATP. The 16mer
fragments were 5'-32P-labeled with T4 polynucleotide kinase and y-32P-ATP. All
the oligonucleotides were annealed and then incubated with T4 DNA ligase at
16 °C for 16 h. The 195mer products were isolated by denaturing PAGE, and
eluted from the gel in elution buffer. The products from the denaturing gel were
then re-annealed and further purified on a non-denaturing PAGE electrophoresis.
__
197
Nucleotide Excision Repair Assay of 195mer DNAs. The platinated
methylated
or non-methylated 195mers were used as substrates for the
nucleotide excision repair assay as described.(13) The AA8 CHO cell-free extracts
were added into the mixture and incubated at 30 C for 2 h. The reaction
products were then treated with proteinase K (15.3 mg/mL) and SDS (10% W/V)
at 30 °C for 15 min, followed by phenol extraction and ethanol precipitation. The
repair products were analyzed on an 8% denaturing PAGE gel.
Gel Electrophoresis Mobility Shift Assay of HMGB1 DomA with ds-16mers. Gel
electrophoresis mobility shift assays (EMSAs) of HMGB1 binding to either
methylated or non-methylated oligonucleotide duplexes were performed in a
similar manner as described elsewhere.(l5) Typically, a portion of either
methylated or unmethylated
dsDNA probes (0.4-5 nM, 5000 cpm) were
incubated with increasing amounts of HMGB1 dom A in 10 Rl EMSA buffer (10
mM HEPES, pH = 7.5, 10 mM MgC12, 50 mM LiC, 100 mM NaCl, 1 mM
spermidine,
0.2 mg/mL
BSA, and 0.05% Nonidet P40) at 30 C for 1 h. The
samples were then analyzed on a 5% non-denaturing PAGE gel at 4 °C.
Results
Synthesis and Purificationof Platinated Oligonucleotidesfor 195mer Repair
Substrates. Platinated oligonucleotides fragments for the synthesis of the 195mer
substrates were successfully prepared and purified, the yield varying from 65%
to 85% yield depending upon substrate (Figure 6.2). The identity of the 16mer
198
substrates was confirmed by mass spectrometry from Biopolymers lab (Table 6.4).
The mass difference between non-platinated and platinated substrates is
2
consistent with the addition of a {Pt(NH3)
2} + group. In addition, the difference
in mass between doubly-methylated and non-methylated substrates matches the
predicted value for two methyl groups. UV/Vis-AA ratios of the platinated
DNAs also indicate one platinum bound to each DNA molecule (Table 6.5).
Sequencingof 16mer and 36mer Substrates.In the cytosine-specific MaxamGilbert reaction, the cytosine ring is attacked by hydrazine; as a result, the base is
cleaved and ribosylurea is formed. The DNA is further cleaved with piperidine
through ,-elimination of the phosphates group. The presence of a methyl group
on the 5-position of the cytosine ring interferes with action of hydrazine.(14) The
efficiency of cleavage at the 5-methylcytosine residue is thus much lower than
cleavage at an unmodified cytosine. Indeed, the Maxam-Gilbert reactions for the
16mers and 36mers revealed a distinct difference between methylated and
analogous non-methylated substrates (Figures 6.3-6.9).The bands corresponding
to the cytosine residues were clearly observed in cytosine-specific reaction,
whereas the corresponding bands of methylated cytosine residues were
diminished. Together with the mass spectrometry data, these results confirmed
presence and the positions of the 5-methylcytosine residues.
In addition, the Maxam-Gilbert reaction also revealed the position of the
platination site. The N-7 position of guanine is methylated by dimethyl sulfate
during the G-reaction, which thereby makes the base susceptible to removal from
199
the sugar ring by hydrolysis. The presence of a platinum adduct bound to the
N(7) position of guanine is expected to block the addition of a methyl group at
that site, suppressing the appearance of a guanine band in the Maxam-Gilbert
chemical degradation of DNA. Indeed, the intensity of the bands corresponding
to the guanine residues of 16mers where the platinum is situated decreased by
approximately two-fold when compared to the intensities of the corresponding
bands in the non-platinated substrates (Figure 6.10). Therefore, the MaxamGilbert G-reaction results together with mass spectrometry and UV/Vis-AA
analysis confirmed the presence of the 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand
cisplatin-DNA cross-links.
Time Course of Platination of 14mer and 16mer Substrates. For both the
single-stranded 16mer substrates and the double-stranded 14mer probes, the
presence of 5-methylcytosine residues reduced the rate at which cisplatin formed
platinum-DNA intrastrand cross-links. The overall extent of platination after
24 h was significantly lower for methylated substrates compared with nonmethylated substrates (Tables 6. 6-7 and Figures 6.11-13).
EMSA Assay of dsDNA with HMGB1 Dom A. To investigate the effect of
DNA methylation on HMGB1 dom A binding to the major cisplatin-DNA
adducts,
the
3 2 P-labeled
site-specifically platinated
16GG-Pt, methylated
platinated 16GGMe-Pt and 16GG-Pt//Me probes were prepared for EMSA
studies (Figure 6.14). The presence of the methylated cytosine residues adjacent
to platinated guanine base moderately reduced the affinity of HMGB1 dom A for
200
cisplatin-modified DNA (16GGMe-Pt), whereas the presence of methylated
cytosine opposite to a platinated guanine (16GG-Pt//Me) significantly abolished
the interaction between the DNA probe and HMGB1 dom A.
Effect of Methylation on NER of 195merSubstrates. Ten different 195mers
containing either 1,2-d(GpG) or 1,3-d(GpTpG) intrastrand platinum-DNA crosslinks were successfully synthesized and purified (Figure 6.1 and 6.15A). The
methylcytosines were located either on the opposite strand of the platinated
guanines or flanking the platinated guanines on the same strand. The 195mer
probes were used as repair substrates to determine whether or not the presence
of adjacent 5-methylcytosine residues would modulate the NER of platinumDNA adducts (Figure 6.15 B). The relative repair efficiencies are reported in
Figure 6.15 C. Although the repair efficiency of methylated DNA is similar to the
efficiency of non-methylated DNA, some modest effects of DNA methylation
occur. First, the effect of methylation depends on the location of methylcytosines
and type of cisplatin lesions. For 1,2-d(GpG) intrastrand cross-link, repair is
slightly stimulated when two methylcytosines are located opposite the platinated
guanines (Figure 6.15, lane 7 vs lane 10), whereas no stimulation is observed
when the methylcytosines are located in flanking positions of platinated
guanines (Figure 6.15, lane 6 vs lane 10). For 1,3-d(GpTpG) intrastrand cross-link,
no change or slight inhibition of repair is observed when two methylcytosines
are located opposite the platinated guanines (Figure 6.15, lanes 2 vs lane 5),
whereas moderate inhibition is observed when the methylcytosines are located
201
in positions flanking the platinated guanines (Figure 6.15, lane 1 vs lane 5).
Inducing mono-methylcytosine in the strand opposite platinated guanines also
modulates excision repair in a location-specific manner (Figure 6.15, lane 3 vs
lane 4; lane 8 vs lane 9).
Discussion
DNA Methylation Modulates the Formationof Platinum-DNA Adducts. The
effect of cytosine methylation on the structure of DNA had been studied through
a variety of means including X-ray crystallography, NMR spectroscopy, gel
electrophoresis,
FT-IR spectroscopy, and molecular dynamics simulations.
Although no gross conformational changes occur by introducing a 5-methyl
group on the cytosine base,(16-20) methylation causes certain local perturbations.
In particular, methyl group alters the backbone dynamics,(21-23) bending
flexibility,(24) and curvature of the DNA.(25,26) Moreover, recent studies
revealed that 5-methylation changes the thermodynamic (free enthalpies) and
kinetic properties of the DNA backbone. Through methyaltion, the GpC step is
stabilized in the BI conformation by about 1.1 kcal/mol, whereas the BI substate
of CpG is destabilized by 0.8 kcal/mol, although still energetically favored. The
BI and BII are two conformations defined by different (C4-C3-03-P) and (C3 '03 '-P-05 ') torsion angles. The barrier for conversion of BI to BII in Gp5smCis
increased by 1.5 kcal/mol compared to the non-methylated step GpC, and the
barrier for 5 mCpGis lowered by 0.8 kcal/mol compared to CpG. (27) In addition,
202
solid-state deuterium NMR analysis revealed a significant reduction in the
phosphodiester backbone mobility at the site of the methylated cytosine.(21)
Substitution of hydrogen with 5-methyl group in cytosine increases melting
temperature.(28) The 5-methyl group of the cytosine base enhances of the base
stacking ability and changes the sugar pucker and the glycosidic bond torsional
angle.(19,29) One X-ray crystallographic study revealed that the methyl group
forms weak C-H-..O hydrogen bond with anionic oxygen atoms of the phosphate
group, which may stabilize the interaction.(30) These factors may contribute to
the changes of DNA structure of methylated DNA and account for differences in
protein-DNA interaction.
The formation of intrastrand cisplatin-DNA cross-links causes the adjacent
guanines bases to roll toward each other, bending the helical axis of the host
duplex in the direction of the major groove resulting a flatter minor groove
opposite the platinum adduct. Moreover, such adducts partially disrupt the
stacking interactions between neighboring base-pairs at and adjacent to the
platination site.(1) Therefore, it is not surprising that methylated DNA is less
reactive to cisplatin compared with non-methylated DNA. Several factors, such
as
reduced
flexibility, increased
base stacking,
significantly
reduced
phosphordiester backbone mobility of methylated DNA may contribute to the
diminished rate and extent of platination for the methylated DNA compared to
unmodified DNA in vitro.
203
DNA Methylation Modulates the Interaction betwzeenHMGB1 Dom A and
Cisplatin-DNA Adducts. Cisplatin modifications distort the structure of the DNA
duplex, bending it significantly toward the major groove and exposing a wide,
shallow minor groove surface for protein binding. Several classes of proteins,
such as high mobility group box proteins, repair proteins, and transcription
factors,
recognize
1,2-d(GpG) intrastrand
cross-linked
platinum-DNA
adducts.(1,31) It is therefore of interest to investigate the effects of cytosine
methylation on the interaction between these proteins and platinated DNA. We
have examined HMGB1 dom A as a model to investigate such behavior.
HMGB1, a well-documented protein that preferentially binds to 1,2d(GpG) cisplatin-DNA adducts, has two tanden HMG box domains and an
acidic C terminus. The structure of the complex of HMGB1 dom A with a
dsl6mer containing a site-specific 1,2-d(GpG) cisplatin-DNA adduct has been
described. A striking feature is the intercalation of the Phe 37 side chain into a
hydrophobic notch in the minor groove formed by the cisplatin-DNA adduct.
This intercalation is important for DNA affinity, since domA F37A mutant
protein has extremely low binding affinity for the platinated DNA probe. (31)
Previous studies revealed that alteration of the nucleotides flanking the
platinum lesion modulated HMG-domain protein recognition of cisplatinmodified DNA.(15) Here we demonstrate that the presence of methyl groups on
cytosines flanking the platinum lesion also modulate HMG-domain protein
affinity in cisplatin modified DNA. Moreover, when the 5-methylcytosines are
204
located in the strand opposite that containing the cisplatin-DNA adduct, the
binding affinity is almost completely abolished. Such a striking effect is
consistent with photo-crosslinking results that indicate significantly less photocrosslinking of HMGB1 with platinated DNA containing methylcytosines in the
opposite strand.(32) Moreover, the effect of adding an exocyclic methyl group
also is consistent with results from a previous study, which revealed that HMGD
binding affinity is stimulated upon a substitution of dT by dU. (33)
The inhibitory effect on HMGB1 binding caused by the exocyclic methyl
group of cytosine has two possible origins. The presence of a methyl group near
a cisplatin-DNA cross-links alter the local structure and thereby reduce the
affinity for HMGB1 dom A. In addition, the methyl group may directly block a
specific interaction of HMGB1 dom A with platinated DNA. The binding surface
of the HMGB1 A domain extends exclusively to 3' side of the bend locaus caused
by platination and covers five base-pair steps. Phe 37 intercalation plays a
determining role in this protein-DNA interaction. It has been proposed that
hydrophobic residues are employed as an interactive wedge to pry open one or
several base pair steps, thereby bending the DNA away from the protein toward
the major groove.(31,34) Methylated cytosines cause a local deformation of the
major groove of DNA, inducing rearrangement of the methylated base pairs,
which are then pushed into the minor groove causing a deeper major groove.
(18,35)Therefore, the presence of methylcytosine may disrupt the intercalation of
Phe 37 and serve as a stereochemical barrier to the compression of the major
205
groove required by the bending induced by HMGB1 domA. Moreover, helix I of
HMGB1 dom A interacts with sugar-phosphate backbone of the bottom strand,
the strand opposite to platinum cross-link, whereas helix II mainly contacts the
backbone of the unmodified
strand.(31) Since methylation changes the
thermodynamic and kinetic properties of the DNA backbone, reducing the DNA
flexibility by increasing stacking interactions, it may indirectly affect this
interaction. An X-ray crystal structure of cisplatin-modified methylated DNA in
complex with HMGB1 dom A would provide more detailed information.
The effect of DNA methylation on the binding affinity of HMGB1 for
cisplatin-DNA adducts may have significant biological consequences. In vitro
nucleotide excision repair studies indicate that HMGB1 shields the 1,2-d(GpG)
intrastrand platinum cross-link from NER by stably binding to this DNA
adduct.(36,37) Moreover, HMGB1 interacts with the nucleosome at the entry and
exit points of the DNA, thus decreasing the compactness of the chromatin fibers
by weakening the binding of H1 to nucleosomes. HMGB1 also facilitates binding
of the remodeling complex ACF to nucleosomal DNA, thereby accelerating its
ability to induce structural changes in the nucleosome.(38-40) Addition or
deletion of the exocyclic methyl group might add another layer to modulate the
affinity of this protein to DNA and thereby its function in vivo.
DNA Methylation Modulates NER of Cisplatin-DNA Adducts. The EMSA
results reveal significant reduction in the binding affinity of HMGB1 dom A to
methylated DNA probes.
Since HMGB1 shields cisplatin-DNA lesions from
206
repair, one would expect that NER might be less inhibited for methylated DNA
probes. However, we only observed a modest stimulation in NER for DNA
methylated on the strand opposite platinated guanine residues. One explanation
is that proteins binding to methylated DNA, such as methyl-CpG-binding
domain
(MBD) family proteins, shield the cisplatin-DNA adducts, like
HMGB1.(41,42)MBD4 has been related to the cellular response to DNA damage
and to affect DNA repair.(8,43-45) A specific role for MBD on NER of cisplatinDNA damage remains to be determined.
The effects of DNA methylation on NER depend upon the lesion type and
the specific flanking sequence and range from strong stimulation to strong
inhibition.(46) The presence of 5-methylcytosine bases could induce differential
conformational alterations of 1,2-d(GpG) and 1,3-d(GpTpG)cisplatin-DNA crosslinks and affect repair protein recognition and NER.
DNA methylation may also modulate repair at the nucleosome level in
vivo, since it affects the affinity and positioning of histone octamer.(12,47,48)
Addition of a 5-methyl group on cytosine changes the rotational positioning
compared with normal nucleosomes. Moreover, DNA methylation and histone
post-translational modification are closely related. In vivo, DNA methylation is
always accompanied with genetically inactive heterochromatin, either by
recruiting HDAC or by interacting with MBD family proteins.(49,50) DNA
methylation often leads to histone deacetylation, whereas histone acetylation
prevents DNA methylation.(4,51) These processes potentially would alter the
207
accessibility of a cisplatin-DNA lesion on the surface of histone octamer.(48)
Previous studies revealed that NER of cisplatin-DNA adducts is modulated by
the nucleosome core particle and by post-translational modifications of the
histones.(l3) DNA environmental changes caused by DNA methylation would
add another layer of control on DNA repair from nucleosomes. Therefore, it
would be interesting to investigate how DNA methylation would affect repair of
cisplatin-damaged DNA at the nucleosomal level.
Implications in Cancer and Processingof Cisplatin. The present results
provide insight into the possible activity of cisplatin in targeting certain
malignant cells.
The presence of 5-methylcytosine residues inhibits the
formation of platinum-DNA adducts. Therefore, a DNA region that is deficient
in 5-methylcytosine residues is more susceptible to platination. In fact, most
cancer cells exhibit global hypomethylation of DNA and hypermethylation in the
promoter regions of certain genes compared to normal cells.(11,52)Such global
hypomethylation might be important for the preferential killing of cancer cells by
cisplatin. Hypomethylation may lead to an increase in the platinum-DNA
adducts level over that in normal methylated DNA, which may contribute to the
activity of this drug against certain cancers.
In addition, the presence of 5-methylcytosines modulates HMGB1 binding
affinity to DNA having the platinum lesion. Increased HMGB1 levels increase
cellular sensitivity to cisplatin.(53) Widespread hypomethylation would provide
208
another possible way to increase interaction of HMGB1 with cisplatin-modified
DNA.
On the other hand, hypermethylation in promoter regions silences the
gene expression. The promoters of several repair genes are hypermethylated in
certain cancers, affecting repair activity.(11,54) Taken together, the combined
effects of increased platinum binding, increased binding of HMGB1 and
subsequent suppression of repair at hypomethylated regions and the silencing of
repair genes or other genes important for cell function by hypermethylation may
cause more efficient cell death under these conditions compared to cells with
normal methylation patterns. DNA methylation thus adds another level of
complexity in understanding the regulation of cellular processing of cisplatin.
The combination of epigenetic drugs with cisplatin could provide
promising effects for clinical use. The DNA methylation inhibitors 2'-deoxy-5azacytosine and 5-azacytosine exhibit synergistic anticancer activity with
cisplatin.(55-57) In addition, co-treatment cisplatin with HDAC inhibitor
increased the cisplatin-DNA adduct formation and cellular sensitivity to the
drug.(58,59) DNA methylation and histone deacetylation are closely related.(6062) A combination of the HDAC inhibitor, trichostatin A (TSA),with 2'-deoxy-5azacytosine caused a synergistic reactivation of expression of silenced genes in
cancer cells.(63,64) Further exploration of "cocktail" combination of DNA
methylation inhibitors, HDAC inhibitors, and cisplatin would be worthwhile for
more efficient anticancer therapy.
209
Acknowledgment
I thank Dr. J. T. Reardon and Dr. A. Sancar for providing AA8 cell extracts
and A. Danford for assistance in synthesizing and characterizing some of the
oligonucleotide fragments. I also acknowledge members of our laboratories for
helpful discussion and comments on the manuscript.
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Table 6.1. The Ion Exchange HPLC Gradient for Isolation of Platinated 16mer
Products
Time (min)
0
25
30
35
% Buffer A
80
65
0
80
% Buffer B
20
35
100
20
Buffer A consisted of 0.025 M ammonium acetate in 10% aqueous acetonitrile
(pH = 6.0). Buffer B contained 0.025 M ammonium acetate and 1 M sodium
chloride in 10% aqueous acetonitrile (pH = 6.0).
Table 6.2. Buffer Conditions for the Time Course of Platination of SingleStranded 16mer Substrates by Ion Exchange HPLC.
Time (min)
0
% Buffer A
72
% Buffer B
28
12.5
15
45
25
65
75
18
72
28
Buffer A consisted of 0.025 M ammonium acetate in 10% aqueous acetonitrile
(pH=6.0). Buffer B contained 0.025 M ammonium acetate and 1 M sodium
chloride in 10% aqueous acetonitrile (pH=6.0).
216
Table 6.3.
Buffer Conditions
for the Time Course of Platination of Double-
Stranded 14mer Substrates by Reverse Phase Ion Exchange HPLC.
Time (min)
% Buffer A
% Buffer B
0
10
15
18
100
65
0
100
0
35
100
0
Buffer A consisted of 1 M ammonium acetate in 7.5% aqueous acetonitrile (pH =
6.0). Buffer B was 50% aqueous acetonitrile (pH = 6.0).
Table 6.4. Electrospray Mass Spectrometry Results for 16mer Substrates.
Mass
Mass
(Calculated)
(Experimental)
(g/mol)
(g/mol)
16GTG
16 GTG Pt
16GTGMe
16GTGMe-Pt
16GG
16GG-Pt
16GGMe
4720.1
4949.1
4720.4 + 0.5
4748.1
16GGMe-Pt
4962.1
4748.4 + 0.6
4975.2+ 0.9
4706.0 + 1.0
4931.4+ 0.5
4733.5 + 0.6
4960.7+ 0.6
DNA
4947.7+ 1.0
4977.1
4705.1
4934.1
4733.1
Table 6.5. UV-vis and AA Spectroscopy Results for 16mer Substrates.
DNA
[Pt]/[DNA] Ratio
16 GTG Pt
16GTGMe-Pt
16GG-Pt
16GGMe-Pt
0.97
1.00
1.00
1.09
217
Table 6.6. Extent of Platination for Single-Stranded 16mer Substrates.*
Time
30 min
1h
2h
4h
6h
8h
24h
16 GG
24.08±0.54%
27.80±0.09%
36.16±0.92%
39.70±1.20%
41.77±1.24%
42.80±1.24%
44.06±1.20%
16 GG Me
17.57±1.60%
21.36±1.49%
26.84±2.24%
31.31+2.78%
33.64±1.90%
34.81±2.06%
35.75±3.00%
16GTG
21.32+0.72%
25.30±1.07%
32.57±0.20%
39.14±0.01%
42.31±0.37%
44.11±0.45%
46.65±0.05%
16 GTG Me
18.96±0.10%
22.10±0.04%
27.05±0.22%
33.33±0.48%
35.97±0.78%
37.83±0.77%
40.31±0.87%
-
*Average of two experiments.
Table 6.7. Extent of Platination for Double-Stranded 14mer Substrates.*
Time
14 CC
14MM
30 min
13.16±0.02%
19.77±1.29%
28.08±1.61%
30.43±1.08%
30.95±1.14%
30.30±1.55%
30.80±1.51%
6.00±0.31%
-
1h
2h
4h
6h
8h
24h
*Average of two experiments.
8.13±0.18%
12.82+0.85%
18.13±0.63%
19.28±1.78%
19.95±0.78%
22.46±1.77%
218
83
1lGCGG"C
~-
195GG-Pt Me
72
36CC
96
-
1ICGrTGC
83
87
16CGTGC
83
16CC=
96
n2
3"C
87
72
3PCA
83
16CGGC
96
83
16CGTGC
96
195GTG-Pt//MAM
195GG-Pt//MM
195GG-Pt/CM
83
36CC
16CGGC
m
C
-
-36!CC
87
72
87
96
-
87
72
36C AC
87
96
83
16CGTGC
96
195GG-Pt//MC
195GG-Pt//CC
195GTG-Pt Me
87
36CAC
83
96
-AI
-
195GTG-Pt//MAC
CAC
72
36CC
87
83
16CGGC
96
83
16CGTCC
96
87
72
36CAC
87
72
36
n
36cc
72
36CuC
195GTG-Pt//CAM
87
195GTG-Pt//CAC
Figure 6.1. Strategy for synthesizing site-specifically platinated 195DNA probes.
Five synthetic oligonucleotides and one bearing a 1,2-d(GpG) or 1,3-d(GpTpG)
intrastrand platinum-DNA cross-link were annealed and ligated as shown.
219
16GGMe-Pt
16GG-Pt
/
A I...,
16GTGMe-Pt
16GTG-Pt
/
'I
ski
'
.l!-
, t~
I: ,h u i xr · u+ v
i.
}'~
rr
r
._,_..
A
.
r
I
r
:,I
.-
P84b-;
;-
*
;
=
c
He
Figure 6.2. HPLC trace of platination products of 16mer DNAs after 18 h
incubation. Peaks corresponding to the desired platinated DNA are identified.
220
16GG
G
G+A C+T
16GGMe
G
C
,e t ,
G+A C+T
4
I
f'Ot
I*
... *.
4-
4-
a... 9_
-
*_
Hlw
YI~ y*
Y,
1.
**
*a0
*M
c*
so
C
a"
I
so
*·
as ~rr
-
_
-~~~..
Figure 6.3. Denaturing PAGE analysis of Maxam-Gilbert sequencing of 16GG
and 16GGMe substrates. The 16GG and 16GGMe probes were sequenced by
chemical degradation. Each lane is labeled according to its contents. The C+T
lane and C lane for the 16GG show two extra bands (arrows) that do not appear
in the corresponding positions in the 16GGMe lanes. These differences indicate
the presence of 5-methylcytosine residues in the 16GGMe sample at these
locations.
221
160
ai
ml
o.
,B
a a
ii
-A
-W
:
16 GGMo
a
~
I
~
m
~
~
l
~
~
-'
-
aO
I --01
1
A4W AJJIL
CIVTCC
ft
in61
".4.
Q
it I·
I
a k'·"" ')
P
¢
k
.
_C1C TC
_
A
% P 4%
ji
Oft %
P
m
aa
T
I: .
·I
.
_)
;t
·v .-,.~h......_~~.
J _h _-
*-,'3s
.
*-3'
e
C
aS
S
C
T
C
I
ft
i
T
*
i
*m
i
m
5-.
Figure 6.4. Maxam-Gilbert sequencing results for the 16GG and 16GGMe. The
intensities of bands in the gel were plotted as a function of distance to generate
the plots.
The 16GGMe has the same sequence as the 16merGG with the
exception of the 5-methylcytosine residues. The 5-methylcytosine residues are
not cleaved, and therefore do not produce bands in the gel (arrows).
222
16GTG
G
G+A C+T
4
16GTG-Me
C
*
fY
G
_
it'
a
4N.
up
*
to
of
,
G+A C+T C
"a
_
.
I0
id----
_"
an
.
-
0
.
40
A
.
Figure 6.5. Maxam-Gilbert sequencing of 16GTG and 16GTGMe substrates.
The
16GTG and 16GTGMe were sequenced by chemical degradation. Each lane is
labeled according to its contents. The C+T lane and C lane for the 16GTG show
two extra bands (arrows) that do not appear in the corresponding positions in
the 16GTGMe lanes. These differences are indicative of the presence of 5-methylcytosine residues in the 16GTGMesample at these locations.
223
16mer GTG Me
siadi
O. .
0
(3
.
.a
T
CC
C
C
CCTCT
4A
T
S-M4
kAAJ-IJ
aCCCC
a.
0
:1
,CCTCTC
Il
CC
''.J'J
C
C
C
T
C
T
5-MC
,
.
C
C
, UC.
C
C-Mc
- ·"-'6...
,*_,n ; 4_
1t
aA;.-.
ti.*,W
l. ,-tw
)3'
5'-
e--3'
S
5'
Figure 6.6. Maxam-Gilbert sequencing results for the 16GTG and 16GTGMe. The
intensities of bands in the gel were plotted as a function of distance to generate
the plots. The 16GTGMe has the same sequence as the 16GTGwith the exception
of the 5-methylcytosine residues that replace the cytosine residues adjacent to the
platinum binding site.
The 5-methylcytosine residues are not cleaved, and
therefore do not show bands in the gel (arrows).
224
36CC
G A+G C+T C
l I11
36MM
36CAC
36MAM
G A+G C+T C
GA+G C+T C
GA+G C+T C
I
I
I
I
1
I
I
I
1111
- _
,Down
~ _ ^.,| IF~~,4k
" -
>
:f
, I
Figure 6.7. PAGE analysis of Maxam-Gilbert sequencing of 36mer substrates.
The 36mers were sequenced by chemical degradation.
Each lane is labeled
according to its contents. The C+T lane and C lane for the 36CC show two extra
bands that do not appear in the corresponding positions in the 36MM lanes.
Similar differences can be seen between the 36CAC and 36MAM lanes. These
differences are indicative of the presence of 5-methylcytosine residues in the
36MM and 36MAM sample at these locations.
225
36mer GG
OG 000G 00
ah
A AA OGAGAG
TTT
-1.
36mer GG Me
*me000000
0 0 O
~
-
u~o
GGOAGA
C
i
AA
C
AoGOQAOAOG
OA
aOd
u, T
C
"
a0
,a
A
CC
00
s
A A
C
T
m-C
e_
4ImC 5NFC
,
.-+-M 3'
-
3
:5'-
5'-
U
S,,
r
IA'
*- 3'
e)m'
5"_
Figure 6.8. Maxam-Gilbert sequencing results for the 36CC and 36MM probes.
The intensities of bands in the gel were plotted as a function of distance to
generate the plots. The 36MM has the same sequence as the 36CC with the
exception of the 5-methylcytosine residues that replace the cytosine residues
located 18 and 19 nucleotides from the 5' end. The 5-methylcytosine residues are
not cleaved, and therefore do not show bands in the gel (arrows).
226
36 OTG
,a
GG
m
36mertGTG Me
G
G
CG
.
43 -
U
.f
i
-
'"1
1 -
- -
4it
m
AOOAOA0OA80
A
A
A
AeA
C T
a
SF
h'"!''.,,*,*,
1*
A"I
ON
I{
0
GGI3AG
';ii
Ai,.,A
C C
I
A
.
',
tij'
.
&i
A
VV, v.
1W
a.
,,
i
PI
t!
AG A
A
-r
Ba
A%
A
r
5IWC
-.
---
.
I
A A
.
I-WBC
:I
I
--
I
n
C
m
3.-MSC Ime-C
I
,
...-.
.
4-
3'
la
-. k '
fW
I
:1 U
i
3
4-3'
i m
'3
5'-+
Figure 6.9. The Maxam-Gilbert sequencing results for the 36CAC and 36MAM.
The intensities of bands in the gel were plotted as a function of distance to
generate the following plots. The 36MAM has the same sequence as the 36CAC
with the exception of the 5-methylcytosine residues that replace the cytosine
residues located 18 and 20 nucleotides from the 5' end. The 5-methylcytosine
residues are not cleaved, and therefore do not show a band in the gel.
227
Counts
[LINE- li [LINE- 21
(width 1)(width 1)
mm
Figure 6.10. Comparison between platinated and non-platinated substrate of
Maxam-Gilbert sequencing. In order to verify the presence of platinum in the
16GG Pt substrate, the products from the G reaction were compared from this
substrate with the products from the G reaction from the 16GG non-platinated
probe. Although the background noise in these two samples is the same, the
intensity of the bands for the 16GG are nearly two-fold more intense than the
corresponding bands for the 16GG-Pt sample. This suppression of signal can be
attributed to the presence of a platinum adduct in the 16GG-Pt samples. The
dotted line above is the data from the 16GG and the solid line represents the data
from the 16GG-Pt sample.
228
t!^
0
0
_
40
U
30
20
10
0
0
5
10
15
20
25
Time (h)
Figure 6.11. Time course of platination of 16GG and 16GGMe. The extent of
platination was evaluated at regular intervals. The 16GG (circles) is more rapidly
platinated than its methylated analog 16GGMe (squares).
__
229
_
_
OA
v
O..
20
10
0
0
5
10
15
20
25
Time (h)
Figure 6.12. Time course of platination kinetics of 16GTG and 16GTGMe. The
extent of platination was evaluated at regular intervals. The 16GTG (circles) is
more rapidly platinated than its methylated analog 16GTGMe (squares).
230
30
cs
25
5
0
0
5
10
15
20
25
Time (h)
Figure 6.13. Time course of platination kinetics of ds14CC and ds14MM. The
extent of platination was evaluated at regular intervals. The ds14CC (circles) is
more rapidly platinated than its methylated analog ds14MM (diamonds).
231
A.
HMGB1
dOmA-'-
16GG
16GG-Pt
16GG-Me
16GG-Pt-Me
16GG//Me
16GG-Pt//Me
B.
16GG
BMt
1
EMSA oftHMGBI dom A with ds16mers
l
......
......
30
.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
25
. .........
.........
.........
20
.i.
15
.......................
10
-i
O
0
19
190
HMOB1dom A
Figure 6.14. EMSA of dsl6mers with HMGB1-dom A. Methylation at cytosines
flanking the GG-Pt adduct slightly reduced the binding of HMGB1 dom A,
whereas methylation at cytosines on the strand opposite GG-Pt lesion
significantly reduced protein binding. B. Quantitative EMSA results of HMGB1dom A with ds16GG (red), dsl6GGMe (blue), and ds16GG/ /Me (green).
232
A.
1
2
3
4
5
6
7
8
9
10
'Vell
4 m
a_
cIsT)NA-.
swaUvt
mC
C.
B.
1 23
4 5 6 7 8 91 10
140
·:
1-
.
.
.
.
.
i
l
.i
l
i
l
i
I
I
120
4, I o -f.............I.
00
'
.o
0
2
Figure 6.15. A. Non-denaturing
3
4
5
5
7
e
9
10
PAGE gel of dsl95mers. B. Excision assay with
methylated and non-methylated DNA probes. Lane 1, 195GTG-Pt Me; lane 2,
195GTG-Pt//MAM; lane 3, 195GTG-Pt//CAM; lane 4, 195GTG-Pt//MAC; lane
5, 195GTG-Pt; lane 6, 195GG-Pt Me; lane 7, 195GG-Pt//MM;
Pt//CM;
lane 9, 195GG-Pt//MC;
lane 8, 195GG-
lane 10, 195GG-Pt. C. The relative excision
efficiencies of ds 195mers. Data in columns 1-5 are normalized by the excision
efficiency of column 5, data in columns 6-10 are normalized by the excision
efficiency of column 10.
233
Appendix 6.1
The following twelve oligonucleotides were synthesized for use as the 195mer
repair substrates. 5-Methylcytosine residues are represented with the letter M.
83mer:
GCTGACAACAAAAAGATTGTCTTTTCTGACCAGATGGACGCGGCCACCC
TCAAAGGCATCACCGCGGGCCAGGTGAATATCA-3'
16mer GG: 5'-CCTCTCCGGCCTCTCC-3'
16mer GTG: 5'-CCTCTCCGTGCTCTCC-3'
16mer GG Me: 5'-CCTCTTCMGGMCTCTCC-3'
16mer GTG Me: 5'-CCTCTTCMGTGMTCTCC-3'
96mer: 5'-GTATTATGAATTCAGCTGCTCGAGCTCAATTAGTCAGACCCCAA
CAGCTGGAGAGAACAGCGACCCGCGGGCCCCGGCAGGCGGCTCAAGCA
GGAG-3'
87mer: GCTCCTGCTTGAGCCGCCTGCCGGGGCCCGCGGGTCGCTGTTCTCT
CCAGCTGTTGGGGTCTGACTAATTGAGCTCGAGCAGCTGAA-3'
36mer CC: 5'-TTCATAATACGGAGAGGCCGGAGAGGTGATATTCAC-3'
36mer MM: 5'-TTCATAATACGGAGAGGMMGGAGAGGTGATATTCAC-3'
36mer CAC: 5'-TTCATAATACGGAGAGCACGGAGAGGTGATTATTCAC-3'
36mer MAM: 5'-TTCATAATACGGAGAGMAMGGAGAGGTGATTATTCAC-3'
72mer: CTGGCCCGCGGTGATGCCTTGAGGGTGGCCGCGTCCATCTGGTC
AGAAAAGACAATCITTTTGTTGTCAAG-3'
The following two self-complementary sequences were synthesized for the
kinetic study of platinating a double-stranded DNA:
14mer CC: 5'-TATAGGTACCTATA-3'
14mer MM: 5'-TATAGGTAMMTATA-3'
234
Chapter 7 Transcription-Coupled and DNA Damage-Dependent
Ubiquitination of RNA Polymerase II in Vitro*
* The work in this chapter was published in PNAS 99, 4239-4244(2002).
235
Introduction
Transcription-coupled repair (TCR) is a cellular pathway for the removal of the
many lesions that block and arrest transcription. TCR is responsible for the rapid and
preferential repair of damage in the transcribed strand of active genes (1-3). Specific
mutations in genes are known to cause defects in TCR, including Cockayne syndrome
(CS) complementation groups A and B which debilitate TCR of UV- and oxidativedamage induced lesions (4, 5), and the breast cancer susceptibility gene BRCA1, which
is defective in TCR of oxidative DNA damage (6, 7). Lesions induced by
chemotherapeutic agents such as cisplatin are also removed by TCR (8, 9), implicating
TCR in the differential response of cells to cancer treatment.
The recent discovery that UV irradiation or cisplatin treatment of cells induces
ubiquitination of the large subunit of RNA Pol II (Pol II LS) and its degradation (10, 11),
suggested
a biochemical link between ubiquitination
and
DNA repair. The
ubiquitinated polymerase is hyperphosphorylated on the C-terminal domain (CTD) of
Pol II LS (10). Ubiquitination of Pol II was not observed in TCR-deficient CSA and CSB
cells but could be restored upon transfection of their respective cDNAs (10). These
results further indicate a relevance of Pol II ubiquitination to the mechanism of action of
cisplatin. This chemotherapeutic agent is routinely used in cancer treatment against
otherwise resistant solid tumors, such as encountered in testicular, ovarian and breast
cancer (12).
Interestingly, Pol II undergoes degradation in cells treated with alpha-amanitin,
an inhibitor of its transcription (13). This amatoxin binds specifically to RNA Pol II (14,
236
15), and arrests the elongating polymerase in a conformation that inhibits incorporation
of a subsequent nucleotide triphosphate to the nascent RNA transcript (16, 17). Pol II
transcription inhibition by alpha-amanitin may resemble its transcription arrest at a
DNA lesion. It is not known whether alpha-amanitin dependent degradation of Pol II is
mediated by the ubiquitin pathway.
In order to understand
the cellular response to DNA damage during
transcription, we have investigated transcription- and DNA damage-dependent
ubiquitination of RNA Pol II in vitro. We significantly modified the original in vitro
assay for ubiquitination of Pol II (18) to increase its sensitivity. In addition, we
compared nuclear extracts from cells at different cell cycle stages in order to optimize
ubiquitination activity.
Materials and Methods
Chemical Reagents
Alpha-amanitin and His-tagged ubiquitin (His-Ub) were purchased from Calbiochem.
Aphidicolin,
hydroxyurea
and
nocodazole
(methyl-5-[2-thienyl-carbonyl]-1H-
benzimidazol-2-yl) carbamate) were purchased from Sigma-Aldrich.
PALA (N-
phospho-N-acetyl-L-aspartate) was obtained from the Drug Biosynthesis and Chemistry
Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National
Cancer Institute (Bethesda, MD). Cisplatin was provided by Johnson Matthey.
237
Preparationof Nuclear Extracts
Nuclear extracts were prepared from HeLa cells as described (19). Cells were
maintained in MEM with 10% horse serum (Gibco BRL)at 370C at a cell density of 0.5 1.0 x 106 cells/mL. To arrest HeLa cells at different stages, cells were either grown in
spinner flasks to 0.5 x 106 cells/mL as above, or on plates in DME containing 10% heatinactivated fetal bovine serum (Gibco BRL)to 30-50%confluency. The cells were then
serum-starved for 16 h by replacing the serum-containing media with serum-free
media. Serum was added back to the media to a final concentration of 10% on the
following day, and cells were incubated for a further 8 h prior to the addition of various
drugs to a final concentration of 1 mg/mL aphidicolin, 750 pM hydroxyurea, 2 tLg/mL
nocodazole and 500 jiM PALA, respectively. Cells were incubated with the drugs for
16 h before harvesting.
For analysis of drug-treated cells, 1 x 106 cells in PBS were permeabilized with
95% ethanol and stained by incubating with RNase I (200 g/mL, Calbiochem) and
propidium iodide (25 iig/mL, Sigma-Aldrich). The stained cells were analyzed in a
fluorescence-activated cell sorter (FACS Calibur, Becton Dickinson) and cell cycle stages
determined with the MODFIT LT program.
Platinationof PlasmidDNA
Plasmids pUC19 (New England Biolabs), pG5MLP-G380 (obtained from D.
Tantin), pUC118-296 (obtained from C. Kneip), pHIV+TAR-G400 and pHIVATAR-G100
(20) were prepared from overnight cultures of plasmid-harboring JM109 (Promega) in
238
LB media using a Promega Wizard Midi-prep kit. pG5MLP-G380 contained the major
late promoter (MLP) with a 380 bp G-less cassette inserted 10 bp downstream of the
initiation site. pUC118-296 contained the MLP and 5S rDNA nucleosome positioning
sequences (21).
The cis-[Pt(NH3)2(H2 0)2 ] cation (activated form of cisplatin) was prepared by
stirring a solution of cis-DDP (6.4 mg, 0.021 mmol) with 1.98 molar equivalent AgNO3
overnight in the dark. Any precipitated AgCl was removed by centrifugation, and the
supernatant was diluted to a final concentration of 1 mM cis-[Pt(NH3)2 (H20)2 ]2 in
water.
Platination of plasmids was achieved by incubating 0.043 nmol DNA with
activated cis-DDP at rf = 0.1 (where rf equals the number of moles of drug added per
mole of DNA nucleotide) in 100 pL 10 mM sodium phosphate (pH 6.8) for 16 h at 37 °C.
The unbound platinum salt was removed by dialysis. The ratio of platinum bound per
nucleotide, i.e. the rb value, was determined by flameless atomic absorption
spectroscopy (Perkin-Elmer HGA-800 Analyst 300) and UV absorption spectroscopy
(Hewlett Packard 8453).
In Vitro TranscriptionAssay
Transcription reactions (25 L) were performed essentially as described (20).
Nuclear extracts (30 gg) were incubated at 300C for 30 min in buffer 1 [10 mM HEPESKOH (pH = 7.9), 10% glycerol (v/v), 60 mM KC1, 7 mM MgC12, 0.1 mM EDTA, 1 mM
ATP, 10 mM creatine phosphate, 12 gg/mL poly IC, 2 g/mL poly dG-dC] containing 7
mM DTT, C/G/UTP
mix (25:200:200
M), 10
Ci [a- 32P]CTP [800 Ci (29,600
239
GBq)/mmol], and 200 ng DNA template. To study the effect of alpha-amanitin, the
reactions were pre-incubated at 300 C for 15 min in the absence and presence of 5 IM
alpha-amanitin under the above conditions, but with the NTPs and creatine phosphate
added subsequently. The reactions were then incubated for a further 30 min. [In order
to observe a complete inhibition of transcription by alpha-amanitin, the pre-incubation
step was included to allow for sufficient drug binding to Pol II. For the in vitro
ubiquitination assay (see below), the results obtained with or without pre-incubation
with alpha-amanitin were similar.] All reactions were subjected to RNase T1 digestion
at 37°C for 5 min and G-less RNA transcripts were isolated and analyzed by urea-PAGE
as described (22). The gels were vacuum-dried and autoradiographed with KODAK XOMAT film at -80°Cfor 24-40 h.
In Vitro UbiquitinationAssay
Reactions were set up as in in vitro transcription assays with 60-70 g nuclear
extract in buffer 1 (which contains 1 mM ATP) with 1 pigDNA template, 200 AM CTP,
UTP and GTP and 1.25 g His-tagged ubiquitin (His-Ub). Saturating levels of alphaamanitin (5 pM) were added to the mix where indicated. Reactions were incubated at
30°C for 45 min, and His-ubiquitinated proteins were isolated by incubating at 40C for
1 h with 20 1 Ni-NTA agarose (Qiagen) in a final volume of 200 AL in buffer 2 [50 mM
sodium phosphate (pH 7.9), 0.3 M NaCl, 0.05% Tween 20 (v/v)] containing 10 mM
imidazole. The Ni-NTA agarose was pre-blocked with 1 mg/mL BSA prior to use.
240
After low-speed centrifugation
(735 g), the Ni-agarose beads containing His-
ubiquitinated proteins were washed twice with 1 mL buffer 2 containing 50 mM
imidazole. The Ni-bound proteins were eluted with SDS-loading buffer [20 mM
Tris.HCl (pH 6.8), 10% glycerol (v/v), 100 mM 2-mercaptoethanol, 1% SDS (w/v),
0.02% bromophenol blue (w/v)] containing 0.1 M EDTA (pH 7.0). To analyze the
protein content, samples were boiled in SDS-loadingbuffer and electrophoresed in SDS7.5% polyacrylamide gels, followed by electro-transfer to Immobilon-P membranes
(Millipore).
Pol II was detected with N20 (Santa Cruz), a non-phospho-specific rabbit
polyclonal IgG. The phosphorylated form of Pol II was detected with H5 or H14
(Covance), a mouse monoclonal IgM which recognizes phospho-serine 2 or 5 of the
CTD heptapeptide repeat.
Results
Alpha-amanitinStimulates Ubiquitinationof RNA PolII
To investigate a possible correlation between ubiquitination of RNA Pol II and
the arrest of transcription, we examined the effect of alpha-amanitin, a Pol II inhibitor,
on polymerase ubiquitination in vitro. Previous results suggested little or no effect of
alpha-amanitin on in vitro ubiquitination of polymerase in nuclear extracts from
unsynchronized cells (18). We prepared nuclear extracts from cells arrested in G1/S
phase by aphidicolin, a DNA a-Pol inhibitor (23), and tested for alpha-amanitin
dependent ubiquitination of Pol II LS (see below for drug and cell cycle dependence).
__
241
Cell cycle stages were verified by FACS analysis (not shown).
To increase the
sensitivity of the assay, His-Ub was added to the reaction, and ubiquitinated proteins
were selected on Ni-NTA agarose (Qiagen) and analyzed by Western blotting. The
reaction conditions were those used for transcription (see Material and Methods).
SDS-PAGE of total unselected extracts and Western blot analysis with a nonphospho-specific antibody to the N-terminus of Pol II LS (N20) revealed that
hypophosphorylated
Pol
IIA (Figure
7.1a,
lane
1)
was
converted
to
hyperphosphorylated Pol IIOduring the reaction (Figure 7.1a, lanes 2 and 4). Upon
addition of alpha-amanitin, a new slow migrating band appeared (marked with an
asterisk; Figure 7.1a, lanes 3 and 5). This slow migrating band was observed in the Nibound fraction only when His-Ub was present in the reaction (Figure 7.1a, lane 10),
indicative of ubiquitination of RNA Pol II. The Pol IIA and II0 bands recovered in the
Ni-NTA fractions in the absence of His-Ub are due to non-specific binding to Ni agarose
(Figure 7.1a, lanes 7-8). When the Western blot was stripped and re-probed with an
antibody to the phosphorylated CTD (H14), ubiquitinated Pol II was also detected
(marked with an asterisk; Figure 7.lb, lanes 3, 5 and 10). H14 recognizes phosphoserine 5 of the CTD heptapeptide repeat. Similar results were obtained using H5,
another antibody which specifically recognizes phospho-serine 2 of the CTD repeat (not
shown).
Thus
alpha-amanitin
stimulates
ubiquitination
of Pol II LS, and
hyperphosphorylated IIo comprise the ubiquitinated species, consistent with previous
results (10). Only a small percentage of Pol II (- 5%) is ubiquitinated since, relative to
242
the input material (Figure 7.1a and b, lanes 1-5),most of the polymerase was recovered
in the fraction not bound to the column (flow-through) (Figure 7.1a and b, lanes 11-15).
To determine whether ubiquitination activity varied at different stages of the cell
cycle when arrested by drug treatment, we compared the activities of extracts from cells
treated with different cell-cycle inhibitors. The fraction of cells at each stage was
determined by FACS analysis (not shown). Extracts from cells treated respectively with
aphidicolin, hydroxyurea (a ribonucleotide reductase inhibitor) (24) and PALA (an
inhibitor of de novo pyrimidine biosynthesis) (25), all showed similar levels of alphaamanitin induced ubiquitination (Figure 7.2a, lane 5-6, 8-9, and 11-12, respectively).
Consistent with previous findings (18),extracts from unsynchronized cells did not show
an appreciable alpha-amanitin induction of ubiquitination (Figure 7.2a, lanes 2-3).
G2/M extracts from cells treated with the microtubule inhibitor, nocodazole (26), gave a
higher proportion of Pol IIo to Pol IIAunder the reaction conditions (Figure 7.2a, total,
lanes 13-15),but showed little or no ubiquitination (Figure 7.2a, lanes 14-15). In parallel
work, we compared in vitro transcription activities of the different extracts using
pG5MLP-G380, which contains a major late promoter (MLP) and a 380 bp G-less
cassette (G380). The amount of G380 transcript produced by G1/S and S extracts after
RNase T1 digestion was approximately two-fold higher (Figure 7.2b, lanes 3 and 5) than
extracts from unsynchronized cells (Figure 7.2b, lane 1), whereas that of G2/M extracts
was considerably lower (Figure 7.2b, lane 7). In all cases, alpha-amanitin inhibited
transcription as judged by disappearance of the transcript (Figure 7.2b, lanes 2, 4, 6, and
8).
243
Dependenceof Ubiquitinationon DNA
Since alpha-amanitin binds to Pol II itself (15, 27), we studied whether
ubiquitination of polymerase due to alpha-amanitin depends on the presence of DNA.
We titrated the ubiquitination reaction against increasing concentrations of template
DNA (Figure 7.3). In the absence of DNA, only low-level or background ubiquitination
of polymerase was observed (Figure 7.3, lanes 3 and 4). When a promoter-containing
DNA (pUC118-296) was present, the extent of ubiquitination with alpha-amanitin
increased with increasing amounts of DNA until 1.2 g template was added to the
reaction (Figure 7.3, lanes 5-10). The level of ubiquitination, however, declined upon
further addition of DNA (Figure 7.3, lane 12). Therefore, alpha-amanitin induces
ubiquitination dependent upon the presence of DNA, probably by interacting with
polymerase-DNA complexes during transcription.
This stimulation could not be
recapitulated by nucleotide depletion or termination of transcription by addition of 3'O-methyl-GTP (not shown). At high DNA concentrations in vitro, total transcription by
Pol II is typically not promoter-dependent and occurs in the absence of a promoter (not
shown).
Consistent with this behavior, duplex plasmid DNA stimulated alpha-
amanitin dependent ubiquitination independent of the promoter sequence (not shown).
Similarly, both supercoiled and linearized plasmid were as effective in stimulating
ubiquitination (not shown).
244
DNA
Ubiquitinationis Inducedby Arrest of Transcriptionon Cisplatin-Damaged
The above data strongly suggest that polymerase-DNA complexes stalled during
transcription are targeted for ubiquitination. To investigate a role for DNA damage in
stimulating Pol II ubiquitination, DNA templates containing lesions that arrest
transcription by Pol II, were added to the reaction.
Plasmid DNAs modified by
cisplatin having an rb value (bound platinum to DNA nucleotide ratio) of 0.055, or an
average of one platinum adduct per 18 DNA bases, were tested for induction of Pol II
ubiquitination. Reactions containing alpha-amanitin were also performed in parallel for
comparison of activities.
As observed earlier, ubiquitination of Pol II LS was negligible in a reaction
lacking DNA or containing unmodified DNA (Figure 7.4a, lanes 2-4). In contrast,
ubiquitination was induced significantly by each cisplatin-modified template (Figure
7.4a, lanes 6, 9 and 12), to similar levels as that induced by alpha-amanitin with
unmodified DNA (Figure 7.4a, lanes 5, 8 and 11). This result indicates that both
cisplatin and alpha-amanitin induce ubiquitination in a similar fashion, probably by
inhibiting elongation of Pol II transcription on DNA.
At this level of cisplatin
modification, transcription from cisplatinated DNA containing a 380 bp G-less cassette
(G380)was abolished, whereas transcription from an unmodified DNA template with a
100 bp G-less cassette (G100) was unaffected in the same reaction (Figure 7.4b, lanes 12). Thus, the absence of transcript from the G380 cassette is specific to cisplatin
modification of the DNA, suggesting that ubiquitination is induced by arrest of
transcription due to the lesions in the DNA. As with alpha-amanitin, ubiquitination
I
245
due to cisplatin-modified DNA is independent of the presence of a specific promoter on
the template DNA. Ubiquitination was observed with cisplatinated pUC19 DNA, albeit
at a lower level (Figure 7.4a, lanes 11-12).
Dependenceof Ubiquitinationon Transcription
The transcription dependence of the ubiquitination reactions was examined
further by using antibodies to a general transcription factor involved in pre-initiation
complex assembly, viz. TFIIB (28).
Addition of an antibody to TFIIB inhibited
transcription (Figure 7.5c, lane 3), as indicated by disappearance of the transcript
present in a normal in vitro transcription reaction (Figure 7.5c, lane 1). As expected,
addition of an antibody to c-myc had no effect on transcription (Figure 7.5c, lane 2).
When the ubiquitination reaction was tested in the presence of an anti-TFIIB antibody,
the ability of either alpha-amanitin
or cisplatin-modified DNA to stimulate
ubiquitination was severely reduced (Figure 7.5a and b, bound fractions, lanes 6-7)
relative to a control without antibody (Figure 7.5a and b, bound fractions, lanes 2-3).
Ubiquitination overall was unaffected by an antibody to c-myc (Figure 7.5a and b,
bound fractions, lanes 4-5). Analysis of the total extracts revealed that phosphorylation
of CTD was largely inhibited by addition of antibody to TFIIB (Figure 7.5a and b, total,
lanes 6-7). This behavior is probably a consequence of inhibition of transcription
initiation, which is accompanied by Pol II phosphorylation.
246
Discussion
We report the first evidence for transcription-coupled and DNA damagedependent ubiquitination of RNA Pol II in vitro. Addition of alpha-amanitin, an
inhibitor
of
Pol
Hyperphosphorylated
II
elongation,
stimulates
ubiquitination
of
Pol
II
LS.
Pol II is ubiquitinated, consistent with an engagement in
elongation. This stimulation requires the addition of template DNA, also indicating a
dependence upon polymerase being engaged in transcription. Moreover, addition of
DNA templates containing cisplatin adducts, which arrest transcription, stimulates
ubiquitination
of Pol II LS as compared with reactions containing unmodified
templates. The transcription dependence of both alpha-amanitin and cisplatin-induced
ubiquitination was further demonstrated by the abrogation of ubiquitination following
addition of antisera to a component of the transcription pre-initiation complex, TFIIB.
DNA Repair
Implicationsfor Transcription-Coupled
Previous in vivo experiments pointed to a possible role for ubiquitination of Pol II
in transcription-dependent
DNA repair. Bregman et al. (10) observed that the
polymerase was ubiquitinated upon DNA damage from UV irradiation or cisplatin
treatment of repair-competent cells and that this ubiquitination was absent in TCRdeficient Cockayne syndrome cells. Our finding that ubiquitination is triggered by
alpha-amanitin as well as cisplatin-DNA adducts in vitro, both of which arrest
transcription, is consistent with a role for ubiquitination in the cellular response to DNA
damage.
The transcription dependence of ubiquitination in vitro suggests that
247
components of the reaction specifically recognize a form of the polymerase generated
by its arrest at lesions or by alpha-amanitin. This same arrested complex is probably a
key structure signalling TCR.
Only a small proportion of polymerase is ubiquitinated (- 5%) in a reaction
containing saturating levels of alpha-amanitin. Strikingly, the levels of ubiquitination
induced by alpha-amanitin and by DNA with an average of one platinum adduct per 18
DNA bases (approximately 1 platinum adduct per helical turn) are qualitatively similar.
Alpha-amanitin binds with high affinity (10a-10
M) (15) to a conserved region of Pol
II LS (27). It is a very specific and potent inhibitor of the elongating polymerase (14, 29).
Biochemical studies reveal that alpha-amanitin neither dissociates the polymeraseDNA-RNA complex nor competes with nucleotide incorporation (14,15). Rather, under
conditions of elongation, it blocks Pol II after formation of a phosphodiester bond, and
probably inhibits a transition in the conformation of Pol II required for its movement
along the DNA template (16, 17). Thus the binding of alpha-amanitin may arrest the
elongating polymerase in a transitional conformation that is recognized by components
in the reaction specifying ubiquitination. Based on the similarities in ubiquitination of
Pol II due to alpha-amanitin and cisplatin-DNA damage, the platinum lesion most
likely also blocks translocation of Pol II along the DNA, stabilizing a similar transitional
conformation. The major cisplatin-DNA adduct is the intrastrand cross-link between
adjacent purine residues, with the 1,2-d(GpG) and 1,2-d(ApG) intrastrand cross-links
being the predominant lesions (30). Both the 1,2-d(GpG) and 1,3-d(GpTpG) adducts are
efficient blocks to transcript elongation in vitro (31, 32) and in vivo (33).
248
Evidence suggests that the inability to remove an RNA polymerase stalled at a
DNA lesion prevents access to repair enzymes, thus enhancing the mutation rate. The
same polymerase would also block elongation by other polymerases and hence interfere
with gene expression. An RNA Pol II stalled at a UV-induced cyclobutane pyrimidine
dimer prevented access of a small bacterial repair protein in vitro (34). Human mutant
cells impaired in TCR of oxidative lesions, viz. CSA and CSB, as well as subgroups of
xeroderma pigmentosum XPB, XPD and XPG that show Cockayne syndrome, exhibit a
high mutation frequency of 30-40% at an 8-oxoguanine lesion compared with a normal
level of 1-4% (35). TCR of oxidative lesions, including thymine glycols, is also defective
in cells derived from brcal-/-mice (6) and the human breast carcinoma cell line HCC1937
(7, 36), which harbors a mutation of the BRCA1 gene inherent in many incidences of
breast and ovarian cancer (37, 38). Transcription was not observed beyond the 8oxoguanine lesion on plasmid DNA transfected into the XPG/CS and HCC1937 cells
(35, 36). Further, TCR-deficient CSA and CSB cells are more susceptible to apoptosis
upon UV irradiation, perhaps due to the inefficient removal of RNA Pol II at UVinduced lesions and subsequent blockage of transcription (39).
The CellularSignals TriggeringUbiquitination
Ubiquitination occurs by covalent attachment of multiple ubiquitin molecules to
the protein substrate.
This process is typically mediated by three enzymes, the
ubiquitin-activating enzyme El, the ubiquitin-conjugating enzyme E2 and the substrate-
249
specific ubiquitin ligase complex E3 (40, 41). Ubiquitination by E3 ligase is highly
regulated and in many cases, substrate recognition requires covalent modification, e.g.
in the phosphorylation of IKB(42) and p-catenin (43). Immunoblot analyses of whole
cell extracts from UV-irradiated/cisplatin-treated
extracts from the in vitro ubiquitination
cells (10) and of Ni-selected nuclear
assay (this work), demonstrate
that
ubiquitinated RNA Pol II is recognized by antibodies specific to phosphorylation of
either serine 2 or serine 5 of the CTD. Previous results using nuclear extracts from
growing cells indicated that a GST-CTDfusion protein was ubiquitinated in a kinasedependent manner (18), suggesting some modification of this repetitive domain may be
important for ubiquitination.
An interesting finding of the present work is that nuclear extracts from cells
arrested with drugs which affect DNA synthesis in the G1/S phase, exhibit significantly
enhanced ubiquitination activity relative to unsynchronized cells. The reason for this
behavior is unclear since ubiquitination of Pol II following DNA damage is observed in
vivo in unsynchronized cells (10), which are primarily in the G1 phase of the cell cycle.
Of possible relevance is that all three drug inhibitors activate or induce proteins
involved in repair of DNA damage.
Included are the DNA damage cell cycle
checkpoint protein p53 (44-47),and the breast cancer susceptibility protein BRCA1 (48),
which in addition to transcription-coupled repair of oxidative damage (6, 7, 36), is
involved in double-strand break DNA repair (49, 50). p53 interacts with proteins
involved in TCR, viz. CSB, as well as the XPB and XPD subunits of TFIIH (51), with the
latter interactions being important for induction of apoptosis (52). BRCA1,on the other
250
hand, undergoes phosphorylation and relocalized into replicating DNA structures upon
treatment of S-phase cells with hydroxyurea or DNA damage agents (48). BRCA1 also
interacts with RNA Pol II holoenzyme (53) and contains a ring finger domain that
interacts with the ring finger domain of another protein BARD1 (54). Ring finger
domains are generally involved in the ubiquitination pathway (55) and BRCA1 can
facilitate E2-dependent ubiquitination in vitro (56). BARD1, which co-localized with
BRCA1 upon DNA damage (48), enhances ubiquitination activity; a breast cancerderived Cys mutation in the ring finger of BRCA1, which is deficient in its interaction
with BARD1, abolishes this activity (57). The same mutation is defective as a tumor
suppressor. The substrate for ubiquitination by BRCA1is currently not known but may
be RNA polymerase II [also see (58)].
Implicationsfor the CisplatinMechanismof Action
Previous studies revealed that specific architectural changes induced upon
formation of the major cisplatin 1,2-intrastrand DNA cross-links lead to binding of
minor groove intercalating proteins including HMGB1 (59, 60) and TBP (61). Such
processes inhibit nucleotide excision repair (NER) in vitro (62) and delay NER in vivo
(63), leaving the adducts intact for subsequent recognition through arrest of Pol II
elongation during TCR. The present results suggest that ubiquitination of RNA Pol II
may be an important event in the repair of cisplatin damage. Because removal of the
stalled polymerase is critical for repair, inhibition of ubiquitination of Pol II is likely to
enhance cell killing by cisplatin.
251
Acknowledgments
I thank Prof. Philip A. Sharp and Dr. Keng-Boon Lee for great collaboration. I am
grateful to C. Kneip and D. Tantin for pUC118-296 and pG5MLP-G380. I thank D.
Tantin for critical review of the manuscript, and members of the Sharp laboratory for
helpful discussions. This work was supported by the U.S. Public Health Service grant
PO1-CA42063 from the National Cancer Institute (NCI) and R01-AI32486from the
National Institute of Health (NIH) to P.A.S.; and by NIH grant CA34992 from NCI to
S.J.L..The work was also partially supported by the Cancer Center Support (Core) grant
P30-CA14051from NCI.
252
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(a) WB: N20
Total
His-Ub:
a-am:
SM
-
Ni-Bound
-
+
+
SM
-
Flow-through
+
+
-
+
SM
-
+
+
-
+
II0
IIA
(b) WB: H14
Iio =I
12 3 45
Figure 7.1.
El
Esdm
6 7 8 9 10 1112131415
Ubiquitination of Pol II is induced by alpha-amanitin in an in vitro assay.
Nuclear extracts from cells treated with aphidicolin were incubated in an in vitro
ubiquitination reaction with pUC118-296 in the absence or presence of His-Ub and
alpha-amanitin (a-am). His-ubiquitinated proteins were selected on Ni-NTA agarose.
Total (lanes 1-5), Ni-bound (lanes 6-10), and flow-through fractions from the Ni-NTA
agarose (lanes 11-15) were analyzed by SDS-7.5%PAGE and transferred to a PVDF
membrane. The immunoblot was probed consecutively with antibodies to, (a) the Nterminus of RNA Pol II (N20), and (b) the phosphorylated CTD of Pol II (H14). Lanes 1,
6 and 11 show untreated nuclear extracts.
258
(a) WB:N20
S
GS
G2M
G1/S
-
(b)
- (aphkidcouin)
(HU) (PALA)
(noodlezoi)
cr-am: SM -
SM - + SM - + SM - + SM -
~~Total
~.
.
.
L
G/S
S G2/IMV
+- .+ a:-am
- +--
_,...
-
__.,,
.,,.
..
1W
G380
Irotrrgo
:od
A
2 3 4 5 6 7 8 9 101112 1314 15
Figure 7.2.
ib I
SUD
E:
'
. :.-
12 3 4 5 6 7 8
Comparison of ubiquitination and transcription activities of various drug-
induced cell cycle stage nuclear extracts. (a) The in vitro ubiquitination assay was
performed as described in Figure 7.1 with nuclear extracts from unsynchronized cells
(lanes 1-3) as well as with cells treated respectively with aphidicolin (lanes 4-6),
hydroxyurea (HU) (lanes 7-9), PALA (lanes 10-12), and nocodazole (lanes 13-15).
Reactions (containing His-Ub) were analyzed by SDS-7.5%PAGE, and the immunoblots
for the total (top) and Ni-bound proteins (bottom) are shown. The immunoblot of total
extracts is shown at a lower exposure time than that for the bound proteins to allow
better resolution of protein bands. Lanes 1, 4, 7, 10 and 13 show untreated nuclear
extracts.
(b) Nuclear extracts from unsynchronized cells (lanes 1 and 2), and cells
arrested at the cell cycle stages of G1/S (lanes 3 and 4), S (lanes 5 and 6) and G2/M
(lanes 7 and 8), were incubated in an in vitro transcription reaction with pG5MLP-G380
containing a 380 bp G-less cassette (G380), in the absence and presence of alphaamanitin respectively. Reactions were subsequently treated with RNase T1, and G-less
transcripts were isolated and analyzed by 8 M urea-6% PAGE. An autoradiograph of
the gel is shown.
259
DNA:
a-am:
0+ +
0
SM
-
+
0.3
0.6
-
-++
1.2
-
2.4
(pg)
+
]Ub
Total
' Iio
-
110
IMIA
IUb
Bound
' 110
1 2 3 4
5 67
8 910
11
12
WB: N20
Figure 7.3.
Alpha-amanitin induction of Pol II ubiquitination is DNA-dependent.
Ubiquitination reactions with nuclear extracts from aphidicolin-treated cells and His-Ub
were titrated against increasing amounts of pUC118-296 (0-2.4 gg, lanes 3-12), and the
effect of alpha-amanitin (a-am) was assayed. Reactions were analyzed as described in
Figure 7.1, and immunoblots of total (top) and Ni-NTA bound proteins (bottom) against
N20 are shown. Lanes 1, untreated extracts. Lanes 2, ubiquitination reaction containing
1 gg DNA, incubated in the absence of both His-Ub and alpha-amanitin.
260
(a) WB: N20
DNA:
c-am:
'
SM
-
+ZQ
nrl""MI
VIIu P nlI\/l,
r' i VT
-G380 TAR-G400 pUC19
+
-
+
cis
-
+
cis
-
(b)
top
- cisPt
cis
Ub
Total
Iio
IIA
Bound
M i
bG0
1 2 34 5 6 7 8 9101112
Figure 7.4.
12
Ubiquitination of Pol II is induced to a similar extent by cisplatin-modified
DNA and alpha-amanitin.
(a) The effects of alpha-amanitin and various cisplatin-
modified DNAs on Pol II ubiquitination were examined in the in vitro ubiquitination
assay. Immunoblots of total (top) and bound proteins (bottom) are shown. Lanes 1,
untreated nuclear extracts. Lanes 2 and 3, ubiquitination reactions performed in the
absence of DNA, with alpha-amanitin (a-am) added where indicated. Lanes 4-5, 7-8
and 10-11, reactions containing unmodified DNA with the addition of alpha-amanitin
where indicated. Lanes 6, 9 and 12, reactions containing cisplatinated DNA (cis). (b) In
vitro transcription reactions were performed simultaneously with pG5MLP-G380
containing a G380 cassette and pHIVATAR-G100containing a G100 cassette. Lane 1,
transcription with unmodified DNA. Lane 2, transcription with cisplatinated pG5MLPG380 but unmodified pHIVATAR-G100. Autoradiograph
of gel is shown.
(a)
antibody:
cr-am:
--
Total
c-rnyc TFIIB
-
SM
261
+
+
-
+
Bound
SM
cisPt:
SM
-
+
-
+
-
S
IIA
(b)
-
-
(c)
ps
c-myc TFIIB
SM
+
-
+
+l
s
-
-
A,
+
+
-
Ub
+
V
-A, =
to
-
top
-
G380
Ub
IIA
4&..MW_.AWMRI
1 2
3 4 5 6 7
1 2
3 4
5
6
7
1 2 3
WB: N20
Figure 7.5.
Stimulation of polymerase ubiquitination by alpha-amanitin or cisplatin-
modified DNA is suppressed by inhibiting transcription. (a) A typical ubiquitination
reaction was carried out in the absence (lane 2) or presence of alpha-amanitin (a-am)
(lane 3). Corresponding reactions were performed with the addition of antibodies to cmyc (lanes 4-5) or TFIIB (lanes 6-7). Figure shows immunoblots of total extracts (left)
and Ni-bound fractions (right), which were developed with antibodies against Pol II LS
(N20). The immunoblot of total extracts is shown at a lower exposure time than that for
the bound proteins.
Lanes 1 show untreated nuclear extracts. (b) Ubiquitination
reactions were performed in the presence of unmodified DNA (lane 2) or cisplatinmodified DNA (cis-DDP) (lane 3). Corresponding reactions were conducted with the
addition of the different antibodies, followed by immunoblotting as described in (a).
Lanes 1 show untreated nuclear extracts. (c) In vitro transcription reactions using
pG5MLP-G380 were performed in the absence (lane 1) and in the presence of an
antibody to c-myc (lane 2) or TFIIB (lane 3). Reactions were analysed as in Figure 7.2b.
Autoradiograph of gel is shown.
262
Part II. Synthesis of Site-SpecificallyCisplatin-Modified DNA
Templates and In Vitro Transcription Study
263
Introduction
Cisplatin, cis-diamminedichloroplatinum
(II), is routinely used for
treatment of testicular, ovarian, breast and several other types of cancers.(1)
DNA is widely accepted as the biologically relevant target of cisplatin.(2)
Cisplatin forms covalent, bifunctional DNA adducts, in which platinum binds to
the N(7) positions of purine nucleotides, forming 1,2-d(GpG) and 1,2-d(ApG)
intrastrand, 1,3-d(GpNpG) intrastrand, interstrand and protein-DNA cross-links.
Because of geometric constraints, the clinically inactive trans-DDPisomer can not
form such 1,2-intrastrand cross-links.(3,4) Therefore, it is likely that the ability of
cisplatin to form these particular DNA adducts accounts for to its unique
anticancer activity.
Platinum-DNA adducts affect many normal biological processes in the cell,
including transcription. Disruption of transcription may be the most significant
contributor to the anticancer activity of cisplatin.(2,5-9) Eukaryotic transcription
is a complex process requiring not only RNA pol II but also a series of general
transcription factors. These factors direct RNA pol II to the appropriate promoter
sequences, facilitating transcription initiation, and promoting transcription
elongation. The basal transcription factors TF IIB, TF IID, TF IIE, TF IIF, and TF
IIH are required for accurate initiation of RNA pol II-catalyzed transcription
from a promoter sequence.
264
Previous studies of the effect of cisplatin on transcription were performed
either in vivo or in vitro by using pure RNA polymerase or cell extracts. In vivo
studies of RNA pol II transcription from a globally platinated plasmid in
mammalian cells demonstrated that RNA polymerase II bypasses cis- and transDDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively.(10)
Cisplatin can substantially reduce transcription from a mouse mammary tumor
virus promoter stably incorporated into mouse cells.(11) On the other hand,
cisplatin can block synthesis of rRNA, transcribed by RNA pol I, while activity of
RNA polymerase II continued to be detected throughout the nucleus of HeLa
cells.(12) These seemingly contradictory data could result from the use of
different detection methods or different cell lines.
Results from studies in vitro also seem very controversial. Pure phage SP6,
T3, T7 and E.Coli. RNA polymerases, as well as wheat germ RNA pol II (no
transcription factors), were investigated for their activity in the presence of
cisplatin.(13-17) Globally platinated DNA and site-specific cisplatin intrastrand
cross-links located at d(GpG), d(ApG) or d(GpTpG) sequenceswere
used as
transcription templates. Polymerases were blocked by cisplatin adducts on the
transcribed strand, but not on the non-transcribed strand. Moreover, the
transcription by these polymerases was blocked more efficiently on cisplatintreated DNA than on trans-DDP-treated DNA.
These previous in vitro studies employing pure polymerases may not
accurately represent the conditions in mammalian eukaryotic cells. Eukaryotic
265
transcription is a highly complex process requiring not only RNA pol II, but also
a series of general transcription factors. These transcription factors direct RNA
pol II to the appropriate promoter sequences, to facilitate initiation and to
promote elongation.
Recent experiments revealed that RNA pol II stalls at a 1,3-d(GpTpG)
cisplatin adduct but not at a 1,2-d(GpG) cross-link. This work employed using
extracts from Xeroderma Pigmentosum complementation group F GM8437
fibroblast cells.(18)The mechanism of bypass is still unclear, and the results seem
in conflict with each other. In order to reconcile these conflicting data and to
investigate the effect of cisplatin on transcription elongation, a 296-bp probe
containing a single, site-specific 1,2-d(GpG) intrastrand cisplatin cross-link either
on the transcribed or the non-transcribed strand was synthesized as the substrate
for in vitro transcription experiments using either recombinant transcription
system or HeLa nuclear extracts.
Materials and Methods
T4 polynucleotide
kinase (PNK), T4 DNA polymerase, T7 DNA
polymerase, T4 DNA ligase, NTPs, dNTPs, NEB buffer 2, NEB buffer 3 and T4
PNK buffer were purchased from New England BioLabs. The T4 gene 32 protein
was purchased from Boehringer Mannheim. RNA Pol II and general factors were
kindly provided from Prof. D. Reinberg at UMDNJ. HeLa nuclear extracts were
obtained from Prof. P.A. Sharp at MIT.
266
y-3 2 P-ATP (10 mCi/mL,
6000 Ci/mmol)
and at-32 P-UTP (40 mCi/mL,
6000Ci/mmol) were obtained from Perkin-Elmer Life Science. A low mass DNA
ladder was purchased from Invitrogen Life Technologies. Phosphoramidites and
chemicals for DNA synthesis were obtained from Glen Research. Agarose, LB
agar, and a phenol/CHCl3/isoamyl
alcohol mixture were purchased from
GibcoBRL. SequaGel diluent and concentrate solutions were purchased from
National
Diagnostics. The ion exchange (IE) high performance
liquid
chromatography (HPLC) column (Dionex DNApac PA100) was purchased from
Dionex and Quick Sephadex G-25 Spin columns from Roche. Other chemicals
were obtained from Sigma or Mallinckrodt.
Synthesis and Purification of DNA Fragments. The DNA fragments (for
sequences, see Appendix 7.1) were synthesized and purified as precursors for
preparing the DNA 296mer. All oligonucleotides were synthesized on a 1 tmol
scale with an Applied Biosystems DNA synthesizer (Model 392) and purified by
conventional methods. The platinated oligonucleotides were prepared, purified
and characterized as described previously.(19)
Platination,Purification,and Characterization
of PlatinatedDNA Primer 20GG.
An aliquot of 179 jtL of activated cisplatin (0.84mM) was mixed with 100 1 L of
oligonucleotide (1.50 mM) in a 1:1 in 0.01 M phosphate buffer (pH 6.75). The
mixture was incubated in the dark at 37 C for 6-7 h. The platinated DNA
products were purified by IE HPLC and characterized by Maxam-Gilbert
267
sequencing, mass spectrometry, UV-vis spectroscopy and atomic absorption
spectroscopy (AAS).
Synthesis of DNA 296-Pt by the Enzymatic Ligation Method. The 296mers
were prepared by a two-phase enzymatic ligation strategy from nine synthetic
DNA fragments (Figure 7.6). In the first phase, 5'-end phosphorylated fragment
TT#9 was annealed with fragment TT#1, and 5'-end phosphorylated fragment
TT#2 was annealed with fragment TT#8. Then these four pieces of DNA were
ligated together to form fragment I. In parallel, fragment TT#3 was annealed
with 5'-end phosphorylated TT#7, and 5'-end phosphorylated fragment TT#4
was annealed with TT#5 and 5'-end phosphorylated TT#6-Pt separately. Then
these five pieces of DNA were ligated together to form fragment II. The products
were then purified on a 5% denaturing PAGE gel electrophoresis. The desired
bands containing DNA fragments I and II were isolated and extracted following
the QIAEX II PAGE gel extraction protocol.
In the second phase, purified
fragments I and II were 5'-phosphorylated, annealed, and ligated to form the
final product 296-Pt. The product was purified on a 5% denaturing PAGE gel
electrophoresis. The desired band containing the DNA 296-Pt was isolated and
extracted following the QIAEX II polyacrylamide gel extraction protocol. The
resulting DNA 296-Pt was ready for the in vitro transcription assay. The
concentration of 296-Pt was estimated by using a low mass DNA ladder
(Invitrogen Life Technologies). The low mass DNA ladder is composed of an
equimolar mixture of six blunt-ended DNA fragments of 2000,1200, 800, 200 and
268
100 bp. Electrophoresis of 4 iL of this DNA results in bands containing 200, 120,
80, 40, 20 and 10 ng of DNA respectively. The sample DNA concentration can be
estimated by comparing its intensity to that of the similar sized band of the low
mass DNA ladder.
Site-Directed-Mutagenesis(SDM). In order to improve the efficiency of the
primer extension reaction, a longer precursor was needed. Two guanines were
mutated to two cytosines. The original and mutated precursors are underlined as
follows:
Before SDM 5' ... GATA CCG GTT CTC GTC CGA TCA CC ... 3'
After SDM
5' ... GATA CCG GTT CTC CTC CCA TCA CC ... 3'
The primers Pr36ml (5' CTT GAC TTC GGT GAT GGG AGG AGA ACC
GGT ATC TTC 3') and Pr36m2 (5' GAA GAT ACC GGT TCT CCT CCC ATC
ACC
GAA GTC
AAG 3') for site-specific
mutagenesis
were
designed,
synthesized, and purified. A PCR reaction with the primers was carried out on
the double-stranded circular DNA template pUC119-296 (constructed by Dr. C.
plasmid was transformed
into E. coli XL1-Blue
cells. A 50 gL portion of transformed
cells was plated on LB-
Kneip). The mutated
supercompetent
Agar plates containing 100 pg/mL ampicillin and grown overnight at 37 C. A
well-isolated colony was picked from a subcloned plate, suspended in 5 mL of
LB media containing 100 plg/mL ampicillin, and grown overnight at 37 C with
agitation to reach saturation. The saturated solution was added to 500 mL of LB
269
media containing 100 glg/mL ampicillin and left to grow overnight at 37 C,
while rotating at 200 rpm. Cells were harvested by centrifugation for 10 min at
6000 rpm in a Sorvall GS3 rotor. The resulting mutated plasmid DNA was
isolated following the Quiagen plasmid preparation kit and submitted to the
MIT Biopolymers Lab for DNA sequence analysis.
Synthesis of DNA 296-Pt by the PrimerExtensionMethod.
The procedure for primer extension is diagrammed in Figure 7.7.
1. Plasmid Transformation.The circular plasmid pUC119-296, containing the
unmodified transcription template for the 296mer sequence, was transformed
into E. coli XL-1 blue cells. The unmodified plasmid DNA was isolated as
described in the Quiagen plasmid preparation kit.
2. ssDNA Production.In the presence of VCS M13 helper phage, ssDNA of
plasmid pUC 119-296 was produced by the transformed E. coli XL-1 blue cells.
Briefly, a 5-250 mL portion of sterile LB containing 100 Pg/mL ampicillin and 10
g/mL tetracycline was pre-warmed to 37 C. A portion of 1/100 volume of
mid-log phase XL 1 Blue cells was mixed with phage VCS M13 stock solution,
followed by incubation at room temperature for 5 min and addition to the prewarmed LB. The infected culture was allowed to grow for 5-6 h at 37 °C, with
rotation at 250-300 rpm. Kanamycin was added to give a final concentration of 70
pg/mL and the culture was allowed to grow for 12-16 h.
The solution of infected cells was centrifuged for 15 min at 9000 X g, 4 °C
twice. The supernatant was transferred to a fresh flask and a 250 pL aliquot of
270
RNase free DNase I was added to give a final concentration of 10 U/mL. After
incubation at 37 C for 15 min, PEG 8000 (10 g) and NaCI (7.5 g) were added,
and the solution was stirred for 1 h at room temperature, followed by
centrifugation for 20 min at 12,000 X g, 4 C. The supernatant was removed, and
the bottle with the pellet was centrifuged for 5 min at 12,000 X g, 4 C, after
which any remaining supernatant was removed. The pellet was resuspended
with 7 mL of 10 mM Tris-HCl and 1 mM EDTA (pH = 8.0), followed by three
rounds of phenol extraction and ethanol precipitation. The resulting ssDNA was
characterized by agarose/EtdBr gel electrophoresis.
3. Primer Extension from Circular ssDNA. Circular ssDNA (35 pmol) was
annealed to the 5'- phosphorylated platinated primer 20GG-Pt (190 pmol) in 100
IiL of 10 mM Tris-HCl (pH = 7.9), 10 mM MgC12, and 50 mM NaCl. Following
annealing, the solution was mixed with 30 units of T7 DNA polymerase, 15 units
of T4 DNA polymerase, and 1200 units of T4 ligase in the presence of 1.5 mM of
each dATP, dCTP, dTTP, dGTP, 1 mM ATP, 50 pg/mL BSA, 7.5 mM DTT, 40
mM Tris-HCl (pH 7.5), 10 mM MgCl2, 12.5 mM NaCl (modified from the
conditions used by Debyser).(20) The reaction was incubated at 37 C for 2 h,
after which a second aliquot containing 15 units of T4 polymerase and 1200 units
of T4 ligase was added. The reaction was incubated at 37 °C for another 2 h. The
resulting product was analyzed by 0.7% native agarose gel electrophoresis.
The primer extension product mixture was separated by CsCl/EtdBr
gradient centrifugation. After centrifugation, the relative specific densities of
271
DNA species, including circular DNA and nicked DNA, gave rise to a banding
pattern visualized by detecting intercalated EtdBr with 366 nm UV light. A
syringe was used to puncture the centrifuge tube and remove the two visible
bands. EtdBr was removed by extracting twice with isoamyl alcohol. CsCl was
removed by spin dialysis with Centricon-100 cartridges. The recovered DNA
sample was analyzed by agarose gel electrophoresis.
4. Sma I Digestionand AgaroseGel Purificationof 296-Pt. The resulting DNA was
subjected to Sma I digestion to get the 296-Pt fragment. The 296-Pt fragment was
separated from the remaining DNA on a 0.7% agarose gel electrophoresis. The
desired band containing 296-Pt was isolated and extracted following the QIAEX
II agarose gel extraction protocol. The resulting DNA was annealed and ready
for the in vitro transcription assay.
In Vitro Transcriptionwith a ReconstitutedTranscriptionSystem. The in vitro
transcription assay was carried out as previous described.(21) Briefly, doublestranded DNA template (80-850 fmol) was mixed with 0.1
L of RNA
polymerase II, 2.6 pmol TBP, 2.6 fmol TF IIB, 0.22 pmol TF IIE, 0.166 pmol TF IIF,
1.5 pL of TF IIH and 12 U RNAse inhibitor in the total volume of 40 ILLbuffer
containing 10 mM Tris-HCl (pH = 7.9), 0.1 mM EDTA, 5 mM P-mercaptoethanol,
0.1 mM PMSF, 50 mM KCl, 8 mM MgC12,100 mM (NH4)2SO4,66 mM DTT, 20
mM HEPES-KOH, 10% glycerol, and 4% PEG 8000. The mixture was incubated at
30 °C for -30-40 min, after which time 5-10 piCi t-32P-UTP, 50 pM UTP and 0.6
mM of ATP, CTP, and GTP were added. The reaction was incubated for an
272
additional 40 min at 30 C. For transcription from the G-less cassette template,
RNAse T1 was added after the reaction, followed by incubation at 37 C for an
additional 5 min. A 100-120 pILaliquot of stop mix, containing 1 mg/mL yeast
tRNA, 10 mM EDTA, 100 mM NaOAc (pH = 5.5), 0.2% SDS and 100 pg/mL
Proteinase K, was added, and the reaction was incubated at 37 °C for 30 min. The
reaction was extracted once with 150 jlL of phenol/CHCl3/isoamyl
(25:24:1, v/v/v)
alcohol
and precipitated with 500 trL of ice-cold ethanol. RNA
transcripts were analyzed by a 6% denaturing urea PAGE gel.
In Vitro TranscriptionAssay with NuclearExtracts.HeLa nuclear extracts (30
gLg)were incubated with 80-200 fmol of DNA template at 30 C for 30 min in a
buffer containing 10 mM HEPES-KOH (pH = 7.9), 10% glycerol (v/v), 60 mM
KC1, 7 mM MgCI2 , 0.1 mM EDTA, 1 mM ATP, 10 mM creatine phosphate, 12
ng/pL poly IC, 2 ng/pL poly dG-dC and 7 mM DTT. A 1 jiLU/G/CTP mixture
(125:5000:5000 plM) and 10 [ICi a- 3 2 P-UTP (40 mCi/mL,
6000Ci/mmol)
were
added, and the reactions were then incubated at 30 °C for an additional 30 min.
For transcription from a G-less cassette, RNase T1 was added after the reaction,
followed by incubation at 37 °C for an additional 5 min. RNA transcripts were
isolated and analyzed by urea-PAGE gel electrophoresis. The gel was vacuumdried and exposed to a phosphorimager screen.
273
Results and Discussion
Platination,Purification,and Characterizationof DNA Primer20GG.
The oligonucleotide
20GG (5'-CCACAACCGGTATCTTCACC-3')
was
synthesized, platinated, and purified. The corresponding cisplatin modified
oligonucleotide was named 20GG-Pt. The platination sites, underlined in the
sequence, were confirmed by a Maxam-Gilbert reaction (Figure 7.8). The ratio of
[Pt] / [DNA] is 1.00, confirming the presence of one Pt atom per DNA strand by
determining
DNA concentration
with
UV spectroscopy
and
platinum
concentration with atomic absorption spectroscopy (AAS). The molecular weight
determined by mass spectrometry also confirmed only one platinum atom on
each DNA strand.
Synthesis of DNA 296-Pt by the Enzymatic LigationMethod.
The DNA 296-Pt was synthesized and purified by a two-phase ligation
strategy. The products were separated by 5% denaturing polyacrylamide gel
electrophoresis (Fig. 7.9). The final DNA 296-Pt was annealed and ready for the
transcription assay. The concentration of 296-Pt was estimated to be 50 mg/L
and 5 mg/L for 296mer and 296-Pt, respectively, by using agarose gel
electrophoresis (Figure 7.10). The total yield was about 1-5 %.
Site-Directed-Mutagenesis.
In order to make the site-specifically platinated precursor longer, thereby
improving the efficiency of the primer extension reaction, site-directed-
274
mutagenesis was carried out to replace G with C in the DNA non-transcribed
strand. The success of the site-directed-mutagenesis reaction was confirmed by
the DNA sequencing analysis.
Synthesis of DNA 296-Pt by the PrimerExtensionMethod.
1. ssDNA Productionand Purifcation. A stock of helper phage VCS M13 was
thawed and plated with E. Coli XL1-Blue cell. Phage were propagated from one
well-isolated phage plaque and then harvested and titered. A titer of 5 x 1012
pfu/mL was prepared and used for subsequent ssDNA production.
Intensive investigations were carried out to optimize the conditions for
ssDNA production. A total of 56 different multiplicities of infection (MOI)
combined with different incubation times of ssDNA production were carried out
and the corresponding products were analyzed on a 0.7% native agarose gel.
Based on those 56 reactions (data not shown), the best MOI was around 100, and
the best incubation time is 10-12 h. In the optimized procedure for ssDNA
production, a portion of 5-250 mL sterile LB containing 100 g/mL ampicillin
and 10 pg/mL tetracycline was pre-warmed to 37 °C. A portion of 1/100 volume
of mid-log phase XL1-Bluecells was mixed with phage VCS M13 stock solution
(MOI=100), incubated for 5 min at room temperature, and added to the prewarmed LB. The infected culture was grown for 1 h at 37 °C with rotation at 250300 rpm. Kanamycin was then added to give a final concentration of 70 g/mL
and the culture was allowed to grow for another 10-12 h. The rest of procedure
275
followed the established method.(22) Further agarose gel purification was
performed followed by extraction of the desired band from the gel. The resulting
ssDNA was characterized by agarose/EtdBr gel electrophoresis (Fig. 7.11).
2. PrimerExtensionReaction.The 5' end of the platinated precursor 20GG-Pt
was phosphorylated with T4 PNK. The resulting phosphorylated DNA was
annealed with ssDNA.
Intensive investigations were carried out in order to improve the product
yield of the primer extension step. For the annealing efficiency, one of the most
important steps, several precursors with different lengths were designed, and the
corresponding site-directed mutagenesis of the plasmid was carried out. The
length of the precursor was increased from 12, 16, 18, to the final 20GG-Pt.
Several different polymerases including T4 and T7 DNA polymerase and
correspondingly different reaction buffers were used. In addition, different
incubation times and temperatures were tried. In the final optimized conditions,
circular ssDNA (35 pmol) was annealed to 5'- phosphorylated platinated primer
(190 pmol) in 100 gtL of 10 mM Tris HC1 (pH = 7.9), 10 mM MgC12 and 50 mM
NaCl. Following annealing, the solution was mixed with 30 units of T7 DNA
polymerase, 15 units of T4 DNA polymerase and 1200 units of T4 ligase in the
presence of 1.5 mM each dATP, dCTP, dTTP, dGTP, 1 mM ATP, 50 ,g/mL
BSA,
7.5 mM DTT, 40 mM Tris HCl (pH 7.5), 10 mM MgCl2, 12.5 mM NaCI (modified
from the condition used by Debyser). (20,22)The reaction was incubated at 37 °C
for 2 h, after which a second aliquot of 15 units of T4 DNA polymerase and 1200
276
units of T4 DNA ligase were added. The reaction was incubated at 37 C for
another 2 h. The resulting product was analyzed on a 0.7% native agarose gel
(Fig. 7.12). The primer extension product mixture was separated by CsC1/EtdBr
gradient centrifugation. The recovered platinated supercoiled plasmid DNA,
then digested with Sma I DNA was separated by agarose gel electrophoresis, and
the desired band corresponding to 296 bp was extracted.
In Vitro TranscriptionAssay.
The ds 296mer with a single site-specific1,2-d(GpG) intrastrand platinated
lesion was used in an in vitro transcription assay by the completely reconstituted
system containing RNA pol II, TF IIH purified from HeLa cells, and recombinant
TBP, TF IIB; TF IIE, TF IIF purified from E. coli. The G-less cassette template was
run as a control of transcription in addition to a ds 296mer containing no
platinum lesion.
Transcription from the unplatinated template produced an RNA product
corresponding to a full-length transcript (around 182 nt), whereas transcription
from the platinated template, produced a truncated product of approximately
121 nt (Figure 7.13).Such a difference in transcript length matches the location of
platinum modification site, indicating that the 1,2-d(GpG) platinum-DNA lesion
blocks RNA pol II transcription efficiently. A similar difference in transcript
pattern was also observed by comparing results from unplatinated 296mer (Fig.
7.14, lane2) and platinated 296mer (Fig. 7.14, lane 3) by incubating with HeLa
nuclear extracts. The RNA transcripts produced from an unplatinated template
277
were about 60 nt longer than the RNA transcripts from the platinated template.
However, the absolute lengths of the RNA transcripts are longer than expected.
It is possible that RNA Pol II binds and starts transcription from the blunt-end or
near the end of linear DNA instead of the normal starting site (promoterindependent or non-specific transcription), thereby producing a longer RNA
transcript. In fact, the RNA transcripts from circular DNA template were
abolished when the nuclear extract was pretreated with TFIIB antibody, while
RNA transcripts from linear DNA templates were only slight affected, indicating
that most of the transcription from linear DNA is TFIIB independent. (Figure 8.9,
Chapter 8)
The difference in RNA transcription indicates that the presence of 1,2d(GpG) intrastrand cross-link on the template strand abolishes the full-length
transcripts, producing only truncated transcripts. These results are consistent
with recent reports using T7 RNA polymerase and RNA pol II from rat liver.
(17,23)
A parallel in vitro transcription experiment was performed with a DNA
template containing a site-specifically platinated coding (non-transcribed) strand
(data not shown). In this case, the full-length transcripts are reduced, but not
totally abolished. Similar to our reports, Tornaletti et al. observed that 1,2-d(GpG)
cisplatin-modification in the transcribed DNA strand constitutes a strong
physical barrier to RNA pol II, whereas 1,2-d(GpG) cisplatin-modification in the
non-transcribed DNA strand does not.(23) We did not observe the almost 100%
278
RNA Pol II bypass of 1,2-d(GpG) adduct as did Culliane, however. This apparent
conflict could be due to differences in the preparation of the modified DNA
template, HeLa nuclear extracts or conditions of the in vitro transcription assay.
In addition, the degradation of the DNA template during incubation with
the transcription system is observed, presumably by exonuclease activity. Such a
degradation problem compelled us to develop the dumbbell DNA template
described in Chapter 8.
In summary, here we show that cisplatin-DNA adducts blocks RNA Pol II
transcription differentially based on strand location. A 1,2-d(GpG) cisplatinDNA cross-link on the transcribed strand blocks RNA pol II more efficiently
compared to the same adduct on the non-transcribed strand. Such RNA
polymerase blockage by platinum-DNA adducts plays an important role in the
cellular processing of cisplatin. Arrested RNA polymerase functions as a damage
recognition factor and triggers several signaling transduction pathways,
facilitating transcription-coupled repair or apoptosis. The detailed mechanisms
of such signaling transduction pathways, such as the nature of the signal during
RNA Pol II blockage at cisplatin-DNA adducts and the propagation of this signal
to downstream processes, deserve further investigation. Such information will
provide valuable clues about how to regulate such processes and rationally
design of more efficient platinum-based drugs and therapeutic strategies.
279
Acknowledgments
I am grateful to Dr. C. Kneip and Dr. D. Tantin for pUC118-296 and pG5MLPG380, Prof. R. D. Reinberg for the pure RNA Pol II and transcription factor
proterins. I also thank Dr. K. B. Lee, Dr. C. Kneip, Dr. Y. Mikata for technical help.
References
1.
Pil, P., and Lippard, S. J. (1997)Cisplatin and Related Drugs. Encyclopedia
of Cancer (Bertino, J. R., Ed.), Academic Press, San Diego
2.
Jamieson, E. R., and Lippard, S. J. (1999) Chem. Rev. 99, 2467-2498
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Eastman, A. (1986) Biochemistry 25, 3912-3915
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282
Fragment II
Fragment I
T1
TT3
TT2
HO
TT9
*
TT4
P
P
W
TT8
TT7
OH
T'6
TT5
I,
/,
T4 DNA ligase
*
HO
OH
296mer
Figure 7.6. Two-phase enzymatic ligation strategy for synthesizing a platinated
296mer.
283
produce ssDNA with
hlpe phage
p-
\
pUC118-296c
u] Mural
insted
plat
pi rimer
pUC118-296t
,a ^ .- .---restriction
enmymes
Jgase mnd nudleotides,
produce dsDNA,
CsCIEtd Br
Centrifugation
N%
Figure 7.7. Procedure for synthesizing a platinated DNA 296mer by the primer
extension method.
284
G
G G+A G+A
G
G
00
Pt:
+
.+
Figure 7.8. Maxam-Gilbert sequencing results for 20GG-Pt. Lane 1 (from left), G
reaction of 20GG-Pt; lane 2, G reaction of 20GG; lane 3, G+A reaction of 20 GG-Pt;
lane 4, G+A reaction of 20GG.
285
A
B
1
1
2
2
3
4
5
6 7
3
-
300
-
-
296mer
200
Figure 7.9. Denaturing PAGE of the two-phase ligation reaction product and
purified 296-Pt. A. Denaturing PAGE of the two-phase ligation reaction product.
Lane 1, 100-bp DNA markers; lane 2, the ligation reaction in which the top strand
is labeled; Lane 3, the ligation reaction with labeled bottom strand. The band
near 300bp is 296-Pt (arrow). B. Denaturing
PAGE of purified
296-Pt and
Fragments I and II. Lanes 1 and 2, the purified fragment II, labeled on the top
strand (TT3) and on the bottom strand (TT6), respectively; lanes 3 and 4, the
purified fragment I, with labels on TT1 and TT9, respectively; lanes 5 and 6, the
purified 296-Pt,labeled on the top strand or on the bottom strand; lane 7: phase 2
reaction product.
286
M 1
2 3 4
?gQ.
Figure 7.10. Concentration of 296mer is estimated by using the low mass DNA
ladder. Lane M, Hi-Lo DNA marker; lanes 1-3, low mass DNA ladder, 2 jtL, 4 [tL
and 8 tL, respectively; lane 4: 2 AtL296mer.
ssDNA
dsDNA
ssDNA
ds DNA
Hi-Lo Marker
3000 bp
i4-
2000 bp
1000 bp
Figure 7.11. Agarose gel electrophoresis analysis of purified single-stranded
DNA product. Lane 1 (from left), ssDNA; lane 2, dsDNA as control; lane 3, Hi-Lo
DNA marker.
287
Nicked dsDNA
Gaped dsDNA
Supercoiled dsDNA
---
1
2
3
ssDNA
4
Figure 7.12. Agarose gel electrophoresis analysis of DNA primer extension
products. Lane 1, ssDNA product; lane 2, dsDNA as control; lanes 3-4, T4, 7
primer extension. The supercoiled, nicked and gaped DNA products from
primer extension reaction are indicated (arrows).
288
M 1 2345
F
121-nt
1l2-nt
Acu
*
Full length transcript 182 nt
-------- Ace·--·
Stalled transcript 121 nt
Figure 7.13. In vitro transcription assay with a cisplatin-modified template.
Lane M, 100-bp ladder; lane 1, transcription products from 296-Pt; lanes 2 and 3,
transcription products from 296mer; lane 4, transcription products from G-less
cassette DNA.
289
1 23
'.?'
300
250
200
A-
_
· e--I
i~
t--<
4
_ ar-
Full length transcript
Stalled transcript
J
-
150
125
I~~~~~~~~~~~
_.
_
i;:
100
.
'~
w
.W
MR*
75
Figure 7.14. In vitro transcription assay with a cisplatin-modified template. Lane
1, 100-bp ladder; lanes 2, transcription from 296mer; lane 3, transcription from
296-Pt.
290
Appendix 7.1
Sequence of 296mer (5'- 3')
Coding Strand (Non-Transcribed Strand):
GGGCATCCAAGTACTAACCGAGCGGAGGACTGTCCTCCGAGCGGAGGAC
TGTCCTCCGGCGAGGGCGATTATGTGGCCTGGGCTATAAAAGGGGGTGG
GGGCGCGTTCGTCCTCACTCTCTTCCTGCTCTTACTTTCCAGGGATTTCTAA
GCCGATGACGTCATAACATCCCTGACCCTTTAAATACCTTAACTTTCATC
AAGCAAGAGCCTACGACCATACCATGGTGAAGATACCGGTTGTGGTCCG
ATCACCGAAGTCAAGCAGCATAGGGCTCGGTTAGTACTTGGATGCCC
Template Strand (Transcribed Strand):
GGGCATCCAAGTACTAACCGAGCCCTATGCTGCTTGACTTCGGTGATCGG
ACCACAACCGGTATCTTCACCATGGTATGGTCGTAGGCTCTTGCTTGATG
AAAGTTAAGGTATTTAAAGGGTCAGGGATGTTATGACGTCATCGGCTTAG
AAATCCCTGGAAAGTAAGAGCAGGAAGAGAGTGAGGACGAACGCGCCC
CCACCCCCTTTTATAGCCCAGGCCACATAATCGCCCTCGCCGGAGGACAG
TCCTCCGCTCGGAGGACAGTCCTCCGCTCGGTTAGTACTTGGATGCCC
DNA fragments to synthesize DNA 296-Pt/TS
Fragment TT#1 (67 mer)
GGGCATCCAAGTACTAACCGAGCGGAGGACTGTCCTCCGAGCGGAGGAC
TGTCCTCCGGCGAGGGCG
Fragment TT#2 (87 mer)
ATTATGTGGCCTGGGCTATAAAAGGGGGTGGGGGCGCGTTCGTCCTCACT
CTCTTCCTGCTCTTACTTTCCAGGGATTTCTAAGCCG
Fragment TT#3 (62 mer)
ATGACGTCATAACATCCCTGACCCTTTAAATACCTTAACTTTCATCAAGC
AAGAGCCTACGA
Fragment TT#4 (80 mer)
CCATACCATGGTGAAGATACCGGTTGTGGTCCGATCACCGAAGTCAAGC
AGCATAGGGCTCGGTTAGTACTTGGATGCCC
Fragment TT#5 (51 mer)
291
GGGCATCCAAGTACTAACCGAGCCCTATGCTGCTTGACTTCGGTGATCGG
A
Fragment TT#6 (20GG-Pt)
CC ACAACCGGTATCTTCACC
Fragment TT#7 (79 mer)
ATGGTATGGTCGTAGGCTCTTGCTTGATGAAAGTTAAGGTATTTAAAGGG
TCAGGGATGTTATGACGTCATCGGCTTAG
Fragment TT#8 (87 mer)
AAATCCCTGGAAAGTAAGAGCAGGAAGAGAGTGAGGACGAACGCGCCC
CCACCCCCTTTTATAGCCCAGGCCACATAATCGCCCTCG
Fragment TT#9 (59 mer)
CCGGAGGACAGTCCTCCGCTCGGAGGACAGTCCTCCGCTCGGTTAGTACT
TGGATGCCC
DNA fragments to synthesize DNA 296-Pt/CS
49 mer
CGATCACCGAAGTCAAGCAGCATAGGGCTCGGTTAGTACTTGGATGCCC
72 mer
GGTCGTAGGCTCTTGCTTGATGAAAGTTAAGGTATTTAAAGGGTCAGGGA
TGTTATGACGTCATCGGCTTAG
73 mer
ATGACGTCATAACATCCCTGACCCTTTAAATACCTTAACTTTCATCAAGC
AAGAGCCTACGACCATACCATGA
78 mer
GGGCATCCAAGTACTAACCGAGCCCTATGCTGCTTGACTTCGGTGATCGG
GTGAAGATACCGGTTGTGGTCATGGTAT
Note: The other 5 pieces are same as fragments 1, 2,6-Pt 8, 9 of DNA 296-Pt/TS.
292
Chapter 8 Synthesis and Characterization of Platinated Dumbbell
DNA for In Vitro Transcription and Repair Studies
293
Introduction
Cisplatin, a potent anticancer drug, has been used to treat several types of
cancers for over 25 years. Once inside cells, cisplatin forms covalent DNA adducts,
mostly 1,2-d(GpG), 1,2-d(ApG) and 1,3-d(GpTpG) intrastrand crosslinks. Several
cellular responses, including transcription inhibition, DNA repair, cell cycle arrest,
and apoptosis were induced by cisplatin-DNA adducts.(1-5)
Circular, dumbbell-shaped DNA consists of two hairpin loops at each end
and a central stem DNA duplex.(6,7) Dumbbell DNA has been proposed as an
intermediate for the in vivo formation of inverted dimers (giant palindromes)
during gene amplification in eukaryotic systems. It has been suggested to play
important roles in genetic recombination and regulation in vivo.(8,9)
The structure of dumbbell DNA brings unique advantages for several
applications. Compared to linear DNAs, dumbbell DNAs are highly resistant to
exonucleolytic degradation due to the absence of free termini.(10-12) In addition, the
dumbbell DNAs are more easily taken up by cells than linear DNA.(8) Therefore,
dumbbell DNAs have been used as minimal-sized gene-transfer vectors in gene
therapy or siRNA expression vectors.(13-17) In addition, dumbbell DNA can
sequester specific DNA-binding proteins, such as transcription factors.(7,15,18-22)
Site-specifically platinated double-stranded DNA probes have been prepared
as valuable templates for in vitro studies of cellular processing of cisplatin, such as
protein-DNA interaction, transcription, and repair.(23-25) However, the blunt-ends
294
of double-stranded probes promote non-specific transcription initiation, proteinbinding, and exonuclease degradation. Here we report the synthesis and
characterization of site-specifically platinated dumbbell DNA probes from a pair of
hairpins with 3' overhangs and three or four middle fragments using enzymatic
ligation. The formation of dumbbell DNA probes was confirmed by exonuclease and
restriction enzyme digestion assays. The dumbbell DNA probes were then used as
templates for in vitro nucleotide excision repair and transcription.
Experimental Section
Synthesis and Characterizationof the DumbbellDNA probe.
Synthesis of Site-SpecificallyCisplatin-ModifiedDumbbell DNA Probes. The site-
specifically cisplatin-modified and unmodified dumbbell DNA were synthesized by
ligating five or six fragments of oligonucleotides using T4 DNA ligase as described
elsewhere.(25) Schemes for the synthesis of the dumbbells are shown in Figure 8.1.
Oligonucleotide fragments for each DNA probe are listed in Appendix 8.1. All
fragments were synthesized on an Applied Biosystems DNA synthesizer (Model 392)
and purified by PAGE and IE-HPLC.The platinated probes were same as those used
in Chapter 2. The ligation products of site-specifically cisplatin-modified dumbbell
DNA probes were separated on an 8% denaturing PAGE gel and eluted with TE
buffer over night.
of DumbbellDNA Productby RestrictionEnzyme Digestion.In a typical
Characterization
reaction, the dumbbell probe was synthesized and purified as described above; one
295
fragment (20-mer) was 5'-32plabeled. The internal
3 2 P-labeleddumbbell
DNA probe
(1 fmol) was incubated with the restriction enzyme (EcoR I, BamH I, Hha I) at 37 °C
for 30 min. The reaction was quenched with SDS and proteinse K and then analyzed
on an 8% denaturing and a 5% non-denaturing PAGE gel.
T7 Exonuclease
Digestion.The internally
32 P-labeled dumbbell
DNA probes or
linear DNA probes (1 fmol) were incubated with 5 U of T7 exonuclease enzyme in
50 mM KOAc, 20 mM Tris HOAc (pH = 7.9), and 1 mM DTT at 25 °C for 30 min. The
reactions were then treated with 10% SDS and proteinase K and analyzed on an 8%
denaturing PAGE gel.
ExcisionAssay of DumbbellDNA.
The dumbbell d(GpG)-Pt and d(GpTpG)-Pt probes were used as substrates
for the nucleotide excision repair assay as described.(25) The AA8 CHO cell-free
extracts were added into the mixture and incubated at 30 C for 1.5 h. The reaction
products were then treated with proteinase K and SDS at 30 °C for 15 min, extracted
with phenol, and precipitated by ethanol. The repair products were analyzed on an
8% denaturing gel.
Reconstitution of Site-Specifically Cisplatin-Modified Nucleosome.
The internally
32 P-labeled
dumbbell DNA probes were assembled into
nucleosomes with a reconstituted histone octamer as described.(25) Briefly, DNA
substrate (1 pmol) was incubated for 15 min at 37 C with the core histones in a 1:1
296
molar ratio in a final volume of 10 CiLsolution containing 1 pg of BSA and 2 M NaC1.
The reaction mixture was serially diluted with portions of 50 mM HEPES (pH = 7.5),
1 mM EDTA, 5 mM dithiothreitol (DTT),and 0.5 mM phenylmethylsulfonyl fluoride
(PMSF)over a period of 2.5 h incubating at 30 °C. The resulting solution were mixed
with a 100 piL of 10 mM Tris HCl (pH = 7.5), 1 mM EDTA, 0.1% Nonidet P-40, 5 mM
DTT, 0.5 mM PMSF, 20% glycerol, and 100 pg/mL of BSA and then incubated for
15 min at 30 C.
In Vitro Transcription Assay with DNA Template Containing a G-less Cassette.
Transcription reactions (25 [tL) were performed essentially as described
using 10 gCi a- 32 P-CTP as the radiolabel, together with a C/G/UTP mix (25:200:200
[tM).(26)The DNA templates used was a BamH I linearized plasmid pG5MLP-G380
(containing a 380 nucleotide G-less cassette and a major late promoter) and a circular
(supercoiled) plasmid pHIV/TAR-G100 containing a 100 nucleotide G-less cassette
and a mutant HIV promoter. Antibodies to TFIIB or c-myc were added where
indicated. RNA transcripts were treated with RNase T1 and G-less transcripts
analyzed on a 6 % urea-polyacrylamide gel electrophoresis.
Assessment of Transcription from Dumbbell DNA using RNase Protection Assay.
Transcription reactions were performed as described above, except that
unlabelled C/G/UTP
(200 M each) was used in the reactions. RNA transcripts
were isolated essentially as described,(27) and analyzed using the RNase protection
297
assay (RPA) kit from Ambion. The radiolabelled probe for the RPA assay was
generated by the polymerase chain reaction (PCR) using the dumb-bell DNA as a
template, 10 gCi a-32P-dATP as the radiolabel. As a negative control, yeast RNA was
included in an RPA reaction in the absence of a transcript.
Results and Discussion
Synthesis and Characterizationof DumbbellDNA Probes
All oligonucleotide fragments were purified by PAGE gels. The purification
and characterization of platinated oligonucleotides DNA 20GG-Pt and 20GTG-Pt
were performed as reported.(25)
To test the ligation efficiency of each fragment of dumbbell DNA, a 5'-32plabeled 65-mer was mixed with either the 89-mer (Figure 8.2, lane 2); the 89-mer and
20-mer (Figure 8.2, lane 3); the 89-mer, 20-mer, and 88-mer (Figure 8.2, lane 4); or the
89-mer, 20-mer, 90-mer and 88-mer (Figure 8.2, lane 5) in the ligation buffer. As
indicated in Figure 8.2, the bands corresponding to the predicated ligation products
were observed in the each lane. Lane 1 shows the presence of the starting 65-mer
and the ligation product 154-mer (65+89).Bands corresponding to starting 65-mer,
the ligation products 85-mer (65+20), 154-mer and 174-mer (65+89+20) were
observed. Lane 3 shows the presence of the starting 65-mer, as well as the ligation
products 85-mer, 154-mer, 174-mer, and 262-mer (65+89+20+88).In lane 4, the band
298
corresponding to full-length ligation product was observed. The structure of
dumbbell DNA allows it to migrate more rapidly than linear DNA of similar length.
Due to the low yield of the 90-mer, a six-fragment strategy was then applied
to synthesize greater amounts of dumbbell DNA (Figure 8.1). The 90-mer was
replaced by a 68-mer and a 22-mer and six parallel ligation experiments were
performed to identify the band corresponding to the dumbbell product. In each
ligation experiment, one of six fragments was labeled by y-32 P-ATP.The six parallel
reactions were analyzed on a denaturing PAGE gel (Figure 8.3). The band
containing dumbbell, if any, should be observed in all six lanes. The top band
observed in each lane was cut out and eluted for further characterization.
A restriction enzyme assay was used to identify further the dumbbell product.
Restriction sites of BamH I, Hha I, and EcoRI are located within dumbbell as shown
in Figure 8.4. A missing fragment would cause the product to lose restriction
digestion site of at least one enzyme. Figure 8.5 show that the ligation product was
digested by all three enzymes. This result indicates that the ligation product contains
all six fragments. Addition of T7 exonuclease did not degrade the ligation product
after 30 min, whereas linear DNA was digested (Figure 8.6). The rapid mobility
band in lane 2 is the partial digestion product in which T7 exonuclease is blocked by
the cisplatin lesion.
Taken together, these results indicate successful synthesis of the dumbbell
DNA probe. It could be used as a probe to study transcription-coupled repair and
global-genome nucleotide excision repair as well utilized in other applications.
299
Nucleotide Excision Repair from Dumbbell Probes. The internally
3 2 P-labeled
dumbbell GG-Pt and GTG-Pt (352 nt) were compared with the linear 146GG-Pt and
146GTG-Pt for efficiency of NER. As shown in Figure 8.7, the dumbbell DNA probes
exhibit a higher repair signals (about 2 fold) than the linear DNA probes. In addition,
the similar stimulation results were observed when comparing the excision of
dumbbell probes and their linear counter parts generated by double restriction
enzyme digestion (data not shown).
The structural or topological change of DNA induced by the formation of
dumbbell ends might account for such stimulated of excision. However, it may not
be the main factor. Although studies of small dumbbell DNA (<10 nt) report that
the two hairpin loops and formation of dumbbell changes the DNA structure,(28,29)
very little change was observed in longer dumbbell DNA due to loop ends on the
DNA (>16 bp)(30) indicating the effect of hairpin loops did not propagate over a
long range.
Alternatively, some protein factors, such as end processing proteins, interact
differentially with dumbbell DNA and linear DNA, thus modulating excision repair
differently. For example, the Ku70/80 protein complex can bind to the ends of DNA
and inhibit the binding of nucleotide excision repair proteins such as XPA and p62TFIIH on linear damaged DNA in vitro.(31-34)Studies of the mode of Ku interaction
with DNA revealed that the addition of two hairpin loops to the same 25-bp linear
substrates change the Kd values from 3.8 nM (linear) to 11.4 nM (dumbbell).(35) The
300
contribution of Ku70/80 on such a stimulation in repair from dumbbell deserves
further investigation.
Nucleosome Assembly of Dumbbell DNA. To test whether the formation of
dumbbell DNA affects nucleosome reconstitution, the dumbbell DNA was
assembled with histone octamer to form a nucleosome. As shown in Figure 8.8, a
significant amount of DNA was incorporated into nucleosomes. The presence of
dumbbell ends does not inhibit the formation of a nucleosome. This is the first
synthesis of a dumbbell nucleosome. This dumbbell nucleosome could be used for in
vitro transcription template or repair template to probe cisplatin processing at
nucleosomal level.
Cisplatin Blocks the Transcription from Dumbbell Probes. The cisplatin-modified
and unmodified dumbbell DNAs were synthesized to investigate the effect of
cisplatin on transcription
elongation. As shown in Figure 8.9, the band
corresponding to the full length RNA transcript (Figure 8.9a, lane 4, 8) had
disappeared in cisplatin-modified DNA (Figure 8.9a, lane 11), indicating that RNA
transcript is blocked by cisplatin-DNA adducts.
TFIIB Dependent Transcriptionfrom Dumbbell Probes. One problem in using
linear probes as transcription templates is that a significant amount of RNA
transcripts form in TFIIB-independent manner. RNA polymerase can bind to the
blunt ends of DNA and start transcription without the help of TFIIB. As shown in
Figure 8.9 b, when HeLa nuclear extracts containing circular or linear DNA were
pretreated with TFIIB antibody, the RNA transcripts were abolished or slightly
301
affected, respectively. When the cell extracts were pretreated with anti-TFIIB, the
RNA transcripts were significantly reduced. This result indicates that the most
transcription from the dumbbell is TFIIB dependent.
We have synthesized and characterized the site-specifically cisplatinmodified dumbbell DNA probes. The unique dumbbell structure makes it a
promising candidate or substrate for a variety studies in vitro or in vivo. For
example, the site-specifically platinated dumbbell is a good template
for
transcription and repair. In addition, a photo-crosslinking study using dumbbell
DNA as probe has identified novel proteins that bind to platinum-DNA adducts.(36)
Furthermore, platinated dumbbell DNA could be transfected into cells to regulate
cellular processes by reducing the availability of proteins, such as HMG family
proteins or TBP in cells.
Acknowledgment
I thank Dr. J. T. Reardon and Dr. A. Sancar for providing AA8 cell extracts
and A. Danford for assistance in purifying some of the oligonucleotide fragments
and some characterization experiments of dumbbell. I thank Dr. K.B. Lee for the
transcription work. I also acknowledge members of our laboratories for helpful
discussion and comments on the manuscript.
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32.
Frit, P., Calsou, P., Chen, D. J., and Salles, B. (1998) J. Mol. Biol. 284, 963-973
33.
Frit, P., Li, R. Y., Arzel, D., Salles, B., and Calsou, P. (2000) J. Biol. Chem. 275,
35684-35691
34.
Calsou, P., Muller, C., Frit, P., and Salles, B. (1996) C. R. Acad. Sci. III 319, 179182
35.
Arosio, D., Cui, S., Ortega, C., Chovanec, M., Di Marco, S., Baldini, G.,
Falaschi, A., and Vindigni, A. (2002) J. Biol. Chem. 277, 9741-9748
36.
Zhang, C. X., Wang, D., Danford, A., and Lippard, S. J. (2004) Unpublished
result
305
Five-Fragment Strategy
I-
53
Y
88 mer
%
5
3
20 irenr
65 mer
Six-Fragment Strategy
68 mer
22 mer
88 mer
89 mer
20 m'3Ier
65 mer
Figure 8.1. Strategies for the synthesis of site-specifically platinated dumbbell DNA
probes. Five (top) or six (bottom) synthetic oligonucleotides were annealed and
ligated as shown. Sequences of the individual strands are listed in Appendix 8.1.
306
12345
4
-
65+89+20+88+90mer
4
65+89+20+88mer
4
65+89+20mer
4-
65+89mer
4-
65+20mer
4-
65mer
Mm
Figure 8.2. Denaturing PAGE gel analysis of dumbbell ligation products. Lane 1, 100
bp DNA ladder; lane 2, 5'-32Plabeled 65mer ligated with the 89mer; lane 3, 5'32p
labeled 65mer ligated with the 89mer and 20mer; lane 4, 5'-32plabeled 65mer ligated
with the 89mer, 20mer, and 90mer; lane 5, 5'-32plabeled 65mer ligated with the
89mer, 20mer, 90mer, and 88mer.
307
1 2
3 4
_.M ..
.
&*
5
, .
00
_
6
_e
7 8
9
'
,I,
=S^
Si-- ^40"W
--
.-L
I
_-'.w
_MIM
6
1--
Avow
Omok,'Wj..
..
11
-*09,
4MMIFW-
.:
- -
111
up
Figure 8.3. Denaturing PAGE gel analysis of dumbbell ligation products (sixfragment strategy). Lane 1, 10 bp DNA ladder; lane 2, 100 bp DNA ladder; lane 3, 5'32 p labeled
89mer ligated with the 88mer, 68mer, 65mer, 22mer, and 20mer; lane 4, 5'-
32p labeled
88mer ligated with the 89mer, 68mer, 65mer, 22mer, and 20mer; lane 5, 5'-
32p
labeled 68mer ligated with the 89mer, 88mer, 65mer, 22mer, and 20mer; lane 6, 5'-
32p
labeled 65mer ligated with the 89mer, 88mer, 68mer, 22mer, and 20mer; lane 7,
5'-32p labeled 22mer ligated with the 89mer, 88mer, 68mer, 65mer, and 20mer; lane 8,
5'-32P labeled 20mer ligated with the 89mer, 88mer, 68mer, 65mer, and 22mer; lane 9,
purified dumbbell DNA ligation product (arrow).
308
BamH
68 mer
I
Hha
EcoR
1
4F 22 mer
I
4
S9 mer
,"
20) rli
BamH I
Hha [
r
65 mer [
EcoR
I
Figure 8.4. Digestion sites of restriction enzymes on the dumbbell DNA. The
restriction locations are indicated.
I
309
1
2 3
..
.. . .
4
5
6
7
8
9
.a
i_ .
a
Figure 8.5. Non-denaturing gel of restriction enzyme digestion of dumbbell DNA.
Lane 1, 100-bp ladder; lane 2, untreated dumbbell DNA GTG; lane 3, EcoR I
digestion of dumbbell DNA GTG; lane 4, BamH I digestion of dumbbell DNA GTG;
lane 5, Hha I digestion of dumbbell DNA GTG; lane 6, untreated cisplatin-modified
dumbbell DNA GTG-Pt; lane 7, EcoR I digestion of cisplatin-modified dumbbell
DNA GTG-Pt; lane 8, BamH I digestion of cisplatin-modified dumbbell DNA GTGPt; lane 9, Hha I digestion of cisplatin-modified dumbbell DNA GTG-Pt.
310
12
#3~
Figure 8.6. Denaturing
3
4
.
5 6
Il
gel of T7 exonuclease digestion of dumbbell DNA. The
internally labeled linear (195GG-Pt) and dumbbell DNA probes (db-GTG and dbGTG-Pt) were incubated with 5 U of T7 exonuclease enzyme (lanes 2, 4, and 6,
respectively), at 25 C for 30 min. Lanes 1, 3, and 5 are untreated 195GG-Pt, db-GTG,
and db-GTG-Pt, respectively.
311
Dumbbell 146mer
1
2
3
4
4
*'ffi0pi
Figure 8.7. Denaturing PAGE gel of repair assays for dumbbell DNA. Lane 1, NER
assay of dumbbell GG-Pt; lane 2, NER assay of dumbbell GTG-Pt; lane 3, NER assay
of 146 GTG-Pt; lane 4, NER assay of 146 GG-Pt.
312
1
2
3
Figure 8.8. Reconstitution of dumbbell into nucleosome. Lane 1, Nucleosome
199GTG; lane 2, product from dumbbell nucleosome assembly reaction; lane 3, free
dumbbell DNA.
313
+ RNase AIT1
f
+ RNase A/T1
(b)
(a)
0M
.0
:=I
1.4
ta
6.
cP
-0
AL4
cn
-
A
ii
+
Z!~~~~4
.f
a
$14
+
+
+
g
4+
-6
100 mer
(RNA)
118
72b
123
4
5
6 7 8 9 10 11
Figure 8.9. Effect of anti-TFIIB on transcription from dumbbell DNA.
123
314
Appendix 8.1
Dumbbell GG and GTG sequences: (5' -, 3')
Fragment 89mer
GCGCGTTCGTCCTCACTCTCTTCCTACGTGCAACTCTCGTGACTCCGATITGGGTGAAGATACC
GGTTGTGGCGGTTAGTCCGAATAGT-
Fragment 65mer
GAGTCGTATTACCGAATTCCCCTCATGGGGAATTCGGTAATACGACTCACTATTCGGACTAA
CCG
Fragment 88mer
CAAATCGGAGTCACGAGAGTTGCACGTAGGAAGAGAGTGAGGACGAACGCGCCCCCACCCC
CTTTTATAGCCCCCCTTCAGGATCCGT
Fragment 68mer
ATTCTATAGTGTCACCTAAATCGCCATCAGGCGATITAGGTGACACTATAGAATACGGATCCT
GAAGG
Fragment 22mer
5'-GGGGCTATAAAAGGGGGTGGGG-3'
Fragment 20mer GG
5'-a atcct cctGG ttttt ccac-3'
Fragment 20mer GTG
5'-a atcct ccGTG ttttt ccac-3'
Fragment 89mer GG
GCGCGTTCGTCCTCACTCTCTTCCTACGTGCAACTCTCGTGACTCCGATTTGGTGGGTGGAAA
AACCAGGAGGATTCGTTAGTCCGAATAGT
Fragment 89mer GTG
GCGCGTTCGTCCTCACTCTCTTCCTACGTGCAACTCTCGTGACTCCGATITGGTGGGTGGAAA
AACACGGAGGATTCGTTAGTCCGAATAGT
315
Appendix Al The Interaction of Human SWI/SNF with
Platinated Nucleosomes and Its Effect on NER
316
Introduction
Cisplatin has been widely used as a potent anticancer drug for many years
to treat several kinds of cancer, including testicular, ovarian, head and neck, and
non-small cell lung cancers. Cisplatin forms covalent DNA adducts, primarily
1,2- or 1,3- intrastrand cross-links and a smaller number of interstrand cross-links
as well as protein-DNA crosslinks.(1,2) These cisplatin-DNA adducts are
responsible for triggering several cellular responses, including transcription
inhibition, DNA repair, cell cycle arrest, and
apoptosis.(1,3-6) Nucleotide
excision repair is a major cellular defense mechanism against the toxic effects of
cisplatin. The NER machinery recognizes the cisplatin lesion, unwinds the
nearby DNA duplex, removes 24 to 32 nucleotides containing the cisplatin-DNA
adducts, and promotes the synthesis and ligation of new DNA to fill the
resulting gap.(7, 8)
In eukaryotic cells, DNA is packaged into chromatin. Nucleosomes, the
fundamental unit of chromatin structure, are composed of 146 bp of DNA
wrapped around a histone octamer in a shallow, left-handed helix. The histone
octamer is a tripartite arrangement assembled from one (H3/H4) 2 tetramer and
two H2A/H2B dimers.(9) Chromatin adds another level of complexity to the
regulation of the DNA related processes such as gene expression, DNA
317
recombination, and repair. DNA that is contained within highly compacted
chromatin is inaccessible to the transcription and repair machinery. Previous
work from our laboratory revealed that the nucleosome significantly inhibits
nucleotide excision repair in comparison with the extent of repair by mammalian
cell extracts of free DNA containing the same site-specific platinum-DNA adduct
in vitro. (10)
Two major classes of factors have been identified to alter DNA
accessibility in chromatin. The first class regulates chromatin structure by
reversible formation of covalent histone modifications.(11,12)Histone acetylation,
phosphorylation, methylation and ubiquitination all have a role in the regulation
of gene expression from chromatin.(13,14) In addition, the post-translational
modification of histones can also modulate nucleotide excision repair from
damaged chromatin in vitro.(10) By changing the chemical characteristics of the
histone tails, histone-DNA interactions are reduced, or alternatively, other
factors are recruited to the nucleosome, thereby altering the accessibility of the
nucleosomes to the repair or transcription machinery.
The second class of factors remodel chromatin in an ATP-dependent
fashion, utilizing ATP hydrolysis to change histone-DNA contacts within the
nucleosome.(15, 16) These complexes can be divided into three groups on the
basis of the similarities of their ATPase subunits, namely, Swi2/Snf2, Iswl, and
Mi-2. All three classes play very important roles in nuclear events such as
transcription, replication, recombination, and DNA repair.(16-18)
318
SWI/SNF,
one of best characterized ATP-dependent
remodeling
complexes, was first identified in the yeast Saccharomycescerevisiae.This 2 MDa
multi-subunit complex is highly conserved in eukaryotes.(19) The mechanism of
nucleosome remodeling by SWI/SNF remains to be fully elucidated, although
several potential mechanisms, such as twist defect diffusion and bulge diffusion,
have been proposed.(20)
Recently, a growing body of evidence has linked the activity of the
SWI/SNF complex to DNA repair and recombination within nucleosomes.
SWI/SNF enhances cleavage of the V(D)J recombination signal sequence in the
mononucleosome by the RAG1/RAG2 recombinase.(21) In addition, association
of BRCA1 with SWI/SNF implies that the SWI/SNF chromatin-remodeling
complex might have a role in DNA repair.(22,23) D irect evidence that yeast
SWI/SNF complexes enhance the excision of UV and AAF lesions from
nucleosomes is available from in vitro studies.(24-26) The stimulatory effect of
SWI/SNF on NER depends upon the nature of DNA lesion. SWI/SNF stimulates
the excision of AAF-adducts and the (6-4) photoproducts from the nucleosome,
whereas it has a negligible effect on the repair of TT dimer.(25) These differential
effects of SWI/SNF on repair of UV photoproducts and the AAF adduct
stimulated our interest to investigate the effect of SWI/SNF complexes on the
repair of cisplatin damage within nucleosome. In this chapter, we address the
effect of SWI/SNF on the repair of cisplatin-DNA adducts in synthetic
mononucleosomes. A moderate stimulation by SWI/SNF on repair is reported.
319
Materials and Methods
Materials. Cisplatin was obtained as a gift from Engelhard. T4
polynucleotide kinase and T4 DNA ligase were purchased from New England
Biolabs. Phosphoramidites and chemicals for DNA synthesis were obtained from
Glen Research. y-3 2P-ATP was purchased from Perkin-Elmer Inc.
Synthesis and Purificationof Site-SpecificallyPlatinatedDNA Probes.The sitespecifically platinated DNA duplexes were prepared by enzymatic ligation of
complementary oligonucleotides as previously described.(7) Briefly, four DNA
fragments and one site-specifically cisplatin-modified oligonucleotide were
phosphorylated using T4 polynucleotide kinase and ATP. All the fragments were
then annealed and ligated together using T4 ligase. The full length ligation
products were then separated from partial ligation products by denaturing
PAGE, and subsequently isolated by non-denaturing PAGE.
ProteinsPreparation.Cell-free extracts were prepared from CHO AA8 cells
by the method as described previously.(27) hSWI/SNF complex was prepared
by Dr. H. Y. Fan as described.(28,29) The four histone proteins were expressed in
E. coli BL21(DE3)cells and purified following the published procedures.(30, 31)
The histone octamer was refolded, dialyzed, and then separated on a 16/60
Superdex 200 gel filtration column (Pharmacia).
320
Nucleosome Assembly of Site-Specifically Cisplatin-Modified Nucleosomes. The
internal
32 P-labeled DNA
probes (146GG-Ptor 146GTG-Pt)were assembled into
nucleosomes with reconstituted histone octamers as described.(10) Briefly, 1
pmol of DNA substrate was incubated with the core histones in a 1:1 molar ratio
with 1 pg of BSA and 2 M NaCl in a final volume of 10 pL for 15 min at 37 °C.
The reaction mixture was serially diluted with portions of 50 mM HEPES (pH
7.5), 1 mM EDTA, 5 mM dithiothreitol
(DTT), and 0.5 mM phenylmethylsulfonyl
fluoride (PMSF)over a period of 2.5 h, incubating at 30 °C. The resulting solution
was brought to 0.1 M in NaCl by adding 100 pILof 10 mM Tris -HC1(pH 7.5),
1 mM EDTA, 0.1% Nonidet P-40, 5 mM DTT, 0.5 mM PMSF, 20% glycerol, and
100 gg/mL of BSA, and incubated for 15 min at 30 °C.
GlycerolGradientPurfication. The nucleosome products were purified from
free DNA by glycerol gradient centrifugation in a similar manner as
described.(28) Briefly, nucleosomes were layered onto a 10-30%glycerol gradient
buffer (50 mM Tris HC1 (pH 7.5), 1 mM EDTA, 100 mM KCl, 10 or 30% glycerol)
and spun at 35,000rpm in a SW-41 rotor (Beckman Instruments) for 16 h. The
fractions were collected and characterized by 5% non-denaturing gel at 4 °C. The
purified nucleosomes were used for the NER assay.
DNase I FootprintingAssay. About 10 fmol of 5'-32p labeled 146GTG or
146GTG-Pt mononucleosomes
were treated with 0.2 U DNase I in 20 AxLTE
buffer (10 mM Tris HCl (pH 8.0), 1 mM EDTA) at 25 C for 1 min or 5 min. The
reactions were quenched by 2 pL of 15.3 mg/mL proteinase K/ 10% (w/v) SDS
321
at 30 °C for 15 min, followed by phenol extraction and ethanol precipitation. The
reaction products were analyzed on a 6% denaturing PAGE gel.
ExcisionAssay for NucleosomesPreincubatedwith SWI/SNF. A portion of 0.1
nM internally labeled mononucleosome substrates was pre-incubated with 320
ng of SWI/SNF in 1.6 mM Tris HCl (pH = 7.9), 33.6 mM Hepes (pH = 7.9), 72
mM KCI, 5.44 mM MgC12, 2.4 mM ATP, 200 ng/ iL BSA, 0.08% NP40, 4 ng/ AL
pUC18, 7.3% glycerol, 0.48 mM DTT, 0.56 mM EDTA, 1.6 mM P-mercaptoethanol
and 0.032 mM PMSF at 30 C for 60 min. The AA8 CHO cell-free extracts were
added into the mixture and incubated at 30 C for another 3 h. The reaction
products were then treated with proteinase K (15.3mg/mL) /SDS (10% w/v)
buffer at 30
C for 15 min, followed by phenol extraction and ethanol
precipitation. The repair products were analyzed on an 8% denaturing PAGE gel.
Restriction Enzyme Characterization.To test the remodeling activity of
SWI/SNF on mononucleosomes, 0.1 nM internal
3 2 P-labeled mononucleosome
substrates were incubated with restriction enzyme (5 U Nla III) and, when
needed, 80 ng SWI/SNF in excision buffer (see above) at 30 C for various time.
The reaction products were then treated with proteinase K/SDS buffer at 30 °C
for 15 min, followed by phenol extraction and ethanol precipitation. The reaction
products were analyzed on an 8% denaturing PAGE gel.
322
Results and Discussion
Characterizationof Nucleosomes by Footprinting. The nucleosomes were
prepared by the salt dilution method. The purified nucleosome was analyzed on
a 5% non-denaturing gel (Figure A1.1). The cisplatin modification site is located
at 70th nucleotide from 5' terminus of the DNA duplex. Analysis by DNase I
footprinting revealed a strong 10-bp periodicity (Figure 4.5). The cleavage rate of
nucleosomal DNA by DNase I is much slower than that for free DNA, indicating
that the core histones protect DNA from DNase I cleavage (data not shown).(32)
Comparison
of the cleavage patterns from platinated and unmodified
nucleosomes reveals a difference over the region from the 30th to the 120th
nucleotide from 5' terminus of the nucleosomal DNA (Figure 4.5).This result
indicates, that although the incorporation of cisplatin-DNA adduct does not
affect the overall structure of nucleosome, there is a local distortion. A more
quantitative measure of this structural change, revealed by hydroxyl radical
footprinting is present in Chapter 4. (33)
Restriction Enzyme Characterization.To test the effect of SWI/SNF on the
nucleosome substrate, Nla III digestion was used to monitor the remodeling
activity. As shown in Figure A1.2, addition of SWI/SNF stimulates the digestion
efficiency of nucleosome substrates from 20.3% to 23.4% (4 min digestion) or
from 33.8% to 39.4% (20 min digestion), whereas the addition of SWI/SNF did
not change the digestion efficiency of DNA substrates (93.6%versus 94.3% for 4
min digestion and 95.6% versus 96.4% for 20 min digestion). The measured
323
hSWI/SNF stimulation is not as dramatic as previously reported for yeast
SWI/SNF.(26) There are two possible reasons for this difference. First, the
excision reaction buffer is different for the two studies, and the activity of
hSWI/SNF might not be optimized under these circumstances. Second, different
restriction enzymes, Nla III and EcoR I, were used in the two studies. Nla III
might not act as efficiently as EcoRI to digest the remodeling DNA on the
nucleosome.
The Stimulatory Effect of hSWI/SNF on NER. The nucleosome substrate
146GTG-Pt was pre-incubated with SWI/SNF for 1 h at 30 °C and then incubated
with cell-free extracts for another 3 h. The repair results reveal that preincubation with human SWI/SNF stimulates excision from the nucleosome
substrate by 2.8 fold (from 0.6% to 1.7%), whereas, addition of SWI/SNF
stimulates excision from free DNA by 1.7 fold (from 2.6% to 4.3%, Figure A1.3).
The better stimulatory effect on DNA may due the slight differences in protein
concentration
between
the
SWI/SNF-containing
and
control
reactions.
Nevertheless, the relative stimulation (from nucleosomes vs DNAs) is 1.7 fold.
The SWI/SNF complex disrupts histone-DNA contacts and thereby increases the
accessibility of the platinated duplex. Previous findings revealed that SWI/SNF
differentially stimulates repair of UV-damaged DNA in nucleosomes. SWI/SNF
stimulates the excision of the AAF-G adduct and the (6-4)photoproduct by about
1.5 -2.0, whereas there was no effect on excision of a TT dimer.(25,26) Therefore,
324
the effect of SWI/SNF on repair of 1,3-d(GpTpG)-Pt is more similar to the results
for the AAF-G adduct and (6-4)photoproduct.
Previous
biochemical analysis has demonstrated several different
outcomes of SWI/SNF remodeling activity in vitro, including nucleosome sliding,
the transferring of a histone octamer to another DNA molecule, and the altering
of nucleosome structure.(19) It is not clear whether these events occur by a single
or by multiple mechanisms. Several possible different mechanical forces,
including octamer sliding, DNA twisting, creating a bulge of exposed DNA, and
translocating
DNA,
may
be
involved
in
SWI/SNF
nucleosome
remodeling.(19,20,34,35) Nevertheless, in each case, histone-DNA contacts are
weakened, thereby allowing the cisplatin-DNA lesion to be more accessible to
the repair machinery. Alternatively, local distortions of cisplatin-DNA adducts
are altered by SWI/SNF, thereby making such lesions much easier to be
recognized for repair.
Although the composition and subunit structure of human SWI/SNF and
yeast SWI/SNF are generally well-conserved, the two complexes have certain
differences. The yeast SWI/SNF complex has only one ATPase and one ARIDcontaining protein, whereas human SWI/SNF has two alternative ATPase
subunits and ARID-containing subunits.(36) The DNA binding affinity of ARID
subunits in human SWI/SNF is much higher than its counterparts in yeast
complexes.(37) Moreover, human complexes also have an additional DNAbinding component, BAF57,for which yeast complexes have no counterpart. This
325
HMG-domain containing subunit can bind selectively to four-way junction
DNA.(38) Because of the HMG domain, which binds 1,2-d(GpG) cisplatin-DNA
adducts, the interaction of BAF57 and its role in remodeling cisplatin-modified
nucleosomes deserve further investigation.
SWI/SNF and Histone Modifications.SWI/SNF complexes cooperate with
histone-modifying enzymes such as histone acetyltransferases (HAT) Gcn5 or
CBP/p300 for chromatin remodeling, leading to transcriptional activation of a
large number of genes.(39,40) Acetylation of histone H4 at K8 mediates
recruitment of the SWI/SNF complexes.(41) The bromodomain in the ATPase
subunit Swi2 of SWI/SNF, which interacts specifically with acetylated histone
tails, is important for such regulation in vivo.(42) It is tempting to speculate that
SWI/SNF and HAT may also act synergistically in some cases to modulate NER
of platinated nucleosomes in vivo.
The Role of SWI/SNF in Cancer.The SWI/SNF complex is implicated in
cancer since several subunits either have intrinsic tumor-suppressor activity or
are required for the activity of other tumor-suppressor genes.(43,44)Moreover,
specific mutation and/or loss of BRG1, an ATPase catalytic subunit of the
SWI/SNF complex, has been identified in pancreatic, breast, prostate, and nonsmall-cell lung cancer cell lines.(45-48) The loss or mutation of subunits of
SWI/SNF in tumor cell lines will alter the remodeling activity of SWI/SNF
complex, the cellular repair activity of cisplatin-DNA adducts will therefore be
326
also affected. This differential activity of SWI/SNF in cancer vs normal cells may
partially contribute to the anti-cancer activity of cisplatin.
Acknowledgment
I thank Dr. J. T. Reardon and Dr. A. Sancar for providing AA8 cell-free
extracts, Dr. H.Y. Fan and Dr. R. Kingston for providing hSWI/SNF, A. Danford
for assistance in purifying and preparing some of the oligonucleotide fragments
and DNA probes I also acknowledge members of our laboratories for helpful
discussion and comments on the manuscript.
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330
Nudeosome
DNA
Figure A1.1. Analysis of site-specifically cisplatin-modified nucleosome on a 5%
non-denaturing PAGE gel electrophoresis.
331
t= 20 min
A
-
hSWI/SNF
4 min
4+
+
w
Nucleosome
C
Cut _ _
Cut%:
+
33.8% 39.4%
B
t= 20 min
hSWI/SNF
-
Free DNA
Cut
-
Cut%:
4
. . .
Nla III
60
203% 23.4%
4 min
-
+
Tp
70-72
d(GpTpG)-Pt
_it"6
WM~r
96.5% 96.4%
93.6% 943%
Figure A1.2. The effect of hSWI/SNF on nucleosome structure characterized by
Nla III digestion. Mononucleosome substrates (A) or free DNA (B) were
incubated with 5 U of Nla III and, when needed, 80 ng SWI/SNF in excision
buffer at 30 C for 4 min or 20 min. The reaction products were analyzed on an
8% denaturing PAGE gel. The digestion efficiencies are indicated. The digestion
site of Nla III and platination site are shown above.(C)
332
-'I
Nuc DNA
SWI/SNF
..
-
rI.
1 r
.
Figure A1.3. The stimulatory effect of hSWI/SNF on nucleotide excision repair.
Lane 1, nucleosomal 146GTG-Pt DNA; lane 2, nucleosomal 146GTG-Pt DNA in
the presence of hSWI/SNF; lane 3, free 146GTG-Pt DNA; lane 4, free 146GTG-Pt
DNA in the presence of hSWI/SNF.
333
Biographical Note
The author was born on June 12, 1975 in Beijing, P. R. China to Hua and
Bao-Guo Wang. He was raised along with one young sister, Wei (Wendy) in
Beijing, P. R. China, and attended the Senior High School Attached to Peking
University, where his interest in chemistry and biology was inspired. After
graduating in 1993, he attended college at Peking University, P. R. China, where
his majored in chemistry and did undergraduate research in the purification,
characterization and crystallization of a novel insect neurotoxin from the venom
of the scorpion Buthus martensii Karsch.under the guidance of Prof. Genpei Li
and Prof. Dacheng Wang. He earned his Bachelor of Science degree in Chemistry
with the highest honor in 1998, and began his graduate work at MIT in 1999. He
received Anna Fuller Fund graduate fellowship in 2002. Following graduation,
he will continue his postdoctoral training in Prof. Roger Kornberg's lab at
Stanford Unviersity.
334
Dong Wang
Education
* Massachussetts Institute of Technology, Cambridge, MA.
Ph.D. in Biological Chemistry, 2004.
Thesis advisor: Prof. Stephen J. Lippard
*
Peking University, Beijing,P.R. China.
B.S.with honors, Chemistry, 1998.
Thesis advisor: Prof. Genpei Li and Dacheng Wang
Publications
Wang, D., Lippard, S.J. Cellular Processing of Platinum Anti-Cancer Drugs Identifying Pathways for Chemogenotherapeutic Drug Design. Nature Rev. Drug
Discov. Submitted for publication.
Wang, D., Lippard, S.J. Histone modification following treatment of cells with
cisplatin: cause and effects. J. Biol. Chem., 279(20), 20622-5 (2004).
Wang, D., Hara R., Singh G., Sancar, A., Lippard, S.J. Nucleotide excision repair
from site-specifically platinum-modified nucleosomes. Biochemistry, 42 (22), 67476753 (2003).
Lee, K.B.,Wang, D., Lippard S.J.,Sharp, P.A. Transcription-coupled and DNA
damage dependent ubiquitination of RNA polymerase II in vitro. Proc.Natl. Acad.
Sci. USA., 99 (7), 4239-4244 (2002).
Guan R.J.,Wang M., Wang, D., Wang D.C. A new insect neurotoxin AngP(1)
with analgesic effect from the scorpion Buthus martensii Karsch: purification and
characterization.
. Pept. Res., 58 (1), 27-35 (2001).
Presentations
Wang, D., Lippard, S.J. Histone modification following treatment of cells with
cisplatin: cause and effects. 9th international symposium on platinum
coordination compounds in cancer chemotherapy, New York, Oct., 2003.
Wang, D., Hara R., Singh G., Sancar, A., Lippard, S.J. Nucleosomes
inhibit
nucleotide excision repair from site-specific platinum-DNA adducts. 225th ACS
meeting, New York, Sept., 2003.
Wang, D., Lippard, S.J. Modulation of histone modification on nucleotide
excision repair from a site-specificplatinum-modified nucleosome, 5th Northeast
student chemistry research conference, Boston, Apr. 2003.
Wang, D., Hara R., Sancar A., Lippard S.J. Nucleotide excision repair from a sitespecifically cisplatin-modified nucleosome. 224th ACS meeting, Boston, Aug.,
2002.