CISPLATIN IN YEAST: REPAIR OF DNA ADDUCTS AND BINDING OF HMG
DOMAIN PROTEINS
by
Megan Moore McA'Nulty
A.B.,Chemistry and Physics
Harvard University
1989
Submitted to the Department of Chemistry
in partial fulfillment of
the requirements for the Degree of
Doctor of Philosophy
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September, 1995
© Massachusetts Institute of Technology, 1995
all rights reserved
Signature of Author
Departi'ent of Chemistry
August
17, 1995
t
I
Certified by
,
Stephen J. Lippard
Thesis Advisor
/
/
Accepted by
Dietmar Seyferth
Graduate
Students
admiecitee on
j;Ba
OF TECHNOL.OGY
SEP 121995
LIBRARIES
Scenie
2
This doctoral thesis has been examined by a Commitee of the Department of
Chemistry as follows:
L
Profe~sr John M. Es 'nann, Chairman
II
'1/I'
ProfessoPStepheJ.
fa-i
Thesis Supervisor
\ I4
V
Professor James R. Williamson
3
CISPLATIN IN YEAST: REPAIR OF DNA ADDUCTS AND BINDING OF HMG
DOMAIN PROTEINS
by
Megan Moore McA'Nulty
Submitted to the Department of Chemistry on August, 1995in partial fulfillment
of the requirements for the Degree of Doctor of Philosophy in Chemistry,
biochemistry subdivision
ABSTRACT
Cisplatin is a potent antitumor drug used to treat many different tumors.
It is believed to kill cells by forming adducts on their DNA that block DNA and
RNA polymerases. A class of transcription factors, HMG domain proteins, binds
specifically to these cisplatin-DNA adducts. The effects of these proteins on
cisplatin cytotoxicity were studied. Ixrl is one such protein from the yeast
Saccharomycescerevisiae.When the IXR1 gene is deleted from yeast cells, the
resulting ixrl strain is less sensitive to cisplatin treatment. The cisplatin
cytotoxicity difference depended on the HMG domain of Ixrl, not its natural
function. The decreased sensitivity of the ixrl strain was dependent upon several
proteins (Rad) involved in the excision repair of cisplatin-DNA adducts. When
the genes for the Rad proteins were deleted, the difference in sensitivity between
the IXR1 and ixrl strains was substantially diminished. The IXR1 and ixrl
strains are equally sensitive to ultraviolet light, which means that Ixrl does not
affect excision repair of other forms of damage. Ixrl most likely shields cisplatin
adducts from repair by binding to the them and blocking the access of the repair
proteins. This theory was also supported by examination of the levels of the
cisplatin-DNA adducts in the IXR1 and ixrl strains. Less cisplatin-DNA adducts
were present in the ixrl strain, which supports the idea that the adducts were
repaired more easily in this strain. The difference in adduct level was dependent
on the presence of the Rad proteins. These data indicate that the level of repair
was higher in the ixrl strain, probably because Ixrl blocks cisplatin adducts from
excision repair. The binding of HMG domain proteins to cisplatin-DNA adducts
could also affect binding to their natural sites, thus disrupting the transcription
of the genes they regulate. This hypothesis was tested using Ixrl, which is a
repressor of transcription from the Cox5b promoter. Treating cells with cisplatin
did not affect the level of transcription from the Cox5b promoter, indicating that
Ixrl is not titrated away from the promoter sufficiently to permit increased
expression. The experiments presented in this thesis indicate that new cisplatin
drugs or therapies could be designed by maximizing the level of HMG domain
protein binding to the cisplatin DNA adducts so as to keep the adducts on the
DNA and thus kill the tumor.
Thesis Supervisor: Dr. Stephen J. Lippard
4
ACKNOWLEDGMENTS
My first and loudest thanks belong to my husband, Peter, who helped me
through the six years I worked on this thesis, first by courting me, then by
wanting to marry me, and finally by cooking and cleaning for me so that all my
attention could go to this all-consuming monster for the past six months or more.
Naturally, the rest of my family was also wonderful--my parents got me started
on science in the first place, and have helped ever since. Carol, Don, and John
McA'Nulty made me feel truly welcome in their family; it's been really
wonderful to have another family on this coast. I also want to thank my aunt
Elizabeth for all the vacations in high school and college, and storing my stuff,
and dinners, and everything. My 'little' brother Scott has loved me and argued
with me for 24 years now, and even puts up with me calling him shrimp now
that he's 6'5".
No Lippard lab acknowledgments page is complete without the
paragraph where you try to list all the people who have helped over the years.
The boss himself, Steve, was indispensable, of course. Steve taught me a lot
about being a scientist and how to organize your lab work. My early years were
dominated by Steve Brown and Pieter Pil, and I want to thank Patti Kellett, Mike
Keck, Steve Bellon, and Ken Comess who helped me remember that not all
scientists are looney-tunes. Laura Pence and Joyce Whitehead were my first
close friends in lab, and they helped make it a place I wanted to come to.
Dave Coufal spent large portions of the last year saying "It will all be OK,
Megan" to me before interviews and other horrible occasions, and talking
computers and Star Wars games. Ann, and later Deb and Karen, came in and
lightened up the upstairs bays, and generally made lab more fun and more
hectic. Deb--good luck with the real human cells, and take more time off. Ann,
thanks for letting me use your wet box, and not freaking when the top agar bottle
broke and turned it into a large jello container. Young Douglas, I hope the nickname list is at an end, but I doubt it! Will you get rid of all those rubber bands
before you graduate? Sonja arrived here having actually grown a yeast cell,
which isn't true for anyone else as far as I know, and from there, has helped with
so many different aspects of life that I can't list them all. Sonja, you come to
Harvard Med every so often, how about snack time? Betsy, thanks for being so
easy to teach and share space with, and actually being cheerful around me when
I was wondering if I'd ever get it all done. Andy and Betsy, thanks for giving me
the whole bay for a couple of weeks, you both know how much I needed it.
'Downstairs' platinum--thanks for letting me borrow equipment and
radioactivity for those frantic weeks after the move. There are, of course, two
other subgroups in the lab, and you can't be here for six years without getting to
know some of the people in them. If I start listing all the past members, we'll be
here all night, so I wont. Joanne, Maria, Susanna, I can't seem to pass your bays
without hearing you guys laughing. Thank you (and Doug) for taking me to
lunch before my defense. Andrew Feig provided an interesting competition to
see who could get out first, and I figure that within two days of each other means
we tied. Rene helped so much in the last stages of this thesis--at least in part by
telling me how much worse her school's requirements for theses are. So, Joyce,
Laura, I'll be a postdoc shortly, do I know everything now? The entire job search
process was a living hell. Joyce, Laura, Kristin, and Paige were my companions
for the on-campus interviews and afterwards commiserated about the miserable
parts. Kitty in headquarters made that whole thing less stressful and has even
managed to convince the Bursar's office that I don't owe them several thousand
dollars--a true miracle.
I've probably gotten more help from other professors both here and
elsewhere than your run-of-the-mill graduate student, since my project was not
exactly classical inorganic chemistry. Prof. John Essigmann also works on
cisplatin, and I hope he knows how much I valued his input in those joint
subgroup meetings those first years. The Essigmann lab in general also helped
with advice and supplies, especially the tissue culture areas--I just hope I never
have to clean one again. Prof. Fink provided a lot of much-needed yeast
expertise, and lent me plasmids and yeast strains whenever I needed them. Prof.
Guarente never really met me, but his lab also helped with the hunt to find a
reasonable expression system for Ixrl. The gurus of yeast excision repair, Louise
Prakash and Errol Friedberg (in the person of Wolfram Siede) made chapter 3
possible. Finally, thanks should go to Prof. Jamie Williamson for being willing to
read what is, in essence, a biology thesis.
Since I spent 4 years of college in the Boston area at that small liberal arts
college up the river, I was lucky enough to have many friends in the area already.
The members of the Society for Creative Anachronism helped by providing a
complete distraction from the lab--I recommend such a thing to everyone. Dante,
especially, kept telling me that it could be done, and her husband Mark told my
husband that it could be done, so we just did it. My college friends Claire,
Debbie, and Susan are all pursuing non-science Ph.D.'s right now, so, now it's my
turn to say "See, it can be done", and maybe now I'll actually write letters more
than once a year. Frank was the best man at our wedding and has since made it
possible for me to finish my thesis by offering advice and a view point no one
else could.
Finally, I'd like to thank whoever decided on the yeast nomenclature
system; whoever you are, I'd like to spell your name all in lower case.
6
TABLE OF CONTENTS
Ab stract ..........................................................................................................................
3
Acknowledgments .......................................................................................................4
Table of Contents ..........................................................................................................6
List of Tables .................................................................................................................9
List of Figures ....................................................
10
Chapter 1. Cisplatin and cisplatin-DNA adduct binding proteins ......................15
Introduction ....................................................
16
Cisplatin cytotoxicity ....................................................
Cisplatin adducts ....................................................
16
16
Cisplatin adducts block DNA and RNA polymerases ...................20
Resistance ....................................................
21
Oncogenes and tumor suppresser genes ..........................................
25
Cisplatin treatment causes changes in the cells ...............................25
Cell cycle and apoptosis ........................................
............
26
Other drugs affect cisplatin cytotoxicity ...........................................
27
Repair of cisplatin-DNA adducts ........................................ ............
28
HMG domain proteins ....................................................
30
HMG1 binds to cisplatin-DNA adducts ........................................... 31
HMG domain proteins, a new class of DNA-binding
proteins ....................................................
33
HMG1 binding to DNA ....................................................
36
The natural function of HMG1 ....................................................
38
HMG1 bends cisplatin-treated DNA ................................................. 40
HMG domain proteins and cisplatin antitumor activity................41
Yeast as a model system .......................................
...............................
43
Summary and Conclusions ........................................................ ....................
45
References ....................................................
...... 47
Figures ....................................................
68
Chapter 2. Cisplatin and yeast cells....................................................
93
Introduction ....................................................
94
Materials and methods ....................................................
97
Results ....................................................
102
Cytotoxicity difference between IXR1and ixrl strains ..................102
Ixrl binding to damaged DNA .................................................... 103
Roxl, an HMG domain protein .................................................... 103
Abf2, an HMG domain protein ....................................................
104
Ixrl overexpression studies ....................................................
104
Cytotoxicity assays on Ixrl mutants ..................................................
106
Cytotoxicity assays on cox5astrains ..................................................
108
Anaerobic cytotoxicity assays .................................................... 109
Cytotoxicity assay conditions ........................................
Discussion ....................................................
............ 109
111
7
The HMG domains of Ixrl cause the difference in cisplatin
sensitivity .........................................................
111
Different cellular conditions cause different responses .................113
Conclusions .........................................................
115
References .........................................................
116
Figures ........................................................................
124
Chapter 3. The HMG domain protein Ixrl shields cisplatin-DNA adducts
from excision repair in vivo ........................................................................................
175
Introduction .........................................................
176
Materials and methods .........................................................
179
Results ................................................................................................................ 181
Construction of rad IXR1 and rad ixrl yeast strains ........................181
Cisplatin cytotoxicity in IXR1and ixrl strains ................................181
Excision repair mutants .........................................................
182
Double-strand break repair mutant .
.................................
Error-prone repair mutant .........................................................
Checkpoint mutant .........................................................
Discussion .........................................................
183
184
184
184
Ixrl affects excision repair ........................................
................. 184
Comparison to previous cisplatin yeast studies ..............................187
Double-strand break repair mutant .
.................................
188
Error-prone repair mutant ........................................
................. 188
Cell-cycle checkpoint mutant ......................................................... 189
Conclusions .........................................................
References .........................................................
Figures .........................................................
189
191
198
Chapter 4. Repair of cisplatin-DNA adducts ......................................................... 219
Introduction .........................................................
220
Materials and methods .........................................................
221
Results .........................................................
228
Levels of cisplatin-DNA adducts on yeast genomic DNA............. 228
Levels of cisplatin-DNA adducts on a transcriptionally active
yeast gene .........................................................
228
Restriction enzyme cleavage of cisplatin- and trans-DDPDNA adducts .........................................................
232
Restriction enzyme based DNA adduct repair assay ....................235
Discussion .........................................................
236
Platinum levels on yeast DNA--general DNA .................................236
Platinum levels on actively transcribed DNA in yeast ...................238
Platinum levels on yeast DNA--Other types of repair ...................239
Comparison to mammalian and E. coli cell adduct levels .............. 240
Restriction enzyme digestion of platinum-treated DNA ...............241
Restriction enzyme based repair assay .............................................
243
Conclusions .........................................................
244
8
References ........................................................................................................ 246
Figures ....................................................
252
Chapter 5. HMG domain proteins binding to cisplatin-DNA adducts in
vivo ....................................................
295.............................
Introduction ....................................................
296
296
Titration Hypothesis ....................................................
HMG1 binds cisplatin-DNA adducts in vivo .................................. 297
Materials and methods ....................................................
298
Results ................................................................................................................ 303
303
Titration Hypothesis experiments ....................................................
Plasmid-based assay ....................................................
303
mRNA based assay ....................................................
HMG1 binds to cisplatin-treated chicken erythrocyte DNA in
304
306
chromatin ....................................................
Discussion ....................................................
307
Titration Hypothesis ....................................................
307
HM G1 binds cisplatin-DNA adducts in vivo .................................. 308
Conclusion
....................................................
References ....................................................
Figures ....................................................
309
...... 311
314
9
LIST OF TABLES
Chapter 2
Table I. Saccharomycescerevisiaestrains ........................................................ 119
Table II. Plasmids used in cytotoxicity experiments ..................................120
Table III. Cytotoxicity assay results ..............................................................
121
Table IV. Ixrl overexpression cytotoxicity assay........................................121
Table V. Ixrl-1 cytotoxicity assay .................................................................
122
Table VI. Different growth conditions .........................................................
122
Chapter 3
Table I. Plasmids used in construction of rad stains .................................. 196
Table II. Quantitation of cisplatin cytotoxicity in yeast ............................. 197
Chapter 4
Table I. (D/N)b levels measured by atomic absorption ............................249
Table II. Platinum-DNA adducts block restriction enzyme activity .......250
10
LIST OF FIGURES
Chapter
1
Figure 1.1. Structure of various platinum compounds..............................68
Figure 1.2. Adducts formed by cisplatin adducts ......................................
70
Figure 1.3. Types of cancer that cisplatin therapy is effective
ag ainst ................................................................................................................
72
Figure 1.4. Excision repair .............................................................................. 74
76
Figure 1.5. Excision repair proteins ..............................................................
Figure 1.6. HMG1 and HMG domain protein DNA binding sites ..........78
Figure 1.7. Structure of HMG1 domain B .................................................... 80
82
Figure 1.8. Repair shielding theory ..............................................................
86
Figure 1.9. Titration hypothesis ....................................................................
Figure 1.10. Interruption of genes in Saccharomycescerevisiae............... 90
Chapter 2
Figure 2.1. Ixrl domains ................................................................................. 124
Figure 2.2. Natural functions of Ixrl and related proteins ........................126
128
Figure 2.3. Ixrl expression plasmids ............................................................
Figure 2.4. Cytotoxicity assays performed on JM43 and JM43Aixrl
130
using standard conditions ..............................................................................
Figure 2.5. Confirmation of construction of BWGAixrl strain ................. 132
Figure 2.6. Cytotoxicity assays performed on BWG and BWGAixrl
136
using standard conditions ..............................................................................
Figure 2.7. Ixrl binds cisplatin damaged DNA but not other forms
138
of damaged DNA .............................................................................................
Figure 2.8. Cytotoxicity assays performed on JM43Aroxl, JM43,
142
and JM43Aixrl using standard conditions ...................................................
11
Figure 2.9. Southwestern blot of abf2 strains .......................................
144
Figure 2.10. Southwestern blots of overexpression of Ixrl by using
the GALl promoter ...................................................
148
Figure 2.11. Cytotoxicity assays performed on BWG and BWGAixrl
cells with the control plasmid pCD43 and the Ixrl overexpression
plasmid pCY ....................................................
152
Figure 2.12. Ixrl overexpression from the Met promoter ......................... 154
Figure 2.13. Cytotoxicity assays performed on JM43 and JM43Aixrl
with the control plasmid pYes2 and the Ixrl overexpression
plasm id pMM ....................................................
158
Figure 2.14. Cytotoxicity assays performed on mutant Ixrl and
cox5a strains ....................................................................................................... 160
Figure 2.15. Cytotoxicity assays performed on Ixrl-1 strains ...................162
Figure 2.16. Cytotoxicity assays performed on JM45, F::5 ........................ 164
Figure 2.17. Cytotoxicity assays performed on JM43 and JM43Aixrl
using anaerobic conditions ...................................................
166
Figure 2.18. Cytotoxicity assays performed on IXR1and ixrl grown
on YPD and YPD with 10% dextrose ....................................................
168
Figure 2.19. Cytotoxicity assays performed on JM43 and JM43Aixrl
grown on YPD with 10% dextrose ...................................................
170
Figure 2.20. Cytotoxicity assays performed on IXR1and ixrl grown
in YPGE....................................................
172
Chapter 3
Figure 3.1. Model for excision repair of cisplatin-DNA adducts ............. 198
Figure 3.2. Ultraviolet light sensitivity of various yeast strains ...............202
Figure 3.3. Cisplatin cytotoxicity assays performed on the excision
repair deficient strains RAD IXR1, RAD ixrl, rad2 IXR1 and rad2
ixrl ......................................................
206
12
Figure 3.4. Cisplatin cytotoxicity assays performed on the excision
repair deficient strains rad4 IXR1, rad14IXR1, rad4ixrl, and radl4
ixrl ......................................................................
208
Figure 3.5. Cisplatin cytotoxicity assays performed on the excision
repair deficient strains radl IXR1, radlOIXR1, radl ixrl and radiO
ixrl ...................................................
210
Figure 3.6. Cisplatin cytotoxicity assays performed on RAD IXR1
and the double strand break repair deficient lines rad52IXR1 and
rad52 ixrl ...................................................
212
Figure 3.7. Cisplatin cytotoxicity assays performed on the errorprone repair deficient strains rad6 IXR1 and rad6 ixrl ................................214
Figure 3.8. Cisplatin cytotoxicity assays performed on RAD IXR1,
RAD ixrl, and the cell cycle checkpoint deficient lines rad9 IXR1
and rad9ixrl ...................................................
216
Chapter 4
Figure 4.1. Gel of PCR product from PGK gene from yeast genomic
DNA treated with various levels of cisplatin ...............................................
252
Figure 4.2. Analysis of PCR products from DNA extracted from
IXR1and ixrl cells treated with increasing levels of cisplatin ..................256
Figure 4.3. Analysis of PCR products from DNA extracted from
IXR1 and ixrl cells treated with cisplatin and permitted to recover
in fresh media for various times ...................................................
258
Figure 4.4. Analysis of PCR products from DNA extracted from
radl4 IXR1and radl4 ixrl cells treated with increasing levels of
cisplatin ...................................................
260
Figure 4.5. Analysis of PCR products from DNA extracted from
radl4 IXR1 and radl4 ixrl cells treated with cisplatin and permitted
to recover in fresh media for various times ..................................................
262
Figure 4.6. Analysis of PCR products from DNA extracted from
JM43 and JM43Aixrl cells treated with increasing levels of cisplatin ...... 264
Figure 4.7. Analysis of PCR products from DNA extracted from
JM43 and JM43Aixrl cells treated with cisplatin and permitted to
recover in fresh media for various times ...................................................266
13
Figure 4.8. Sac restriction enzyme digestion of trans-DDPtreated
M13mpl8 virus DNA ....................................................
270
Figure 4.9. SmaI restriction enzyme digestion of cisplatin and transDDP treated pUC19 plasmid DNA ...................................................
274
Figure 4.10. EcoRI restriction enzyme digestion of cisplatin and
trans-DDP treated pUC19 plasmid DNA ...................................................278
Figure 4.11. Construction of trans-DDP-M13-Xhol2C ............................... 282
Figure 4.12. Digestion of trans-DDP-M13-Xhol2C by XhoI ...................... 284
Figure 4.13. Test of repair assay substrates ................................................. 288
Figure 4.14. Restriction enzyme based repair assay performed in
HeLa cell extracts ...................................................
292
Chapter 5
Figure 5.1. Titration hypothesis ....................................................
314
Figure 5.2. -galactosidase activity of JM43 and JM43Aixrl strains
treated with cisplatin ....................................................
318
Figure 5.3. -galactosidase activity of JM43 and JM43Aixrl strains
treated with cisplatin and permitted to recover in fresh media for 16
hours ....................................................
320
Figure 5.4. -galactosidase activity of JM43 and JM43Aixrl strains
treated with cisplatin over time ...................................................
322
Figure 5.5. mRNA analysis of IXR1and ixrl strains treated with
low levels of cisplatin ...................................................
324
Figure 5.6. mRNA analysis of IXR1 and ixrl strains treated with
higher levels of cisplatin ....................................................
328
Figure 5.7. mRNA analysis of IXR1and ixrl strains treated with
cisplatin and permitted to recover in fresh media for 16 hours ................330
Figure 5.8. mRNA analysis of IXR1and ixrl strains treated with
332
cisplatin after they were grown to saturation ..............................................
Figure 5.9. mRNA analysis of IXR1and ixrl strains treated with
cisplatin for various times .............................................
334
14
Figure 5.10. HMG1 is present in the 0.35 M NaCl extract of chicken
erythrocyte nuclei ............................................................................................. 336
Figure 5.11. HMG-1 was bound to DNA in cisplatin treated chicken
erythrocyte nuclei ............................................................................................. 340
15
CHAPTER
1
Cisplatin and cisplatin-DNA adduct binding proteins
16
INTRODUCTION
Cisplatin is a potent antitumor drug. It is especially effective against
testicular tumors and is used in combination with other drugs against a number
of other tumors. Cisplatin works by binding to the DNA, forming both
intrastrand and interstrand adducts. These adducts are widely believed to kill
the cells by blocking DNA and RNA polymerases. The HMG domain is a DNA
binding motif that binds to cisplatin-DNA adducts. HMG domain proteins are
primarily transcription factors. There are currently two main theories regarding
how HMG domain proteins might affect cisplatin cytotoxicity. The proteins may
block the repair of the adducts or the transcription of the genes the proteins
regulate may be disrupted. Investigation of HMG domain proteins in general,
and these theoretical models in particular, may lend insight into the design of
better cisplatin-based antitumor drugs.
The pursuit of these improved cisplatin analogs is important because
cisplatin is not a perfect antitumor drug. The dose of cisplatin necessary to affect
tumors causes many unpleasant and potentially fatal side effects. Ovarian
tumors, which do not have effective alternative treatments, have a particular
problem with intrinsic and acquired resistance. In order to treat such tumors,
better cisplatin analogs or combination therapies need to be designed, taking into
account the new knowledge of how cisplatin-DNA adducts affect cells.
CISPLATIN CYTOTOXICITY
Cisplatinadducts
Cisplatin, or cis-diamminedichloroplatinum (II) (Fig. 1.la) can bind to
DNA, RNA, and proteins. Many studies have demonstrated that the DNA
adducts are the cytotoxic agent (reviewed in Lepre and Lippard, 1990). Studies
with radioactive cisplatin have shown that the lethal dose was related to the
binding of cisplatin to DNA, not to protein or RNA (Akaboshi et al., 1992).
17
Cisplatin forms both interstrand and intrastrand adducts on DNA (Fig. 1.2). The
major cisplatin adduct has the platinum bound to the N7 of two adjacent guanine
bases. Cisplatin d(GG) and d(AG) adducts comprise about 90% of the DNA
adducts (Fig. 1.2a). The minor adducts consist of intrastrand cross-links between
two guanines that are not adjacent (d(GXG))and interstrand cross-links between
two guanines (Lepre and Lippard, 1990) (Fig 1.2 b,c). Cisplatin also forms a
small number of adducts between DNA and proteins (Fig. 1.2d). Monofunctional
adducts are formed at lone G residues (Fig 1.2e);diethyltriaminechloroplatinum(II) or [Pt(dien)C1]+ forms only monofunctional adducts (Fig. 1.le)
(Lepre and Lippard, 1990). The cisplatin analog ethylenediaminedichloroplatinum(II) or [Pt(en)C12](Fig. 1.lb) forms the same adducts as cisplatin.
trans-Diamminedichloroplatinum(II), or trans-DDP, the stereoisomer of
cisplatin, is not an active antitumor drug (Fig. 1.ld). trans-DDPalso kills cells by
forming DNA adducts (Akaboshi et al., 1993),but the DNA adduct spectrum
formed by trans-DDPis substantially different from that of cisplatin. The 1,2intrastrand d(GG) and d(AG) adducts cannot be formed because the distance
between the two Cl ligands on trans-DDPis too large. trans-DDPpredominantly
forms 1,3-intrastrand adducts at d(GXG) and d(AXG) sites (Lepre and Lippard,
1990). Interstrand adducts are formed by trans-DDPbetween complementary
guanine and cytosine residues at d(GC) sites (Brabec and Leng, 1993). Recently,
it has been suggested that trans-DDPdoes not form bifunctional intrastrand
adducts (Boudvillain et al., 1995). This result contradicts the findings of several
different laboratories (for review see Lepre and Lippard, 1990). The recent
results were based on digesting trans-DDPtreated DNA with the 3'-5'
exonuclease activity of T4 DNA polymerase. The exonuclease was stopped by
trans-d(GXG) adducts in studies on short oligonucleotides. When doublestranded DNA was treated with trans-DDP,no exonuclease stops were visible, so
18
the authors concluded that no trans-d(GXG)adducts were formed. There are
several problems with this experiment. First, the DNA was only treated with
trans-DDPfor 24 hours; formation of trans-DDPbifunctional adducts is not
complete until 48 hours have past (Eastman and Barry, 1987). Furthermore, the
DNA was treated with thiourea after the trans-DDPreaction, which is known to
remove 80-85% of the adducts (Eastman and Barry, 1987;Boudvillain et al., 1995).
Since the authors state that interstrand adducts comprise 15% of the pre-thiourea
adducts, the thiourea treatment may be removing all non-interstrand adducts. In
addition to these problems, the actual exonuclease gel figure shows a fairly dark,
diffuse band of undigested material in the trans-DDPlanes; since the final
digestion product (mononucleotides) was not shown in this picture, it is
impossible to determine how complete the digestion actually was. The results
from this paper are inconclusive since the authors did not treat the DNA in a
manner similar to what would occur in cells, and the gels themselves were
inadequate. The nature of the trans-DDPadducts is of interest because
comparisons between the activities of cisplatin and trans-DDPhelp to identify
which aspects of cisplatin activity are important to tumor cytotoxicity.
The DNA adducts formed by cisplatin and trans-DDPdistort the normal
DNA structure. The platinum-DNA adducts bend and unwind the DNA to
varying degrees. The cisplatin d(GG) and d(AG) adducts bend the DNA by 3234° and unwind it by 13° according to gel electrophoretic studies (Fig. 1.2a). The
cisplatin d(GTG) adduct bends the DNA similarly, but unwinds it by 23° (Bellon
and Lippard, 1990;Bellon et al., 1991). X-ray crystallography and NMR studies
confirmed that adducts distort the DNA (Sherman et al., 1985;van Garderen and
van Houte, 1994). This distortion was also apparent in scanning tunneling
microscopic studies of cisplatin treated DNA (Jeffrey et al., 1993). Studies with
anti-nucleoside antibodies indicate that there is more single-stranded character in
19
cisplatin treated DNA than in normal DNA (Sundquist et al., 1986). DNAse I
footprinting also indicated that the helix is distorted in the adduct region
(Schwartz and Leng, 1994). T4 Endonuclease VII, which cleaves 4-way junction
DNA, also cleaved cisplatin-DNA adducts but not normal DNA, indicating that
the adducts form a structure more closely related to the 4-way junctions than to
normal DNA (Murchie and Lilley, 1993). trans-DDP-DNA adducts do not distort
the DNA in the same way as cisplatin. Gel electrophoretic studies indicated that
the d(GXG) adducts caused a hinge joint in the DNA instead of a directed bend,
and that the DNA was unwound by 6-13 degrees (Bellon and Lippard, 1990;
Bellon et al., 1991). The anti-nucleoside antibody study and the T4 endonuclease
VII study gave different results for trans-DDPDNA adducts (Sundquist et al.,
1986;Murchie and Lilley, 1993). There was more single-stranded character in the
trans-DDP treated DNA at a lower platination level, but trans-DDP-d(GTG)was
not cleaved by T4 endonuclease VII. Cisplatin and trans-DDPinterstrand
adducts are different from the intrastrand adducts. The cisplatin interstrand
adduct enormously distorts the DNA, causing gel electrophoresis studies to give
a bending angle of 45° and an unwinding angle of 79° (Malinge et al., 1994).
trans-DDP interstrand adducts bend the DNA by 26° and unwind it by 12°
(Brabec et al., 1993). DNAse I footprinting studies indicated that the distortions
in the DNA extend over a greater area for the interstrand cross-links than for the
cisplatin intrastrand adduct (Schwartz and Leng, 1994).
Cisplatin adducts also disrupt chromatin structure. DNA in cells is
wound around the histone core particle to form nucleosomes, which further
organize into chromatin. Cisplatin treatment of cells altered the arrangement of
nucleosomes, but neither trans-DDPnor [Pt(dien)Cl]+, which forms
monofunctional adducts, affected chromatin structure (Houssier et al., 1983).
Nucleosome placement is affected by other forms of damage, including
20
ultraviolet light induced adducts (Suquet and Smerdon, 1993). The mouse
mammary tumor virus (MMTV) promoter is regulated by the glucocorticoid
receptor, which alters the chromatin around the promoter. This alteration was
reduced by cisplatin but not by trans-DDP (Mymryk et al., 1995). The location of
nucleosomes affects transcription of genes by excluding transcription factor and
polymerase binding (Levin, 1994). Cisplatin may affect transcription by
changing the placement of nucleosomes on promoter regions.
Cisplatinadducts blockDNA and RNA polymerases
Early studies with cisplatin indicated that DNA synthesis was inhibited by
cisplatin treatment of cells. It has also been shown that cisplatin-DNA adducts
block DNA and RNA polymerases in vitro (Comess, 1991;Lemaire et al., 1991;
Murray et al., 1992;Corda et al., 1993;Huang et al., 1993). Several studies using
different DNA polymerases have shown that the cisplatin d(GG) adduct
effectively blocks DNA synthesis in vitro (Comess et al., 1992;Murray et al., 1992;
Corda et al., 1993;Huang et al., 1993). The cisplatin d(GXG) adduct does not
form as effective a block, permitting up to 25% of the newly synthesized strands
to extend past the adduct (Comess et al., 1992). Similar studies on trans-DDP
adducts have led to conflicting results since some show that such adducts do not
block polymerases as effectively as cisplatin DNA adducts, while others show
that they block equally well (reviewed in Bruhn et al., 1990). The blocking of the
DNA polymerase can also lead to misincorporation across from the lesion in
those strands that are synthesized past the lesion. This phenomenon was
investigated by examining the mutation rates of various adducts replicated on
episomes in E. coli (Naser et al., 1988; Bradley et al., 1993; Yarema et al., 1994).
These studies indicate that all the platinum adducts cause mutations in episomal
DNA, although the rate and type of mutation vary from adduct to adduct. In
21
conclusion, cisplatin and trans-DDPadducts block DNA replication and thus
probably prevent the cells from duplicating their DNA without errors.
RESISTANCE
Cisplatin and its analog carboplatin are used by themselves and in
combination with other drugs against metastatic testicular tumors, metastatic
ovarian tumors, advanced bladder cancer, small cell lung cancer, endometrial
cancer, leukemia, head and neck cancers, and cervical carcinoma (Fig. 1.3)
(McEvoy, 1994;Olin, 1994). Acquired or intrinsic resistance to chemotherapy in
any of these diseases can prevent effective treatment resulting in death.
Acquired resistance is a particular problem in ovarian cancer (Arky, 1994). In
two large studies, the initial response rate to cisplatin therapy was about 60% in
ovarian tumor patients. Approximately half of these patients remained tumor
progression free after 2 years, and one third after 3 years. Seventy percent of the
patients died within 3 years of cisplatin treatment. Either a new cisplatin analog
that treats these patients, or a way to tell which patients are responding to
therapy before the tumors grow back would be helpful in prolonging and
improving their lives. The causes of resistance to cisplatin have been studied in
many different systems. According to theory, the major causes of resistance will
be ones which affect the formation or removal of cisplatin-DNA adducts, or the
ability of those adducts to kill the cell. Changes in drug accumulation, removal,
repair of the cisplatin-DNA adducts, and levels of sulfur-containing proteins that
bind cisplatin have been investigated. One issue hampering these studies is the
difficulty in acquiring matched normal and resistant cell lines from patients
undergoing cisplatin chemotherapy. Many of the studies were performed by
using cell lines that were selected for cisplatin resistance by in vitro growth in
media containing cisplatin. Unfortunately, studies with other drugs indicate that
22
resistance mechanisms characterized in this manner are not necessarily the same
as those in actual patients.
One major cause of resistance is increased repair of cisplatin-DNA
adducts. Many different cell lines have been tested for this increase in repair
activity; almost all tested cell lines show some increase. Cisplatin resistant
ovarian cell lines have an increased level of cisplatin-DNA adduct repair. This
result has been demonstrated by comparing adduct levels in cells, by measuring
the rate of adduct removal, by measuring the gene-specific repair of cisplatin
interstrand cross-links, and by performing excision repair assays using cell
extracts (Dempke et al., 1992;Zhen et al., 1992;Schmidt and Chaney, 1993;
Johnson et al., 1994a; Johnson et al., 1994b; Jones et al., 1994; Mellish and Kelland,
1994). The increase in DNA repair has even been confirmed in vivo using lines
acquired from a patient's original and secondary tumors (Ali-Osman et al., 1994).
The in vivo experiment showed that a resistance difference of only 5.9 fold at 5
gM cisplatin could cause clinically important resistance. The difference in repair
between the two strains was 2.8 fold, which indicates the importance of increased
repair in real ovarian tumors. Differences in repair have also been found in
resistant cell lines from colon carcinomas, HeLa, fibrosarcoma, and squamous
cell carcinomas of the head and neck (Chao et al., 1991b; Parker et al., 1993;
Schmidt and Chaney, 1993;Chao et al., 1994;Moorehead et al., 1994;Oldenburg
et al., 1994). Multiple studies were performed by using both normal and resistant
L1210 cell lines, which showed increases in repair of cisplatin-DNA adducts by
several different methods (Sheibani et al., 1989;Gibbons et al., 1991;Olinski and
Briggs, 1991; Calsou et al., 1993). Unfortunately, the "normal" L1210 cell line is
not quite normal because it has a defect in a vital DNA excision repair gene, so
none of these studies are necessarily related to real tumor situations since most
people have functional excision repair systems (Vilpo et al., 1995). Platinum
23
DNA adduct levels have been associated with patient response in many different
diseases (Blommaert et al., 1993;Reed et al., 1993). All of these data support the
conclusion that increased DNA repair is an important method by which cells
develop cisplatin resistance.
Another widely studied resistance mechanism is increased levels of
glutathione (GSH) and related enzymes. Theoretically, when the sulfhydryl
groups on glutathione bind cisplatin, it is unable to form bifunctional adducts on
DNA, so the cells can survive higher levels of cisplatin treatment. Increased
levels of GSH have been found in head and neck carcinomas, ovarian carcinoma
cell lines, testis cancer lines, mouse L1210lines, and colon carcinoma (Behrens et
al., 1987;Hamaguchi et al., 1993;Ishikawa and Ali-Osman, 1993;Schmidt and
Chaney, 1993; Timmer-Bosscha et al., 1993; Oldenburg et al., 1994; Yellin et al.,
1994). Increases in GSH-related enzymes such as glutathione reductase have
been found without any increase in GSH in small cell lung cancer lines
(Twentyman et al., 1992;Hao et al., 1994). Naturally, not every study found such
differences, including one study on ovarian carcinoma lines (Mellish and
Kelland, 1994). The amount of GSH increase varied widely from cell line to cell
line, and was not strictly correlated with cisplatin sensitivity. Overall, these data
support the idea that raising the GSH levels increases cisplatin resistance,
although it is clear that other changes within the cells have been made.
Some studies measured the uptake and accumulation of cisplatin and how
well the drug could make DNA adducts. Decreased uptake or accumulation of
the drug was found in some resistant strains such as ovarian carcinoma cells,
lung cancer cells, and colon carcinoma lines (Twentyman et al., 1992;Schmidt
and Chaney, 1993;Jekunen et al., 1994;Mellish and Kelland, 1994;Oldenburg et
al., 1994). Other strains, including a testis line, an ovarian cancer line, and a
small cell lung cancer line show no such difference (Twentyman et al., 1992;
24
Timmer-Bosscha et al., 1993;Mellish and Kelland, 1994). Ovarian and testis
resistant lines showed slower reaction of cisplatin with DNA (Timmer-Bosscha et
al., 1993;Jekunen et al., 1994). These studies indicate that, whereas decreased
uptake can be part of a resistant phenotype, it is not as frequent a mechanism as
either increased DNA repair or increased GSH levels.
Many other possible mechanisms of resistance have been theorized and
investigated in one cell line or another, indicating the complexity of cisplatin
resistance. One study demonstrated that resistant ovarian cells could alter the
ability of their polymerases to bypass the cisplatin adducts and duplicate the
DNA (Mamenta et al., 1994). Cisplatin resistant lung cancer cells increased
phosphorylation of several nuclear proteins. Differential phosphorylation of
proteins often modulates their activity in many different ways (Nishio et al.,
1992). In another study, the level of ATP was increased in resistant human
ovarian cells; such a change could affect many systems (Berghmans et al., 1992).
The plasma and mitochondrial membrane potentials were decreased in a
fibrosarcoma resistant line (Moorehead et al., 1994),while the activity of
cytochrome c oxidase was increased in cisplatin resistant ovarian, breast, and
squamous head and neck carcinomas (Ara et al., 1994). Differences in
chromosome 12 distribution in the nucleus were found in a resistant testicular
tumor cell line (Reilly et al., 1993). One study of bladder cancer suggested that
increased levels of metallothionein may also cause tumors to become resistant
(Wood et al., 1993). Nevertheless, metallothionein levels did not change in
resistant human lung cells (Twentyman et al., 1992). Some potential mechanisms
cause resistance when examined by changing the levels of expression of proteins,
but do not appear in real resistant cell lines. Topoisomerase II expression
affected repair of cisplatin interstrand cross-links (Ali-Osman et al., 1993).
Treatment of cells with a topoisomerase I inhibitor does not sensitize the cells to
25
cisplatin, however (Teicher et al., 1993). Two studies showed no difference in the
levels of topoisomerases, so this mechanism may not occur in resistant cells (AliOsman et al., 1993; Hamaguchi et al., 1993; Timmer-Bosscha et al., 1993). The
mechanism of cisplatin resistance is apparently very complicated. In order to
improve anticancer treatments, it is important to know which mechanism of
resistance is the most important in each type of cancer, or to investigate only
mechanisms that are common in all types of cancers.
Oncogenesand tumor suppressergenes
The formation of tumors often involves the activation of oncogenes and
inactivation of tumor suppresser genes. The effect of several of the common
oncogenes and tumor suppresser genes on cisplatin resistance has been studied.
Increased levels of the ras oncoprotein lead to increased resistance, as did
increased levels of c-myc (Isonishi et al., 1991;Sklar and Prochownik, 1991;Levy
et al., 1994). Testis and small-cell lung cancer cell lines have an increased level of
embryonic forms of myc, N-myc and L-myc (Saksela et al., 1989). These proteins
may help cause the tumors since they are not normally expressed in nonembryonic tissues. A study of a resistant testicular strain found no increase in cmyc levels (Timmer-Bosscha et al., 1993). The tumor suppresser protein p53 was
not mutated in testicular tumors (Peng et al., 1993), whereas levels of a different
tumor suppresser, Rb, were lowered (Saksela et al., 1989). These results indicate
that the effects of these proteins on cisplatin resistant cell lines are worth
investigating, but they do not guarantee that altered levels of these proteins are
an important determinant of resistance.
Cisplatin treatment causes changes in the cells
The effect of cisplatin on various proteins and cellular systems has been
investigated. Some proteins, such as heat shock protein 25 (HSP25) and the
growth arrest and DNA damage-inducible protein GADD153, were induced, but
26
others, such as CTP synthetase, were inhibited (Ganeva et al., 1991;Oesterreich et
al., 1991;Gately et al., 1994). Cisplatin treatment inhibited parts of the ubiquitin
system and the self-splicing activity of the self-splicing rRNA ribosyme
(Danenberg et al., 1991; Isoe et al., 1992). The levels of poly(ADP-ribosyl)ation,
cAMP, and the dNTPs were raised by cisplatin treatment (Just and Holler, 1991;
Burkle et al., 1993). Cisplatin treatment also affected DNA processing enzymes,
inhibiting DNA helicases and activating a magnesium-dependent nuclease
(Hibino et al., 1994; Villiani et al., 1994). Not all alterations are necessarily related
to the antitumor activity of cisplatin. For instance, the slowing of microtubule
assembly by cisplatin treatment is probably related to the peripheral neuropathy
that affects some patients (Boekelheide et al., 1992). Which of these and the many
other alterations cisplatin causes in cells actually affect its ability to destroy
tumors cannot be determined without many more studies.
Cell cycle and apoptosis
Cisplatin treatment also alters the progression of the cell cycle and sends
cells into a form of apoptosis, or programmed cell death. The cell cycle proceeds
from G1 to S (DNA synthesis) to G2 to M (mitosis) phases. Cisplatin arrested
cells in G2 and at the G2/M border (Sorenson and Eastman, 1988b;Sorenson and
Eastman, 1988a; Sorenson et al., 1990; Inoue et al., 1992). Cells synchronized in
the same place in the cell cycle were more sensitive to cisplatin in the G1 phase
than in the S phase (Krishnaswamy and Dewey, 1993). Cisplatin treatment in
combination with another drug, pentoxifylline, caused accumulation in the S
phase, but these cells were not actually synthesizing DNA (Perras et al., 1993).
Cells that are treated differently show different cisplatin sensitivity and cell cycle
characteristics. Apoptosis, or programmed cell death, occurs in many types of
cells. Cisplatin seems to induce apoptosis normally in some cell types and oddly
in other cell types. Thymocytes need to proceed through the cell cycle in order
27
for cisplatin to cause apoptosis (Evans and Dive, 1993;Evans et al., 1994). CHO
cells arrest in G2, but then enter aberrant mitosis which leads to apoptosis after
the cells lose contact with the extracellular matrix (Demarcq et al., 1994).
Cisplatin causes apoptosis in ovarian carcinoma cells without the usual hallmark
of apoptosis, DNA degradation (Ormerod et al., 1994). Cisplatin-induced
apoptosis and cell cycle breaks vary from cell type to cell type. These studies
need to be expanded to define the involvement of the various proteins involved
in apoptosis and cell cycle arrest.
Other drugs affectcisplatincytotoxicity
The effects of other drugs on cisplatin cytotoxicity may give insight into
the important determinants of that cytotoxicity. Many different drugs have been
used in combination with cisplatin, either in human patients or in mouse or cell
studies. Only those drugs which show substantial synergy with cisplatin will
provide data indicating anything about their mechanisms of action. The reasons
for synergy with most of those drugs are unknown. Tamoxifen helped cisplatin
kill malignant melanomas in human patients. Tamoxifen had no effect on
cisplatin uptake, intrastrand adduct formation or repair, or GSH or
metallothionein levels, so the cause for this synergy is unknown (McClay et al.,
1992). The antibiotic amphotericin B sensitized cell lines to cisplatin by
increasing the intracellular accumulation and DNA adduct formation (Kikkawa
et al., 1993; Morikage et al., 1993). Activation of protein kinase C (PKC) by TPA
(12-O-tetradecanoyl-phorbol-13-acetate) caused cells to become more sensitive to
cisplatin without affecting GSH or metallothionein levels and with only a small
increase in adduct levels and decrease in DNA repair level (Isonishi et al., 1994).
Other PKC activators such as epidermal growth factor (EGF) and transforming
growth factor-ac(TGFa ) sensitized cells to cisplatin by increasing the uptake and
accumulation but not the removal of cisplatin (Basu and Evans, 1994). The
28
increase in uptake caused by EGF was blocked by a PKC inhibitor, but the
increases in accumulation and cisplatin sensitivity were not blocked. Contrary to
these results, anti-EGF receptor antibodies sensitized carcinoma cells grafted into
mice to cisplatin treatment by blocking the action of EGF (Fan et al., 1993).
Presumably, the cells in the latter experiment had an alteration in the EGF
induced pathways which caused the cells to proliferate abnormally. Another
protein used as a drug, interleukin-l1t, caused cells to become more sensitive to
cisplatin by increasing the cellular accumulation of cisplatin and decreasing the
DNA repair in ovarian carcinoma cells (Benchekroun et al., 1993). The various
drugs may all affect different aspects of the cisplatin chemotherapeutic process.
Presumably, other drugs yet to be studied may help target the cisplatin to the
tumor. These studies indicate that the some of the same factors causing
resistance are also important in the drug-cisplatin synergy.
REPAIR OF CISPLATIN-DNA ADDUCTS
Cisplatin-DNA adducts are removed by excision repair (Fig. 1.4). This
process excises a 29 nucleotide piece of single-stranded DNA, and requires 10 or
more proteins (Hanawalt, 1995). These proteins are highly conserved from
humans to yeast (Fig. 1.5). Many of the mammalian proteins were found by
complementing UV-sensitive human xeroderma pigmentosum (XP) or rodent
excision repair cross-complementing (ERCC)strains, which are deficient in
excision repair. Cisplatin-DNA adducts were repaired by this system in cell
extracts (Hansson and Wood, 1989;Sibghat-Ullah et al., 1989;Calsou et al., 1992).
The excision repair system repairs primarily the cisplatin intrastrand adducts.
The interstrand adducts are most likely repaired by a different system, which is
also the case for psoralen interstrand adducts (Hang et al., 1993). Excision repair
is related to transcription; transcribed areas of DNA are repaired more readily
than the remainder (Hanawalt, 1995).
29
The first step of excision repair is recognition of the DNA adduct, which is
predominantly performed by XPA in humans. XPA is a zinc finger protein that
binds to DNA damaged with UV light, cisplatin, and other damaging agents
(Guzder et al., 1993;Jones and Wood, 1993). XPA recruits much of the rest of the
DNA repair complex to the damaged site (Park et al., 1995). There is another
protein that may be a damage recognition protein, called UV-DRP, DDB, or XPE
complementing factor. This protein complements the repair deficiency in some
but not all XPE cell lines (Keeney et al., 1994). UV-DRP binds to UV adducts and
cisplatin-DNA adducts, but not trans-DDPDNA adducts (Chu and Chang, 1988;
Patterson and Chu, 1989;Chu and Chang, 1990;Chao et al., 1991a; Chao, 1992;
Keeney et al., 1993;Reardon et al., 1993;Payne and Chu, 1994). It was induced
by cisplatin treatment in ovarian and other carcinoma cells (Vaisman and
Chaney, 1995). It may be part of the excision repair system, perhaps an
unnecessary protein that assists with the repair of certain types of adducts. The
human single-stranded DNA binding protein, which is involved in excision
repair, is part of a complex of proteins that binds to cisplatin-DNA adducts
(Clugston et al., 1992).
Some of the excision repair proteins have been tested for their effect on
cisplatin cytotoxicity. Raising the levels of the ERCC1 protein in ERCC1 deficient
cells caused the cells to become more resistant to cisplatin, as would be expected
(Lee et al., 1993). When ERCC1 was overexpressed in normal CHO cells, it
caused them to become more sensitive to cisplatin; the authors theorized that it
may be suppressing a pathway for the repair of cisplatin lesions (Bramson and
Panasci, 1993). The expression of ERCC1 and ERCC2 was related in nonmalignant brain tissue, but not in malignant brain tumors, indicating that
expression of one of these proteins was disturbed (Dabholkar et al., 1995). In a
separate study, elevated ERCC1 levels were found in resistant testis teratoma cell
30
lines but not in resistant ovarian cell lines, whereas the levels of ERCC3/XPB
were the same in all the lines (Taverna et al., 1994). Ovarian cancer patients that
did not respond to cisplatin chemotherapy had raised levels of ERCC1 but not
ERCC2 (Dabholkar et al., 1992). These studies indicate that ERCC1 may be
particularly important in determining cisplatin cytotoxicity.
Cisplatin adducts were repaired more readily in actively transcribed
regions of DNA (Bohr, 1991;May et al., 1993). This phenomenon was dependent
on active excision repair. Some excision repair proteins were not involved in this
type of repair. XPC cells are sensitive to UV light but still performed repair on
transcriptionally active regions of DNA (Venema et al., 1991;van Hoffen et al.,
1993;Shivji et al., 1994). The yeast strains deficient in the repair proteins Rad7
and Radl6 also behaved in this manner; oddly, strains missing the XPC homolog,
Rad4, did not (Verhage et al., 1994). At the other end of the spectrum,
Cockayne's syndrome cells were deficient in this transcription specific repair,
although they still had some repair capacity for general DNA (van Hoffen et al.,
1993). XPA, XPD, and XPFwere required for gene specific repair (Evans et al.,
1993;Zhen et al., 1993), as was the yeast homolog of ERCC3/XPB (Sweder and
Hanawalt, 1994). The XPC cell line repair activity occurred more readily when
the cells were not dividing (Kantor and Elking, 1988). In general, gene specific
repair occurred particularly rapidly in early G1 phase (Rampino and Bohr, 1994).
Gene specific repair does not require exactly the same proteins as general DNA
repair, nor does it seem to occur with the same timing in the cell cycle. It is
unclear how important gene specific repair may be in determining which tumors
will be more sensitive to cisplatin.
HMG DOMAIN PROTEINS
The search for proteins that bind to cisplatin-DNA adducts started when
researchers realized that these proteins might have a large effect on cisplatin
31
cytotoxicity. Initially, such proteins were identified by Southwestern blot and
bandshift assays, which detect proteins that bind to cisplatin-treated
radiolabelled DNA. Human cell extracts examined by Southwestern blotting and
bandshift assay contained several such proteins (Chu and Chang, 1988;Toney et
al., 1989; Donahue et al., 1990), some in the 87-100 kDa range and two in the 25-28
kDa range. To identify these proteins, a human cDNA expression library was
screened with cisplatin-modified DNA. The screen yielded the gene for a protein
of unknown function, structure specific recognition protein 1, SSRP-1,which had
an HMG domain (Bruhn et al., 1992). This finding suggested that the 25-28 kDa
band might contain HMG1, another HMG domain protein. The UV-DRP protein
was also initially identified in the bandshift assay. HMG1 binds to cisplatinDNA adducts more tightly than either UV-DRP or XPA (Pil and Lippard, 1992;
Jones and Wood, 1993;Payne and Chu, 1994). Finding a class of cisplatin-DNA
adduct binding proteins led to the study of these proteins and their potential
effects on cisplatin cytotoxicity and resistance.
HMG1 binds to cisplatin-DNA adducts
The 25-28 kDa bands were shown to be HMG1 and HMG2 by selective
immunoprecipitation with anti-HMG1 antibody and by performing parallel
Southwestern and Western blots (Pil and Lippard, 1992). HMG1 contains 2
HMG domains and an acidic tail (Fig. 1.6a). HMG2 is highly homologous to
HMG1; in the mouse, HMG1 and HMG2 genes are 73% identical (Stolzenburg et
al., 1992); Rat HMG1, expressed in Escherichiacoli, bound selectively to
platinated DNA in a bandshift assay (Pil and Lippard, 1992). HMG1 bound
specifically to cisplatin-DNA 1,2-(GpG) and -(ApG) adducts, but not to 1,3(GpTpG) adducts or to monofunctional [Pt(dien)Cl]+-d(G) adducts. Moreover,
HMG1 bound to a site-specific 1,2-(GpG) adduct with a dissociation constant of
3.7x10-7 M, and to the corresponding unplatinated sequence with a Kd of
32
3.4x10-5 M. Another group independently demonstrated that HMG1 and HMG2
bound to cisplatin-modified DNA cellulose, providing further evidence for the
specificity of this interaction (Billings et al., 1992; Hughes et al., 1992).
The fact that HMG1 binds to DNA containing cisplatin adducts is consistent with its interactions with other types of structurally modified DNA, such as
4-way junctions (Fig. 1.6b). As described previously, cisplatin 1,2-intrastrand
cross-links cause the DNA to bend and unwind. Binding of HMG1 was sensitive
to the specific type of bending and the degree of unwinding that occurs for these
different adducts, interacting most selectively with the 1,2-intrastrand crosslinks. HMG1 contacted at least a 15 nucleotide region of DNA in DNAse I
footprinting assays (Locker et al., 1995).
The region of HMG1 responsible for the cisplatin-DNA adduct binding
activity has been investigated. Proteolytically digested HMG1 was used to
determine that HMG domain B, or the combined HMG domains A and B, will
both bind selectively to cisplatin-treated DNA in a Southwestern blot (Chow et
al., 1995). A fragment containing all of domain B plus the acidic tail did not bind
cisplatin-modified DNA, probably because of its negative charge (pI - 4.5).
HMG domain B was separately cloned and found to bind to cisplatin-treated
DNA with the same affinity as HMG1 itself; however, its binding to unmodified
DNA was substantially higher, so the selectivity of domain B for cisplatinmodified DNA was diminished. Similar studies were performed with HMG2
(Lawrence et al., 1993). In this case the amino terminus was found to be essential,
because a fragment containing all but 51 amino terminal residues did not bind to
cisplatin-damaged DNA cellulose. Domain B of HMG2 was also necessary;
removing as few as 30 amino acids from a fragment containing both HMG
domains decreased its binding affinity compared to that of either HMG2 or an
HMG2 fragment lacking only the acidic tail. Both HMG2 domains A and B
33
bound separately, but with very low affinity. These results differ from those for
HMG1, either because of differences in the proteins or in the assay conditions
employed.
HMG domain proteins, a new class of DNA-binding proteins
When a new DNA-binding motif is first announced, it is not unusual to
find it in a large number of related proteins. Such is the case for HMG domain
(Landsman and Bustin, 1993), which has recently joined the list of previously
identified DNA-binding motifs, the zinc finger, helix-turn-helix, and leucine
zipper. The first HMG domain to be identified in a protein other than HMG1
occurred in hUBF, a transcription factor for rRNA (Jantzen et al., 1990). Proteins
containing the HMG domain bend DNA and many of them are transcription factors. HMG domain proteins have been divided into two subfamilies based upon
their ability to bind DNA in a sequence-specific versus a structure-specific
manner (Grosschedl et al., 1994). HMG1 itself typifies the latter category, recognizing structures such as four-way junctions (Fig. 1.6b) and cisplatin-DNA
adducts rather than a specific DNA sequence.
LEF-1, lymphoid enhancer binding protein 1, contains one HMG domain,
binds to the T-cell antigen receptor
(TCRa) enhancer, and participates in gene
expression and differentiation (Travis et al., 1991). The protein bent DNA by
130° when bound to its target site, as revealed by the circular permutation assay
(Giese et al., 1992). LEF-1 contacts AT base pairs primarily in the minor groove;
replacement of AT with IC base pairs, which are identical in the minor groove
but differ in the major groove, had no affect on binding (Giese et al., 1992). Giese
et al. hypothesized that LEF-1might not be a traditional transcription factor.
Instead of activating transcription by interacting with other proteins, LEF-1
might play an architectural role. The bend in DNA induced by LEF-1may bring
distant elements of the promoter into closer contact, permitting proteins bound
34
to those elements to interact and activate transcription. Further experiments
demonstrated that the HMG domain of LEF-1could not substitute for the entire
protein in activating transcription at the TCRocenhancer (Giese and Grosschedl,
1993). In addition, replacement of the HMG domain by the LexA DNA binding
domain and of the LEF-1binding site by the LexA binding site diminished but
did not abolish TCRLoenhancer activity. Since LexA does not bend DNA, the
non-HMG domains of LEF-1 must participate in activating transcription. Thus,
LEF-1 activates transcription both by bending DNA, a property of its HMG
domain, and by interacting with other proteins.
Another extensively studied HMG domain protein is hSRY,the sex
determining factor in humans. When the mouse analogue, mSRY,was expressed
in a female embryo it developed into a male mouse (Koopman et al., 1991). hSRY
bound to four-way junctions (Fig. 1.6b) as well as to AACAAAG and related
sequences (Ferrari et al., 1992;Giese et al., 1994). Human SRYbound predominantly in the minor groove, as shown by methylation interference experiments
and by replacing AT with IC base pairs (van de Wetering and Clevers, 1992).
Mouse SRYhas a significantly different amino acid sequence and formed more
extensive major groove contacts than hSRY (Giese et al., 1994). Two dimensional
NMR studies of hSRY revealed partial intercalation of an isoleucine residue
between two AT base pairs in the minor groove (King and Weiss, 1993). SRY
bends DNA by 83-85° when it binds to its recognition sites (Ferrari et al., 1992;
Giese et al., 1992;Giese et al., 1994). Binding of the HMG domains of mSRY and
hSRY to two different sequences was compared, the former bending DNA by 2030° more than the latter (Giese et al., 1994). A family of proteins related to SRY
called Sox bound the same sequence and, in one case, activated transcription
(Denny et al., 1992;van de Wetering et al., 1993). Sox5 bends DNA by about 75°
(Connor et al., 1994). SRYitself bound to upstream elements in the promoters of
35
two sex-specific genes (Haqq et al., 1993). Mutations in SRY that cause sex
reversal affect the ability of SRY to bind DNA properly (Pontiggia et al., 1994;
Werner et al., 1995). One of these mutant SRYproteins could still bind to 4-way
junction DNA, so only the site-specific binding activity was abolished (Peters et
al., 1995). Thus, much like LEF-1, SRY binds DNA in the minor groove, bends it,
and activates transcription.
In addition to SRY, there is another testis-specific HMG domain protein in
mice, tsHMG1 (Boissonneault and Lau, 1993). tsHMG1 was isolated by screening
a testis library with the testis-specific promoter from PGK-2. It contains two
HMG domains and binds to 5'-AGGTTTTTACAT-3'.This protein was expressed
in postpuberal testis, whereas SRYwas expressed only in early development.
tsHMG1 binds to cisplatin-DNA adducts (Raju, NL, Whitehead, JP, unpublished
data).
hUBF is a transcription factor for rRNA production which consists of five
HMG domains and an acidic tail (Jantzen et al., 1990). As found in other HMG
domain proteins, one or more of the boxes was necessary for DNA binding
(Maeda et al., 1992). hUBF interacted with a specific subunit of RNA polymerase
I (Schnapp et al., 1994),and the second HMG domain affected the role of hUBF in
activating transcription and counteracting histone H-mediated repression
(Kuhn et al., 1994). hUBF also bends DNA, possibly blocking H1 repression
because DNA is wrapped around hUBF in the opposite direction from the way it
is wrapped around the histones (Putnam et al., 1994).
The HMG domain protein SSRP-1(structure specific recognition protein
1), originally cloned because it binds to cisplatin-modified DNA (Bruhn et al.,
1992), interacts with a variety of different sequences. The mouse SSRP-1(also
known as T160) bound to recombination signal sequences used when cells are
performing V-D-J recombination (Shirakata et al., 1991). The chick and rat homo-
36
logues of SSRP-1 bound to the collagen II gene promoter (Wang et al., 1993). The
true function of SSRP-1is uncertain. Not only was SSRP-1present at significant
levels in all tissue types that were screened (Bruhn et al., 1992),but it was also
well conserved across the species; the Drosophila homologue is 54% identical to
the human protein (Bruhn et al., 1993;Hsu et al., 1993). Thus it seems unlikely
that SSRP-1 is a specific factor for collagen II gene transcription or for V-D-J
recombination, since these functions are specific to certain cell types. SSRP-1 also
binds to the c-Myc oncoprotein, further complicating its potential function
(Bunker and Kingston, 1995).
A variety of other HMG domain proteins bind and bend DNA (Diffley
and Stillman, 1992;Fisher et al., 1992;Dooijes et al., 1993). The bending seems to
be a property of the HMG domain, irrespective of whether it occurs in a
sequence- or a structure-specific recognition protein. Any differences between
these various proteins must result either from the exact degree of the bending or
from other domains.
HMG1 binding to DNA
HMG1 is a 25 kDa protein with two HMG domains (A and B) and an
acidic tail (Bustin et al., 1990) (Fig. 1.6). The structure of HMG1 domain B (Fig.
1.7), determined by two dimensional NMR spectroscopy (Read et al., 1993;Weir
et al., 1993), is comprised of three
helices folded into the shape of the letter L.
The DNA-binding properties of HMG1 have been extensively investigated
(Landsman and Bustin, 1993; Grosschedl et al., 1994). Briefly, HMG1 bound to
single-stranded DNA, mono- and di-nucleosomes, cruciforms, 4-way junctions,
and cisplatin intrastrand cross-links (Fig. 1.6). The common element among
these varieties of structurally deformed DNA is unclear, in part because many of
the DNA-binding experiments were carried out under different conditions. Most
37
of the DNA motifs that bind HMG1 have single-stranded character, regions
where the DNA is somewhat underwound, or bent double-stranded structures.
Several experiments have revealed that HMG1 alters the structure of
DNA. HMG1 binding changed the linking number of negatively supercoiled
DNA and prevented its relaxation by topoisomerase I (Sheflin and Spaulding,
1989). A proteolytic fragment of HMG1, missing only the acidic tail, supercoiled
DNA in the presence of topoisomerase; this fragment was more active than
HMG1 itself at high ionic strength (Stros et al., 1994). Electron microscopic
experiments confirmed this result and demonstrated that high levels of the HMG
domains alone compact DNA in much the same manner as histones (Stros et al.,
1994). The acidic tail of HMG1 decreased the ability of the protein to bend DNA,
or at least its ability to bind to the DNA. Sheflin et al. (Sheflin et al., 1993)determined that HMG1 unwinds DNA by 58°, whereas the proteolytic fragment
lacking the acidic tail unwound it by only 10°. The DNA binding sites of the two
proteins were estimated by increasing their concentrations to the point above
which they failed to increase the superhelicity. At this amount of bound protein,
which corresponds to saturation binding, HMG1 covered approximately 20 base
pairs whereas the proteolytic fragment only spanned 4 base pairs. In these
experiments, the acidic tail interfered with binding to DNA.
Modifying the cysteine sulfhydryl groups of HMG1 with N-ethylmaleimide prevented HMG1 from binding as effectively to negatively supercoiled DNA (Sheflin et al., 1993). Since there are no cysteines in the acidic
domain, this result indicated that cysteine residues in the HMG domains are
involved in the interaction of HMG1 with negatively supercoiled DNA. Binding
of HMG1 to single-stranded DNA, and of the proteolytic fragment to any form of
DNA, were unaffected by cysteine modification, so the acidic domain must
38
influence whatever change is created by modifying the cysteine sulfhydryl
residues within the HMG domain.
The naturalfunction of HMG1
The possible functions of HMG1 have been extensively debated in the
literature. One hypothesis is that HMG1 affects the location of nucleosomes on
DNA, thus regulating the regions accessible to transcription. Several experiments support this theory. HMG1 suppressed nucleosome assembly at physiological ionic strength, probably due to its binding the DNA (Waga et al., 1989).
HMG1 also bound to histone H1, as monitored by the quenching of HMG1
tryptophan fluorescence (Kohlstaedt and Cole, 1994). Histone H1 interacted with
the nucleosome core to compact the DNA. The exact state of the HMG1 substantially affected its binding to H1, as did pH changes. At pH 6.0, HMG1 bound as
a tetramer with a dissociation constant of 3.4x10-8 M , whereas at pH 7.5
monomeric HMG1 bound with a dissociation constant less than 10-9 M. Histone
H1 inhibited the transcription but not the replication of DNA (Halmer and Gruss,
1995). HMG1 and HMG2 competed with histone H1 for binding to 4-way
junctions. Four-way junction DNA is a reasonable model for the crossover of
DNA at the entry and exit of nucleosomes. This result indicates that HMG1 may
replace H1 to activate transcription (Varga-Weisz et al., 1994). In early Drosophila
embryogenesis, the HMG1 analogue, HMG-D, was expressed instead of histone
H1 (Ner and Travers, 1994). As the embryo developed, the volume of the
nucleus decreased and the length of the nuclear cycle increased. This alteration
in chromatin occurred concomitantly with increased expression of H1 and with a
decrease in HMG-D expression. The less dense chromatin structure associated
with HMG-D expression may be required for the rapid nuclear cycles in early
development. These experiments reveal the ability of HMG1 to modulate
chromatin structure.
39
HMG1 has also been implicated as a regulator of gene transcription. Since
other HMG domain proteins are transcription factors, HMG1 might affect
transcription by preventing their binding to DNA (Landsman and Bustin, 1993).
It has also been proposed that HMG1 itself activates transcription. In an attempt
to evaluate this hypothesis, the ability of the protein to function in one of the
standard transcriptional activation assays was investigated. An HMG1-LexA
binding domain fusion protein could not activate transcription from the lexA
operator (Landsman and Bustin, 1991). This result does not prove that HMG1 is
not a transcriptional activator. The fusion protein interacted non-specifically
with DNA through its HMG domain, so it was impossible to ascertain whether
the construct bound correctly to its promoter. Other evidence suggests that
HMG1 is required for transcription of a number of genes. Incubation of HeLa
nuclear cell extracts with antibodies to HMG1 inhibited run-off transcription of
four different promoters (Singh and Dixon, 1990). A related study indicated that
the protein was required for initiation of transcription rather than elongation of
the RNA (Tremethick and Molloy, 1988).
HMG1 may promote transcription by helping other factors to bind DNA
and activate RNA polymerase. HMG1 increased the rate of binding of MLTF to
its site on the adenovirus major late promoter (Watt and Molloy, 1988). It also
increased the ability of the progesterone receptor (PR) to bind DNA and activate
transcription (Onate et al., 1994). Addition of HMG1 increased the DNA affinity
of PR by at least an order of magnitude for perfectly palindromic binding sites
and by much more for imperfect sites. PR and HMG1 co-immunoprecipitated
from the binding complex, although HMG1 was not apparent in gel mobility
shift assays. Onate et al. (1994)also tested various domains of HMG1 and other
HMG domain proteins for similar activity. The B HMG domain of HMG1 was
nearly as effective as HMG1 itself, whereas the A domain only weakly enhanced
40
PR binding. A construct including both the A and B domains but not the acidic
tail failed to enhance binding. LEF-1and SRYsimilarly did not enhance PR
binding, even though they did bind to the DNA. Thus, HMG1 facilitated the
binding of PR by inducing a structural change in the target DNA, and this
activity was specific to HMG domain B of the protein. HMG1 stimulated
transcription in COS-1 cells, but only if the acidic tail was intact (Aizawa et al.,
1994). The closely related HMG2 protein interacts with the octamer transcription
factors Octl and Oct2 to assist in their sequence-specific binding, which results in
increased transcription (Zwilling et al., 1995). These data indicate that HMG1
and HMG2 may activate transcription by assisting other proteins.
In summary, these experiments are consistent with the notion that HMG1
is a kind of nuclear chaperone protein which, perhaps by bending and unwinding the duplex, creates an enhanced site for transcription-factor binding and activation of gene expression (Ner et al., 1994).
HMG1 bendscisplatin-treatedDNA
Since HMG1 bends unplatinated DNA, it is no surprise that it also bends
cisplatin-modified DNA. This behavior was first measured by the ligasemediated circularization assay (Pil et al., 1993), an experiment in which it is not
feasible to quantitate the bend angle. In order to obtain this information, it was
necessary to study HMG1 binding to single, site-specific cisplatin-DNA adduct.
Experiments examining the bend angle induced by HMG1 and other HMG
domain proteins binding to a single cisplatin-1,2-(GpG) adduct were performed
by using the circular permutation assay (Chow et al., 1994). Site-specific probes
were successfully employed to calculate the bend angles for HMG1 and several
HMG domain containing proteins bound to the cisplatin-modified DNA. The
bend angles ranged from 70° to 85°;most of the HMG domain proteins had
values closer to 70°, whereas HMG1 itself bent the platinated DNA by 85°. HMG
41
domain B bent the DNA to a degree similar to that of the HMG domain proteins.
These bend angles are of the same magnitude as that found previously for SRY
(Ferrari et al., 1992; Giese et al., 1992; Giese et al., 1994).
HMG domainproteinsand cisplatinantitumor activity
There are several ways in which the binding of HMG1 and other HMG
domain proteins to cisplatin-DNA adducts might affect the biological activity of
cisplatin (Donahue et al., 1990;Lippard, 1994). First, the HMG domain proteins
may themselves be part of the excision repair apparatus of the cell. In order for
excision repair to occur, some protein or other cellular component must
recognize the damage created by the cisplatin adducts. None of the excision
repair proteins which have been sequenced to date have an HMG domain. A
second possibility is that the HMG domain protein may block the cisplatin-DNA
adducts from recognition by the repair apparatus (Fig. 1.8). If the HMG domain
protein does prevent repair of the adduct, then cells could become resistant to
cisplatin by lowering the levels of the protein. A third possibility is that the
cisplatin titrates or "hijacks" the protein away from its natural binding sites (Fig.
1.9). Assuming that the hijacked protein is in some manner vital to the tumor
cell, its diversion could sensitize the cells to cisplatin. Alternatively, the hijacked
protein may cause transcriptional activation or repression in the area of the
cisplatin binding sites, thus disrupting normal gene regulation of the genes in
their vicinity. A few studies have reported that the levels of cisplatin-DNA
adduct-binding proteins increase or decrease when cells become more resistant
to cisplatin treatment. No such effect has yet been observed in actual tumor cells,
however, and the results reported by different laboratories are inconsistent
(Chao, 1991; Bissett et al., 1993; McLaughlin et al., 1993).
These hypotheses, which are not mutually exclusive, have been subjected
to a number of experimental tests. Treiber et al. (Treiber et al., 1994) evaluated
42
the hijacking hypothesis in vitro by using the HMG domain protein hUBF. The
dissociation constant for hUBF binding to a site-specific cisplatin intrastrand 1,2(GpG) cross-link was measured to be 60 pM by analysis of a DNAse I footprint
assay. The Kd for the binding of hUBF to its normal site on DNA, the rRNA
promoter, was of the same order of magnitude, 18 pM. Additional footprinting
experiments revealed that hUBF binds cooperatively to the rRNA promoter. Any
decrease in hUBF levels could have a significant effect on rRNA gene transcription. Footprinting analysis was performed on the rRNA promoter element in the
presence of increasing amounts of cisplatin-treated DNA. At concentrations of
cisplatin substantially lower than those in treated cells, the binding of hUBF to its
promoter site was abolished. In cells treated with cisplatin, hUBF may be
diverted from its natural target sites. Since rRNA is a vital element for cell
survival, down-regulation created by cisplatin treatment might contribute to the
toxic effects of cisplatin. Such a conclusion assumes, of course, that the cell does
not respond by increasing the levels of hUBF expression. Experiments to test the
hijacking hypothesis in vivo are required to evaluate further these possibilities.
The repair shielding hypothesis has been tested in two sets of
experiments, one employing cell extracts and the other, yeast cells. The in vitro
studies were performed with HeLa and other human whole cell extracts (Huang
et al., 1994). A 156-bp linear piece of DNA radiolabeled near a site-specific
platinum intrastrand 1,2-(GpG)or 1,3-(GpTpG) adduct was incubated with the
cell extracts and the resulting mixture was resolved on a polyacrylamide gel.
Bands of approximately 29 bp form for both adducts only when the cell extracts
had all the components of the excision repair excinuclease system. When
relatively high levels of HMG1 were added to the extracts, less repair of the
cisplatin 1,2-(GpG) adduct occurred, presumably because the protein bound to
the platinated DNA and blocked repair. HMG1 had no effect on repair of the 1,3-
43
(GpTpG) adduct, which is reasonable since HMG1 does not bind this adduct.
Previous in vitro excision repair studies employing the repair synthesis assay did
not reveal the ability of the system to excise the 1,2-(GpG) adducts (Szymkowski
et al., 1992). The repair synthesis assay measures the incorporation of
radiolabelled nucleotides into repair patches on the damaged strand of sitespecifically platinated plasmid DNA. The background in this assay can be higher
due to random nicking events which may have obscured the signal. In addition,
the difference may reflect some difference in the cell extracts. In any event, it is
clear from the excision assay that the major adducts of cisplatin on DNA are
effectively removed by the human excinuclease system. More importantly, this
work established the feasibility of the repair shielding hypothesis in a defined in
vitro investigation.
Yeast as a model system
Perhaps the best system yet discovered for revealing how HMG domain
proteins might affect the activity of cisplatin is in yeast. Several yeast HMG
domain proteins have been identified, one by using the same expression library
screening methodology previously employed to identify human SSRP-1(Brown
et al., 1993). The yeast protein, designated Ixrl for intrastrand cross-link recognition, was identified in Saccharomycescerevisiaecell extracts by a Southwestern blot
using cisplatin-modified DNA as the probe (Brown et al., 1993). Ixrl binds to
both globally platinated DNA and site-specific cisplatin 1,2-(GpG) cross-links in
gel mobility shift assays (Whitehead and Lippard, 1995). It bent DNA containing
a site-specific cisplatin adduct to the same extent as other HMG domain proteins,
as determined by the circular permutation assay (Chow et al., 1994).
Experiments were performed to determine exactly how Ixr might have
altered the response of yeast cells to cisplatin. S. cerevisiaeis a useful eukaryote
because it is relatively easy to delete its genes (Fig. 1.10). When the IXR1 gene
44
was interrupted, the resulting ixrl strain was significantly less sensitive to
cisplatin than the wild type, IXR1, strain (Brown et al., 1993). This finding is
inconsistent with the theory that Ixrl might be an adduct-recognition component
of the excision repair apparatus since removing an component of DNA repair
would make the cells more sensitive to cisplatin. The result could be explained if
Ixrl regulates repair proteins and its absence lead to an increase in their
expression, but this is unlikely since the UV sensitivity of the two lines was the
same. The result supports the theory that Ixrl may block the access of repair
proteins to the platinum adducts on DNA. In such a case, deletion of the gene
encoding the Ixrl protein would lead to greater repair and enhance the survival
of cells treated with cisplatin.
Saccharomycescerevisiaehas several different HMG domain proteins. It has
structure specific proteins like HMG1 in both the mitochondria (ABF2)and the
nucleus (NHP6a/b) (Kolodrubetz and Burgum, 1990;Diffley and Stillman, 1992).
These proteins can substitute for each other if they are expressed in the correct
location (Kao et al., 1993). Additionally, the bacterial HU and human mtTFA
proteins can substitute for ABF2,indicating that ABF2does not function as a sitespecific DNA binding protein (Megraw and Chae, 1993;Parisi et al., 1993).
NHP6a/b have been shown to bend DNA in the same way HMG1 does (Paull
and Jonson, 1995). NHP6a/b act in the MAPK pathway which responds to
extracellular signals; they may help activate transcription in a manner similar to
HMG1 (Costigan et al., 1994). Yeast contain other nonspecific DNA binding
proteins with HMG domains, such as SIN1, which is involved in regulation of
HO transcription, (Kruger and Herskowitz, 1991). Roxl is a site-specific DNA
binding HMG domain protein. It binds to the sequence ATTGTTCTCand
represses transcription of hypoxic genes in an oxygen and heme dependent
manner (Balasubramanian et al., 1993). It is not clear whether Ixrl is a sequence
45
or structure specific protein; it binds to the Cox5b promoter region, but the exact
site of binding has not yet been determined (Lambert et al., 1994). Ixrl, also
known as ORD1, represses transcription of COX5b from the Cox5b promoter
region.
SUMMARY AND CONCLUSIONS
The HMG domain is a DNA binding motif that bends DNA. Many of the
proteins that harbor this domain are transcription factors whereas others are
thought to regulate structural properties of DNA in the nucleus. The HMG
domain proteins also bind to cisplatin-modified DNA and, as a class, may affect
the cytotoxicity of the drug. Cisplatin-DNA adducts at therapeutic levels can
titrate hUBF away from its normal binding site in vitro. HMG1 blocks excision
repair in human cell extracts. Removal of the gene encoding Ixrl desensitizes the
yeast to cisplatin. These results and the results detailed in the following chapters
suggest that one strategy for improving the antitumor activity of platinum drugs
would be to synthesize cisplatin derivatives that can form DNA adducts which
would bind HMG domain proteins with greater affinity. These compounds
should be more toxic because of the diminished capacity of the cells to repair the
adducts. It is not yet clear that this approach would selectively sensitize cancer
cells. Since one of the main diseases treated by cisplatin is testicular cancer, it is
interesting that the testis-determining factor SRYis an HMG domain protein.
Perhaps the state of SRY or other HMG domain proteins in solid tumors of the
testes differs from that in normal cells. Investigation of the various HMG
domain proteins and their ability to influence excision repair in testis and other
tissues should lead to better and broader cisplatin based chemotherapy. Before
any of the complicated mammalian cell culture and tumor tissue experiments are
performed, it is important to check the various hypothesis in a simpler system. S.
cerevisiaeis one of the simplest eukaryotes, and its genetics have been well
46
defined. Many of its vital systems contain proteins with homologous
mammalian counterparts, including the excision repair system. The yeast HMG
domain protein Ixrl affects cisplatin cytotoxicity. The use of this system to
investigate models for how HMG domain proteins affect cisplatin cytotoxicity is
described in subsequent chapters.
47
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67
68
Figure 1.1. Structure of various platinum compounds. a) Cisplatin or cisdiamminedichloroplatinum(II). b) [Pt(en)C12]or ethylenediaminedichloroplatinum(II). c) Carboplatin. d) trans-DDPor trans-diamminedichloroplatinum(II). e) [Pt(dien)C1]+or diethylenetriaminechloroplatinum(II)
69
Active antitumor complexes
a)
b)
H3 N
C1
/-7
H2N
NH2
Pt
H3N
Pt
C1
C1
C1
Cisplatin
Pt(en)C1 2
O
c)
H3N
Pt
H3N
O
O
0
Carboplatin
Inactive complexes
d)
+
C1
H3N
e)
H2 N
Pt
C1
NH
Pt
NH3
trans-DDP
C1
NH 2
Pt(dien)Cl+
70
Figure 1.2. Adducts formed by cisplatin adducts. a) GG and AG intrastrand
adducts. Structure was adapted from Sherman et al. (1985)and represents
cisplatin bound to single-stranded d(pGpG). b) GXG intrastrand adducts. c)
Interstrand adducts. d) DNA-protein cross-links. e) Monofunctional adducts.
71
a) GG, AG crosslinks
G
GI
G
A
)NA
32-34°
130
_NH 3
Pt
N NH
K
/
3
/NH 3
Pt
NH3
b) GXG crosslinks
Intrastrand adducts (90-95%)
c) Interstrand adducts (1-5%)
N [H3
/NH 3
Pt
NH 3
G,
G
I
G
NH 3
Pt-NH
3
I
G
Pt
ili:2.>
,.,.,,,::,,.-...
NH3
. -.
d) DNA-protein crosslinks (< 1%)
H20
e) Monofunctional adducts (<1%)
72
Figure 1.3. Types of cancer that cisplatin therapy is effective against. Cisplatin is
used on its own or in combination with other drugs to treat all the marked types
of cancer.
73
and
Cancer
reast
7ancer
Small Cell
Lung Cance
Ovarian
Cancer
Advan(
Bladdel
Cancer
Cer
Carcinoma
dometrial
.ncer
sticular
ncer
Leukemia
74
Figure 1.4. Excision repair. The excision repair process begins with adduct
recognition and incision of the DNA; these steps are performed by the proteins
detailed in Fig. 1.5. The resulting gap is filled by DNA polymerases and closed
by DNA ligase.
75
-~-~
Cisplatin
GG
.0-
GG
dduct is recognized
air proteins
-4 >
Adduct is excised by
repair proteins
d
Gap is filled in by polyl
and closed with
GG
76
Figure 1.5. Excision repair proteins. Human, rodent and yeast proteins are
highly similar; not all proteins in each species have been cloned. The picture at
the bottom gives some indication about the interaction of the yeast excision
repair proteins. These interactions are described in more detail in chapter 3.
77
Human
Rodent Yeast
Radl4
XP-A (XPAC)
Comments
XPACbinds cisplatin and uv
damaged DNA. Rad 14 binds
uv damaged DNA.
XP-B (XPBC)
ERCC3
Rad 4
XP-C (XPCC)
XP-D (XPDC)
Rad25
ERCC2
Rad3
Bandshifts cisplatin damaged
DNA.
XP-E (XPEC)
XP-F
XP-G (XPGC) ERCC5
Rad2
CS-A
CS-B
ERCC6
ERCC1
Rad 10 Removing ERCC1 affects
cisplatin adduct repair.
Rad 1
Proteins for
yeast DNA
excision repair
'Components
of transcription
factor TFIIH
78
Figure 1.6. HMG1 and HMG domain protein DNA binding sites. A) a schematic
view of the three domains of HMG1. B. The various types of DNA bound by
HMG domain proteins. Not all types of DNA are bound equally well.
79
A.
HMG domains
A
acidic
tail
B
E'//////////7//17/I//
B.
four-way junction
single-stranded DNA
AA CAAAG
TT
specific sequence
Pt
H3 N
small circular DNA
NH 3
cisplatin-DNA adducts
80
Figure 1.7. Structure of HMG1 domain B as determined in a two-dimensional
NMR spectroscopic study. The amino terminus is in the upper right-hand
corner. The drawing was made with Molscript (Kraulis 1991) from coordinates
kindly supplied by C. Read and C. Crane-Robinson.
81
\ .::
U~
82
Figure 1.8. Repair shielding theory. HMG domain proteins may bind the
cisplatin-DNA adduct and shield the adduct from the repair protein complex.
83
Repair
--------P-
1
Repair
Complex
I
HMG
domain
protein
Repair
Blocked
85
86
Figure 1.9. Titration hypothesis. HMG domain proteins bind to their normal site
and activate or repress the transcription of gene X. When cells are treated with
cisplatin, HMG domain proteins may be titrated away from their natural binding
sites and the transcription of gene X is not properly regulated.
87
,.
HMG
domain
protein
-10 4 addu
per cell
Small number
of normal
binding sites
Transcription activation or
repression of gene X
Protein not bound to
normal site so the
transcription of gene X is
not properly regulated
89
90
Figure 1.10. Interruption of genes in Saccharomycescerevisiae.The yeast cell used
lacks a particular selectable marker, in this case the gene for Leu2, a protein
involved in leucine biosynthesis. These cells require leucine to grow. An
interrupted copy of the IXR1 (or any other) gene was constructed on a plasmid,
which was cut to give a piece of DNA that the yeast cells could not replicate. The
plasmid fragment was transformed into the yeast strain. Recombination between
the plasmid fragment and the yeast genomic DNA occurred at a low level. Yeast
strains where recombination has occurred have a viable copy of the LEU2 gene.
These cells are selected by growth on media lacking leucine.
91
Yeast cell (leu2)
/
7- x
IXR -IXR1
"4,24
Plasmid fragment
ixrl
Transform
with plasm
' IXR1
LEU2
occurs
LE U2
Select for yeast cells with LEU2 gene
by growing on media lacking leucine
3' IXR1
92
93
CHAPTER 2
Cisplatin and yeast cells
94
INTRODUCTION
The difference in cisplatin sensitivity between yeast cells with the gene for
HMG domain protein Ixrl (IXR1) and those without (ixrl) indicates that Ixrl
mediates cisplatin cytotoxicity (Brown et al., 1993). Further investigation of Ixrl
(Fig. 2.1a) and other HMG domain proteins may help in designing better
cisplatin-related antitumor drugs. A series of experiments was performed to
determine what aspect of Ixrl causes the difference in cisplatin sensitivity. The
lack of sensitivity of the ixrl strain could be due to the cisplatin-DNA adduct
binding properties of Ixrl or it could be related to the actual function of Ixrl.
Yeast HMG domain proteins
The ability of two other SaccharomycescerevisiaeHMG domain proteins to
bind to cisplatin-DNA adducts was investigated. ABF2is made in the
mitochondria, but it is present in both nucleus and mitochondria (Diffley and
Stillman, 1992;Megraw and Chae, 1993). Roxl (Fig. 2.1b) belongs to the other
class of HMG domain proteins, those that bind specific sequences and activate or
repress transcription (Balasubramanian et al., 1993). Roxl represses transcription
from the sequence YYYATTGTTCTCon a number of proteins used for
respiration under hypoxic conditions (Lowry and Zitomer, 1988;Lowry et al.,
1990).
Naturalfunction of Ixrl
Ixrl and Roxl both regulate the transcription of cytochrome c oxidase
subunit V (CoxV) (Zitomer and Lowry, 1992;Lambert et al., 1994). There are two
isoforms of CoxV, CoxVa, which is used under aerobic conditions, and CoxVb,
which is used under hypoxic conditions (Hodge et al., 1989). The genes encoding
the CoxV proteins are called COX5a and COX5b.
Expression of the CoxV isoforms is regulated by several different proteins
(Fig. 2.2). Ixrl represses the expression of the COX5b gene but does not affect the
95
expression of COX5a (Lambert et al., 1994). The conditions which relieve the Ixrl
repression are unknown. Roxl represses the expression of COX5b in an oxygendependent manner (Zitomer and Lowry, 1992). Both ixrl and roxl mutants
express COX5b. Both genes are repressed by glucose, COX5a by HAP2/3/4 and
COX5b directly by MIG1 (Zitomer and Lowry, 1992;de Winde and Grivell, 1993).
TUP1 and SSN6 are involved in both oxygen and glucose regulation of proteins;
they activate different proteins based on which stimuli are present (Williams and
Trumbly, 1990;Zhang et al., 1991;Trietel and Carlson, 1995).
There are other proteins used under hypoxic conditions in the respiration
pathway, which indicates how important such proteins are to the cells.
Cytochrome c is encoded by two isoforms, CYC1 and CYC7 (Laz et al., 1984).
CYC1 is used under normal conditions whereas CYC7 is used under hypoxic
conditions. These proteins are regulated by using some of the same systems as
COX5a and COX5b (Zitomer and Lowry, 1992). Neither these two nor any of the
other hypoxic or aerobic respiration genes investigated were regulated by Ixrl
(Lambert et al., 1994). The cell seems to need different isoforms of some proteins
when the oxygen level is low. This phenomenon is also present in mammalian
cells (Wang and Semenza, 1993). Yeast CoxVb stimulated both higher turnover
rates and higher rates of heme oxidation by cytochrome c oxidase (Waterland et
al., 1991), probably because subunit V affects ligand binding at the oxygen site
and alters the heme a environment (Allen et al., 1995). When the pairs of
cytochrome c-cytochrome c oxidase proteins used under aerobic and hypoxic
conditions were compared, they had very similar activities (Allen et al., 1995).
The changes that Cyc7 caused counterbalanced the changes that CoxVb caused.
It is possible that the cell only wants to make small, subtle changes in this
pathway under hypoxic conditions.
96
Growthof yeast
S. cerevisiaecan grow in several different manners (Ausubel et al., 1994),
and such differences may affect the cisplatin cytotoxicity assay. Yeast can be
either haploid or diploid; all of the strains examined in this thesis were haploid
strains. Yeast can either ferment carbon sources such as glucose, or it can
aerobically respire by using its mitochondria. When there is glucose (dextrose) in
the media, many genes are repressed, including the COX5 genes (Fig. 2.2). Ixrl is
present in cells grown on dextrose. Yeast can also ferment other carbon sources
such as galactose, but they prefer glucose. Aerobic respiration only occurs if the
yeast are grown on non-fermentable substrates such as lactate, glycerol, and
ethanol. Cytochrome c oxidase is required for aerobic respiration. When yeast
are grown in liquid media with dextrose, they enter and go through exponential
growth by fermenting the dextrose. When the dextrose is exhausted, they
undergo a diauxic shift to aerobic respiration. The postdiauxic phase then lasts
for several days during which time the cells grow substantially more slowly.
After 7-8 days, the yeast cells enter stationary phase, which is characterized by
cell cycle arrest (Werner-Washburne
et al., 1993).
The difference in cisplatin sensitivity between IXR1 and ixrl strains was
seen only when cells were grown past exponential phase (S. Brown, personal
communication and data presented here). The cells were not grown long enough
to have entered into stationary phase. Therefore, it is important to consider what
changes may have occurred between exponential growth and the time when the
assay is conducted. The conditions under which the difference in cisplatin
sensitivity is measurable may give some important indications as to how Ixrl
affects cisplatin cytotoxicity. Even if no such information is found, it is important
to understand under what conditions this assay functions so that other studies
can be appropriately designed.
97
Ixrl experiments
Experiments were performed to investigate what features of Ixrl and the
yeast cells are involved in the making ixrl cells less sensitive to cisplatin than
IXR1 cells. The effect of interrupting IXR1 on the cisplatin sensitivity of yeast
cells was confirmed in two different backgrounds of yeast. Other yeast HMGdomain proteins were investigated for cisplatin binding. Ixrl was overexpressed
in ixrl cells to show that it is actually the lack of Ixrl that is causing the
difference. Cytotoxicity assays were performed on cells with mutant forms of
Ixrl, Cox5a interruptions, and Roxl interruptions, demonstrating that the
difference in cisplatin cytotoxicity is not due to the ixrl cells having higher levels
of CoxVb. Finally, cytotoxicity assays were performed under a number of
different growth conditions to determine what effect the state of the yeast cells
might have on the cisplatin sensitivity of IXR1 and ixrl strains. These
experiments indicate that it is the HMG domain of Ixrl that is crucial, not its
function in repressing COX5b expression.
MATERIALS AND METHODS
Yeast Strains and Plasmids
The yeast strains in Table I were obtained or constructed. Plasmid pMet is an
Escherichiacoli plasmid that includes the Met promoter in the polylinker and was
obtained from the laboratory of G. Fink, M.I.T.. The ABF2 knockout plasmid,
pAMI::Trpl, was obtained from the laboratoryof B. Stillman, Cold Spring
Harbor. The IXR1 interruption plasmid pSB5 and pSB4, the E. Coli plasmid
containing the IXR1 gene were described in Brown, et al. (1993). pCD43,
obtained from the laboratory of L. Guarente, M.I.T., and pYes2 (Invitrogen) were
yeast expression plasmids. Plasmid pSL301 (Invitrogen) was a plasmid with a
superlinker containing 50 restriction enzyme sites. pCox5a was a yeast
98
expression plasmid containing the COX5a gene linked to its genomic promoter,
and was obtained from the laboratory of M. Cumsky, U.C. Irvine.
Constructionof IXRl Aabf2and BWGAixrl strains
The two genes were disrupted by transforming the wild type strains with
plasmid fragments containing the gene interrupted by a selectable marker
(Ausubel et al., 1994). Cells with the gene disruptions were selected by growing
the transformed cells on synthetic media lacking either leucine or tryptophan
such that only the presence of the interrupting gene permitted cell growth. The
construction of the BWGAixrl strain was confirmed by Southwestern blot (Pil
and Lippard, 1992), which examines the level of protein present by running an
SDS-PAGE gel, transferring the proteins to nitrocellulose and probing the
nitrocellulose with radiolabelled DNA modified by cisplatin. The strain was also
examined by Southern blot (Ausubel et al., 1994). The blot was probed with a
fragment of IXR1 that is present in both the interrupted and wild-type genes.
The construction of IXRlAabf2was confirmed by Southwestern blotting.
Bandshift assay
Bandshift assays were performed with a 123 bp probe treated with various
DNA damaging agents in the presence and absence of Ixrl. The 123 bp probe
was treated with cisplatin to an (Drug/Nucleotide)bound ((D/N)b) of 0.022, with
trans-DDPto an (D/N)b of 0.053, with [Pt(dien)Cl]+ to an (D/N)b of 0.07, and
with UV light by irradiating on ice with 10,000Joules/m 2 at 254 nm with a
germicidal lamp. The [Pt(dien)Cl]+ and UV light treated probes were
constructed by Pieter Pil (thesis, 1993). The bandshift was performed (Whitehead
and Lippard, 1995) and run on a 7.5% non-denaturing polyacrylamide gel.
Cytotoxicity assays
Yeast cells were grown to saturation in 20 mL of YPD media (Ausubel et
al., 1994) for approximately 64 hours at 30°C. Portions containing 2x10 7 cells of
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each strain were centrifuged to remove the media and resuspended in 1 mL of
SD media, pH 6.5 (Ausubel et al., 1994) plus 0 to 1 mM cisplatin. The cells were
incubated in an Eppendorf tube with gentle rocking for 2 hours at 30°C. The
medium containing cisplatin was removed by pelleting the cells, which then
were resuspended in SD medium, diluted, and plated at 20 to 200 cells per plate.
Three identical YPD plates were made for each level of platination. The plates
were grown for 3 days in a 30°C incubator. Colonies on the plates were counted
and the percent survival at the various concentrations was calculated relative to
controls treated identically except for the lack of cisplatin. Each strain was
assayed in three to five separate experiments and results from a representative
experiment are shown in the figures. The error bars in the percent survival plots
show a 2a level derived from colony counts on the three identical plates and do
not represent multiple experiments. The data were evaluated by fitting a line to
the log of the percent survival. The ln(% survival) was used because, when a
least squares fit was attempted to percent survival data, points below 1%
survival were insufficiently weighted. The slope of the line corresponds to k in
the expression % survival=exp(-k[cisplatin]) used to fit the data. The reported
values for k are an average of three to five separate experiments. Variations to
this procedure are detailed below and in the Results section. The same medium
was always used for growing cells and for plating the cells. Experiments where
the cells were grown in media without dextrose were treated in SD media using
the appropriate sugars instead of dextrose.
Constructionof Ixrl expressionplasmids
pYY (pYes2-Ixrl) and pCY (pCD43-Ixrl) were constructed by placing the
IXR1 gene downstream from the GALl promoter (Fig. 2.3, Table II). pCD43 has a
CEN origin and is present at 1-2 copies per cell. pYes2 has a 2g origin of
replication and is present at 1-50 copies per cell; this plasmid is lost more readily
100
than pCD43 but produces a higher level of expression. The IXR1gene was cut
out of pSB4 with AccI and SnaBI,and the piece was isolated by agarose gel
electrophoresis. pSL301 was digested with EcoRV and SacI, and the vector
fragment was isolated. The two fragments were ligated together to form pSL301Ixrl, with the IXR1gene in the polylinker. The large BamHI-SacIfragment of
pCD43 was ligated to the BamHI-SacIfragment of pSL301-Ixrl containing IXR1
to form pCY. Similarly, pSL301-IXR1and pYes2 were digested with BamHI and
XhoI and the appropriate fragments were isolated and ligated together to form
pYY.
pMM1 has IXR1 expressed by the Met promoter (Fig 2.3, Table II). The
Met promoter was cut out of pMet with XbaI and EcoRV and isolated on an
agarose gel. pYY (pYes-Ixrl) was cut with BamHI which was filled in with
Klenow. The linear plasmid was then cut with SpeI. This procedure removed
the Gal promoter from pYY. The Met promoter was then ligated to this vector.
The resultant plasmids were analyzed for the correct size and restriction enzyme
cleavage patterns, which confirmed that pMet had been inserted into the correct
location.
Cytotoxicityassays using Ixrl expressionplasmids
Cytotoxicity assays were performed by growing BWG and BWGAixrl cells
with either the control plasmid pCD43 or the expression plasmid pCY. The cells
were grown to exponential phase in SCAUmedia (2% lactate, 3% glycerol)
(Ausubel et al., 1994),and induced by adding 2% galactose. The cells were
treated with cisplatin in SD (2% galactose) media (Ausubel et al., 1994). The cells
were never permitted to enter late log phase, where small daughter cells cause
the cell population to become heterogeneous. The cell density and growth rate of
the cells were also monitored, and the different strains used in the cytotoxicity
assays were all brought to the same state. Unless this operation was performed,
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the results were highly variable. Cells were plated onto SCAU(2% galactose)
plates.
Anaerobic cytotoxicity assays
All solutions used were degassed by bubbling argon through them for 0.5
hr for 5 mL volumes to 1 hr for larger volumes. Cells were grown by inoculating
the media aerobically into 16 mm glass screw cap tubes, then degassing the cells
plus media with a sterile cannula through a sterile septum. The tubes were
pumped into the wet box so that the screw cap could be put on the tube; parafilm
was then wrapped around the cap. The cells were grown in a shaking incubator
at 30°C for three days. The ability of this system to keep the media oxygen-free
was tested by using oxygen sensitive probes (BBLGas Pak disposable anaerobic
indicator). The cisplatin treatment was performed in an anaerobic chamber (wet
box). The cells were treated as stated above, except that the platinations were
performed on a rocker in the wet box, at room temperature, about 25°C. The cells
were centrifuged with a microcentrifuge in the wet box. They were plated as
detailed above, except that all procedures were carried out in the wet box. Prior
to being brought into the box, the plates were treated overnight in the BBLGas
pak oxygen removal system; the removal of the oxygen was confirmed with
oxygen sensitive probes (BBLGas Pak disposable anaerobic indicator). The
plates were pumped into the box still in the BBLGas pak apparatus (Becton,
Dickinson, Inc.), which is basically a large jar that can hold the appropriate
atmosphere. After the cells were put on the plates, they were transferred back
into the BBLGas pak apparatus, removed from the box, and grown for three
days at 30°C. After those three days of anaerobic growth, they were removed
and grown aerobically so that the colonies could be easily counted. The oxygen
indicators were always present while the plates were growing, and did not
change color until the plates were removed from the apparatus.
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RESULTS
CytotoxicitydifferencebetweenIXR1 and ixrl strains
The difference in cisplatin cytotoxicity between IXR1 and ixrl was present
in two different strains of yeast, both of which are significantly different from the
original IXR1strain. JM43 and its related mutants were constructed originally by
the laboratory of M. Cumsky, U.C. Irvine. These cells differed from the IXR1
cells in genotype and they grew significantly more rapidly. The JM43 strain was
not as sensitive to cisplatin as the IXR1 strain, as can be seen from the smaller k
value (Table III). The relative sensitivities of strains to cisplatin was quantified as
detailed in the materials and methods section; the exponential slope of the
equation %Survival = exp(-k[Cisplatin]) was found. The slopes corresponding to
two strains were compared, because it is incorrect to compare the relative
sensitivities at a specific cisplatin concentration (S. Tannenbaum, personal
communication). The slope can be easily converted into LD50 values, or any
other similar measure (LD50 = ln(0.5)/k). The JM43Aixrl strain was significantly
less sensitive to cisplatin than the JM43strain (Fig. 2.4). The difference between
these two strains was even greater than that for the IXR1and ixrl strains; the
ratio of the slopes was as high as 6.5, whereas the ratio for the IXR1and ixrl
strains only went as high as 2.1 (Table III).
The other strain of yeast examined, BWG1-7A,was obtained from the
laboratory of L. Guarante, M.I.T.. These cells have a different genotype than the
IXR1 or JM43 strains; they grew at about the same rate as the IXR1 cells. The
BWG strain was more apt to lose its mitochondria when grown on YPD plates,
resulting in small colonies that could not respire. The ixrl mutant of BWG,
BWGAixrl, was constructed by interrupting the IXR1gene with LEU2. The
interruption of IXR1 was confirmed by Southwestern and Southern blots (Fig.
2.5). The Southwestern blot showed that no Ixrl was present. The Southern blot
103
verified that the IXR1 gene had an insert of the appropriate size. The BWG strain
was significantly more sensitive to cisplatin than the other two strains (Table III).
The BWGAixrl strain was less sensitive to cisplatin than the BWG strain by 1.65
fold (Fig. 2.6, Table III). This difference was approximately the same as that in
the IXR1strain. The results with the BWG and JM43strains confirmed the
difference between IXR1 and ixrl strains in two different backgrounds of yeast.
These results indicate that the difference in cisplatin sensitivity between the IXR1
and ixrl strains was not due to an unrelated mutation that may have occurred
when constructing the ixrl strain.
Ixrl binding to damagedDNA
A bandshift assay demonstrated that Ixrl bound specifically to cisplatin
treated DNA (Fig. 2.7). Ixrl did not bind to DNA treated with trans-DDP,
[Pt(dien)C1]+ , which forms monofunctional adducts, or UV light.
Roxl, an HMG domain protein
Roxl is an unusual HMG domain protein because there are five amino
acids in the middle of the domain, dividing it into two separate pieces that are
homologous to other HMG domains (Fig. 2.1b). A JM43Aroxl strain was
obtained and treated with cisplatin (Fig. 2.8). There was no significant difference
between JM43 and JM43Aroxl (Fig. 2.8, Table III). The unusual structure and the
unexpected behavior of this HMG domain protein led us to obtain a Roxl
expression vector and determine whether it bound to cisplatin-DNA adducts.
Up to this time, all HMG domain proteins that had been analyzed had been
found to bind to cisplatin. Results from the Lippard laboratory (N. L. Raju,
unpublished data) and the Cumsky laboratory (J.Lambert, personal
communication) indicate that Roxl does not bind to cisplatin-DNA adducts.
Furthermore, no band of the correct molecular weight is visible in Southwestern
blots performed on yeast cell extracts of ixrl strains (Fig. 2.5, for example). These
104
results indicate that Roxl does not actually bind to cisplatin-DNA adducts, and
thus, no difference in cisplatin sensitivity could be expected. Roxl is currently
the only HMG domain protein known not to bind to cisplatin-DNA adducts.
Abf2, an HMG domain protein
An abf2mutant was constructed in the IXR1strain by interrupting ABF2
with TRP1. This mutant was characterized by Southwestern blotting (Fig. 2.9).
Since this protein is so much smaller than Ixrl, a higher percentage SDS-PAGE
gel was run and the Ixrl bands do not resolve well at the top of the gel, so they
were not included in the figure. The two abf2strains tested were both missing
the same band, indicating that the band is Abf2 and that Abf2 does indeed bind
cisplatin-DNA adducts. Unfortunately, when cisplatin cytotoxicity assays were
attempted, it became clear that abf2cells did not grow as well as ABF2 cells, a
result previously noted in the literature (Diffley and Stillman, 1992). The exact
state of growth of cells makes a large difference in their cisplatin sensitivity.
ABF2 and abf2 cells could not be compared because they could not be grown
similarly enough that any results would be valid.
Ixrl overexpressionstudies
Ixrl was expressed in yeast cells to demonstrate further that it is
responsible for the cisplatin cytotoxicity difference between IXR1 and ixrl
strains. It is also possible that if enough Ixrl could be expressed in an IXR1 cell,
the cell would become even more sensitive to cisplatin. There are several ways of
expressing proteins in yeast using a number of types of vectors. One common
system uses the galactose-inducible promoter GALl. pCY was constructed by
inserting IXR1 in front of the GALl promoter of the plasmid pCD43 (Table II).
Expression off this plasmid was generated by growing the cells in media lacking
dextrose, and then adding galactose to induce expression. When the cells were
induced in exponential growth, expression of Ixrl was strong 1 hour after
105
induction (Fig. 2.10a). Cisplatin cytotoxicity assays are usually performed when
the cells have been grown to saturation or reach the post-diauxic phase.
Galactose-induced Ixrl expression did not occur in cells that were at saturation,
either in cells induced after they were at saturation (Fig. 2.10 B), or in cells that
were continually induced through late exponential phase. Therefore, the
cytotoxicity assays performed with this plasmid had to be carried out with cells
in exponential growth.
Cisplatin cytotoxicity assays were performed on exponential stage BWG
and BWGAixrl cells with either the control plasmid pCD43 or the expression
plasmid pCY. Since the cells were in exponential phase, the difference between
BWG and BWGAixrl was substantially diminished (Fig. 2.11). The expression of
Ixrl from the pCY plasmid did indeed cause the cells to become more sensitive to
cisplatin, both in the BWGAixrl strain and in the BWG strain. This result
supports the hypothesis that it is indeed Ixrl which causes the difference
between IXR1 and ixrl strains.
Other expression systems were investigated since the GAL promoter did
not express protein in cells grown past the exponential phase. Proteins can be
expressed by using the genomic promoter. This strategy was attempted for Ixrl
and for a mutant form of Ixrl, Ixrl-1, which contains the HMG-domain (Fig.
2.1a). Expression of Ixrl or Ixrl-1 from a yeast plasmid containing the gene with
its own promoter occurred in cells in exponential phase, but not once the cells
had reached saturation. Therefore, this system would not function any better
than the GAL system. A newer promoter called the Met promoter can be
induced by growing the cells in media lacking methionine. This promoter was
chosen because cells that have been grown to saturation have exhausted the
dextrose in the media, and may be exhausting other components as well. The
vector was constructed by placing the Met promoter upstream from the Ixrl
106
gene. Cells were grown on SCAUraAMet(dextrose) media to preserve the
plasmid and to express Ixrl off the promoter. The cells do not need exogenous
methionine to grow. Ixrl expression was present in cells grown to saturation
(Fig. 2.12). JM43 and JM43Aixrl were grown with either the control plasmid
pYes2 or the expression plasmid pMM and treated with cisplatin (Fig. 2.13).
There was a strong difference between the JM43+pYes2 and JM43Aixrl+pYes2
strains (slope ratio of 2.7, Table IV). The JM43Aixrl+pMM strain was still less
sensitive than JM43+pYes2,but it was substantially more sensitive than the
JM43Aixrl+pYes2 strain. This result indicates that Ixrl can make cells more
sensitive to cisplatin. There was no difference between the two JM43strains,
probably because the level of Ixrl expression was not sufficiently high. In the
Southwestern (Fig. 2.12), the difference between the JM43 strains is not nearly as
high as the difference between the two JM43Aixrl strains. Unfortunately, there is
no method for raising the levels of expression from this promoter, and this
method has already proven itself better than other more common methods such
as the GAL promoter.
Cytotoxicityassayson Ixrl mutants
There are several strains of yeast isolated by the Cumsky laboratory that
contain mutant forms of Ixrl. These mutants act like ixrl strains in that they
express CoxVb in the presence of oxygen. The best characterized is Ixrl-1 (Fig.
2.1a), which contains most of the sequence of Ixrl, including the HMG domains,
but is lacking the C-terminal tail. Ixrl-1 is in a JM43Acox5abackground. When
Ixrl-1 was tested for cisplatin sensitivity, it was found to be substantially more
sensitive to cisplatin than JM43Acox5a(Fig. 2.14a) The ratio of slopes k(Ixrl-1) /
k(Acox5a) = 2.8 + 0.6, with the values ranging from 2.0 to 3.4. Ixrl-1 was also
more sensitive than JM43 itself (Fig. 2.14a, Table III) by about 1.3 fold. This result
led us to wonder if there was something about the Ixrl-1 protein that caused it to
107
make cells more sensitive to cisplatin. The Ixrl-1 strain, however, is a cox5a
mutant, so it cannot be strictly compared to the wild-type strain. The increased
sensitivity could have been due to the cox5amutation. Since there was no strain
with Ixrl-1 in a wild-type background, and attempts to express Ixrl-1 from a
plasmid in saturated cells failed (see above), CoxVa was expressed from a
plasmid in the Ixrl-1 cells. CoxVa, expressed from its genomic promoter, may be
more stable in cells than Ixrl, and thus more easily expressed. Four strains,
JM43+pCD43,JM43Aixrl+pCD43, Ixrl-1+pCD43, and Ixrl-1+pCox5a were
grown to saturation in SCAUra (dextrose) media (for plasmid descriptions, see
Table II). The difference in cisplatin cytotoxicity between JM43+pCD43 and
JM43Aixrl+pCD43 was significant (Fig. 2.15, Table V), but the two Ixrl-1 strains
were just as sensitive as the JM43+pCD43 strain. This result indicates that the
Ixrl-1 protein does not cause cells to become more sensitive to cisplatin. The
increased sensitivity of the Ixrl-1 strain was dependent on the media conditions;
the previous experiments were done in YPD media. The Ixrl-1 mutant protein
does not cause the cells to become less sensitive to cisplatin even though strains
with that protein express CoxVb. These experiments demonstrate that it is the
HMG domain of Ixrl that is crucial to the cisplatin sensitivity of the strain, not
the function of the protein for suppressing CoxVb expression.
Another Ixrl mutant, Ixrl-9 was also briefly investigated. This mutant has
not been sequenced yet; it was tested primarily to see if there were any
interesting results that might make it worth sequencing. Cisplatin cytotoxicity
assays revealed no significant difference between Ixrl-9 and the most related
strain, BMH200Acox5a,or the parent strain BMH200 (Fig. 2.14b). Since Ixrl-9
does not suppress CoxVb expression, that function cannot be related to the
cisplatin cytotoxicity difference between IXR1 and ixrl strains.
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Cytotoxicity assays on cox5a strains
CoxVa is the isoform of cytochrome c oxidase subunit V that is used under
aerobic conditions. It is required for aerobic respiration if CoxVb is repressed by
both Roxl and Ixrl. The cisplatin sensitivity of the JM43Acox5astrain fell
between that of the JM43 strain and the JM43Aixrl strain (Fig. 2.14a and Table
III). This phenomenon
was significantly less pronounced in the BMH200
background (Fig. 2.14b). The difference in cisplatin cytotoxicity between COX5a
and cox5astrains probably arises because these assays were performed at
saturation, where the cells were required to respire aerobically. A cox5acell
cannot aerobically respire and thus cannot pass through the diauxic shift, which
means that the cell population does not increase to as high a level as in the
COX5a strains. A JM43Acox5aAixrlstrain treated with cisplatin was more
sensitive than the JM43Acox5amutant (Fig. 2.16). The two forms of cisplatin
desensitization were not additive, instead, they were subtractive.
A strain called F::5 was also analyzed in order to determine whether it
displayed any interesting differences in cisplatin cytotoxicity. F::5is
Acox5aAixrl,with both JM43 and JM45 parents. JM45 is exactly the same as
JM43, except that it is the other mating type, and it responds to cisplatin as JM43
does. JM43Acox5aAixrlcells grow very slowly on YPGE media, which contain
only non-fermentable sugars. If the cells are grown on those plates for about 1
week, fast-growing colonies arise at a rate far greater than the background
mutation rate and these form the F::5 strain. The difference seems to be due to a
cellular adaptive response, not a gene mutation (J. Lambert, personal
communication). The F::5 strain was just as sensitive to cisplatin as JM43 itself
(Fig. 2.16, Table III).
These data, taken as a whole, indicate the complexity of the cellular
response to cisplatin and the need to make sure that cell lines being compared
109
are in all other respects identical. cox5na
strains do not respire aerobically in an
efficient manner, and they have a different cisplatin sensitivity because the assay
requires cells to grow until they respire aerobically. Any small differences in cell
growth can have a large effect on measured cisplatin cytotoxicity.
Anaerobic cytotoxicity assays
Since Ixrl regulates the expression of a protein used only under hypoxic
or anaerobic conditions, cytotoxicity assays were performed under the latter
conditions. Cells and plates grown without oxygen were monitored with oxygen
indicator strips. The cisplatin treatments themselves were performed in an
anaerobic chamber at room temperature. The difference in cisplatin sensitivity
between the JM43 and JM43Aixrl cells was still present (Fig. 2.17, Table VI). The
difference was a bit less pronounced than in the aerobically grown cells, with a
slope ratio of 2.4 instead of 3.9, but this result is probably more an indication of
differences in cell growth conditions than anything else. This result indicates
that the increased expression of CoxVb in ixrl cells does not cause the cisplatin
cytotoxicity difference, and that the latter is not function of oxygen metabolism.
Cytotoxicity assayconditions
Both the results with the cox5astrains and the fact that cells need to be
grown to saturation for a large cisplatin cytotoxicity difference to be observed
inspired a series of experiments examining the cytotoxicity assay under a variety
of conditions. The platination media did not seem to make much difference; SD
media with dextrose, galactose, or no sugars at all were all used. SC media (SD
plus amino acids) have also been used successfully, although the added amino
acids caused the cells to become less sensitive to cisplatin. IXR1 cells were still
more sensitive than ixrl cells when cisplatin treatment was lengthened to 4
hours. Cells treated for 4 hours were, of course, more sensitive to cisplatin than
ones treated for two hours. The only change that seemed to affect the difference
110
in cytotoxicity strongly was the state of the cells at the time of platination, which
depended on the media and on how long they were grown. For each different
type of media and plasmid reported in the above experiments, it was necessary
to determine how long the cells took to grow to saturation.
Some experiments examining the effect of different media upon the
cisplatin cytotoxicity difference were performed in hopes that they would shed
some light on the reason the cells needed to be grown to saturation for the
difference to be seen. One possibility is that the dextrose supply was exhausted,
and thus the cells were forced to respire aerobically. Cells were grown on YPD
with 10% dextrose instead of 2% so that the dextrose would not be exhausted
when the cells reached saturation. The IXR1and ixrl cells grown on YPD+10%
dextrose no longer showed the difference in cisplatin cytotoxicity (Fig. 2.18).
This result was confirmed in the JM43 background (Fig. 2.19a). When the JM43
cells were permitted to grow a bit longer, exhausting more of the dextrose, the
difference began to return (Fig. 2.19b).
If the only reason that cells need to be grown to saturation is to exhaust
the dextrose, then cells grown on media lacking dextrose should show the
difference between IXR1 and ixrl without being grown to saturation. YPGE
contains glycerol and ethanol, both of which are non-fermentable carbon sources
and thus force the yeast cells to respire. When the IXR1and ixrl cells were
grown to mid-exponential phase on YPGEmedia, the difference in cisplatin
cytotoxicity was not present (Fig. 2.20a). When the same cells were grown to
saturation, the difference returned, and was even greater than the difference
when the cells were grown on YPD media (Fig. 2.20b, Table VI). The sensitivity
of the strains to cisplatin also changed; cells grown to saturation were far less
sensitive than those in exponential phase. These results indicate that the cells
need to be dense enough before the cisplatin cytotoxicity difference appears.
111
It is possible that the expression of Ixrl changes as cells grow to
saturation. If the expression level of Ixrl were to increase in cells at saturation
the cytotoxicity difference would naturally be larger. Aliquots of cells were
removed as the cells grew to saturation, and Southwestern blots were performed
on these cells. The blots showed no increase in Ixrl expression at all as the cells
reach saturation; if anything, the level of Ixrl decreased. Thus, the need to grow
the cells to saturation for the cytotoxicity difference to become sufficiently large
cannot be explained by an increase in the level of Ixrl. It is possible that Ixrl
was modified in some way that raised its binding capacity for cisplatin-DNA
adducts; this potential alteration did not dramatically change its electrophoretic
mobility.
DISCUSSION
The HMG domainsof Ixrl cause the differencein cisplatinsensitivity
The difference in cisplatin sensitivity between IXR1and ixrl strains was
examined in more depth because this result was the first indication that HMG
domain proteins actually affect cisplatin cytotoxicity. Experiments were
performed in order to determine whether it is the HMG domains of Ixrl or the
function of the protein that causes the difference. First, the difference in
sensitivity was confirmed in two other strains of yeast, in order to be sure the
effect was not caused by some unrelated mutation. Ixrl was overexpressed in
ixrl strains to demonstrate that the difference in cisplatin sensitivity is due to the
missing Ixrl protein. In spite of difficulties encountered in expressing protein in
cells that have been grown to saturation, it was shown that JM43Aixrl plus
overexpressed Ixrl is more sensitive to cisplatin than the JM43Aixrl plus control
plasmid strain. These results indicate that the difference in cytotoxicity between
IXR1 and ixrl strains is caused by the lack of the Ixrl protein.
112
Several experiments were performed that demonstrate that increased
expression of CoxVb in the ixrl strain was not the cause of the decrease in
sensitivity to cisplatin. Two strains with mutant Ixrl proteins were examined,
Ixrl-1 and Ixrl-9. Both of these strains express CoxVb, so if CoxVb expression
affected cisplatin cytotoxicity, these strains would be less sensitive than the
corresponding wild-type strains. Neither Ixrl-1 or Ixrl-9 was less sensitive to
cisplatin, indicating that the change in CoxVb expression does not affect cisplatin
cytotoxicity. Roxl is also a transcriptional repressor of CoxVb expression; CoxVb
is expressed when either Ixrl or Roxl is missing. JM43Aroxl was as sensitive to
cisplatin as JM43 (Fig. 2.8). Finally, the cytotoxicity assay was performed under
anaerobic conditions. CoxVb is expressed under such conditions because the
Roxl repression is alleviated. The JM43Aixrl strain was still less sensitive than
the JM43 strain. This result indicates that no protein that is expressed differently
in anaerobic cells is responsible for the cisplatin cytotoxicity difference. These
results conclusively demonstrate that the increased CoxVb expression in the ixrl
strains is not causing the difference in cisplatin cytotoxicity.
There are several experiments that demonstrate that the missing HMG
domains cause the difference in cisplatin cytotoxicity. Ixrl binds to cisplatin
DNA adducts, as can be seen in any of the Southwestern blots, or Fig. 2.7. Its
binding has been quantitated and demonstrated by footprint analysis by J.
Whitehead (Whitehead and Lippard, 1995). As demonstrated in Fig. 2.7, Ixrl can
bind to cisplatin-DNA adducts, but not to trans-DDP, [Pt(dien)Cl]+ , or UV
adducts. IXR1and ixrl strains were equally sensitive to trans-DDPand UV light
damage (data not shown) (Brown et al., 1993). A homolog of cisplatin which
forms similar bifunctional DNA adducts, [Pt(en)C121,also shows the cytotoxicity
difference (data not shown). If the mere act of damaging the DNA were
sufficient to cause the differential cytotoxicity, then the ixrl strain should have
113
been less sensitive than the IXR1strain to either trans-DDPor UV light. Only
damaging agents that cause adducts to which HMG domain proteins bind cause
ixrl strains to be less sensitive than IXRI strains. Furthermore, mutant Ixrl
proteins that still bind cisplatin-DNA adducts did not affect cellular cisplatin
sensitivity. The Ixrl-1 strain is not less sensitive than its corresponding wild-type
strain, JM43 (Fig. 2.14, 2.15). The Ixrl-1 protein is missing the end of the protein
which contains a long stretch of glutamine residues (Fig. 2.1a), and is thought to
be related to protein-protein interactions (Latchman, 1991). Since the HMG
domains are still intact, Ixrl-1 can still bind the cisplatin-DNA adducts.
Therefore it is the absence of the HMG domains, not the absence of a putative
protein-protein interaction domain, that causes the decrease in cisplatin
cytotoxicity in the ixrl strain. The binding of the HMG domains to the cisplatinDNA adduct is most likely the cause of the difference in cisplatin cytotoxicity.
Exactly how the binding causes the cytotoxicity difference is explored later in this
thesis.
Different cellular conditions cause different responses
Many experiments have been performed that indicate the complex nature
of the effect of different conditions on the IXR1 /ixrl cisplatin cytotoxicity
difference. Cisplatin cytotoxicity assays only show a strong difference between
IXR1 and ixrl strains when the cells have been grown to saturation. The initial
cytotoxicity experiments were all performed in YPD media. The difference was
not present if the cells were grown in YPD media with 10% dextrose instead of
2% dextrose. This result indicated that the difference was repressed by glucose.
Perhaps the cells need to respire aerobically or ferment alternative sugar sources
in order for the difference to appear, because there are many genes that are
repressed by glucose. Cytotoxicity assays were performed on cells grown in
YPGE,which only contains non-fermentable carbon sources. Exponential phase
114
cells grown on YPGEdid not show a difference in cisplatin cytotoxicity between
the IXR1 and ixrl cells, indicating that alleviation of dextrose inhibition was not
the only reason cells had to be grown to saturation. When the YPGE cells were
grown to saturation, the difference was substantially larger than in the YPD cells,
supporting the theory that dextrose does inhibit the cytotoxicity difference.
There was no rise in Ixrl expression in saturated cells that could explain these
results.
Cells that have been grown to saturation, or post-diauxic phase, are
different from exponential cells in several ways. Post-diauxic phase cells are, by
definition, growing substantially more slowly. Several studies have been
performed on cells in the post-diauxic phase or in the stationary phase.
Unfortunately, because the difference is not well defined, papers sometimes refer
to post-diauxic phase as stationary phase leading to confusion about which phase
to assign a given alteration in cellular function (Werner-Washburne et al., 1993).
Cyclic AMP concentrations are lower in post-diauxic shift cells, and the mRNA
levels drop by 50% (Werner-Washburne et al., 1993). The chromatin folds up
more tightly in a TOP1 dependent fashion (Wemer-Washburne et al., 1993). The
diauxic shift activates transcription of several proteins, including CYC7 (Pillar
and Bradshaw, 1991), several heat shock proteins, several proteolysis proteins
including ubiquitin-conjugating proteins, and, of course, proteins involved in
aerobic respiration such as alcohol dehydrogenase (ADH2) (Wemer-Washburne
et al., 1993). CYC1, the other isoform of cytochrome c, is down-regulated in the
stationary phase, and perhaps in the post-diauxic phase (Pillar and Bradshaw,
1991). In some cases, the diauxic shift is caused by nitrogen, sulfur or carbon
starvation (Drebot et al., 1990a), and the Ras pathway is involved in these cases
(Werner-Washburne et al., 1993). The transition from stationary or post-diauxic
phase cells to exponential growth is also of interest, since the cisplatin-treated
115
cells undergo this shift on the plates in order to form colonies. Histones H2A
and H2B are made during the transition (Drebot et al., 1990b),and a zinc finger
protein, GCS1, is involved in regulating the transition (Ireland et al., 1994). It is
clear in the literature that not only are these phenomena complicated, but they
have not been fully investigated yet. Therefore, it seems unlikely that we can
understand the reason why cells need to be grown to saturation in order for the
cisplatin cytotoxicity difference between IXR1 and ixrl cells to appear before
other researchers define the system more completely.
CONCLUSIONS
In conclusion, the differential sensitivity of IXR1 and ixrl strains is due to
the HMG domains of Ixrl, not to its ability to repress COX5b expression. The
reason that Ixrl binding to cisplatin-DNA adducts affects cisplatin sensitivity is
investigated more in subsequent chapters.
116
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Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and
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Latchman, D. (1991).Eukaryotic transcription factors. Academic Press.
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Laz, T.M., D.F. Pietras and F. Sherman (1984) Differential regulation of the
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Lowry, C.V., M.E. Cerdan and R.S. Zitomer (1990)A hypoxic consensus operator
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Megraw, T.V. and C.-B. Chae (1993)Functional complementarity between the
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Pil, P.M. and S.J. Lippard (1992) Specific binding of chromosomal protein HMG1
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Pillar, T.M. and R.E. Bradshaw (1991)Heat shock and stationary phase induce
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Trietel, M.A. and M. Carlson (1995)Repression by SSN6-TUP1is directed by
MIG1, a repressor/activator
protein. Proc. Natl. Acad. Sci. (U.S.A.) 92, 3132-3136.
Wang, G.L. and G.L. Semenza (1993)Characterization of hypoxia-inducible
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Werner-Washburne,
M., E. Braun, G.C. Johnston and R.A. Singer (1993)
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Whitehead, J.P. and S.J.Lippard (1995)Purification of the yeast protein,
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Zhang, M., L.S. Rosenblum-Vos, C.V. Lowry, K.A. Boakye and R.S. Zitomer
(1991) A yeast protein with homology to the f3-subunit of G proteins is involved
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119
Table I
Saccharomyces cerevisiae strains
Strain
Genotype
IXR1
MATa, ura3-52, ade2, trpl-1, Iys2-201, his3-200, leu2-3,112
ixrl
IXR1 ixrl::LEU2
IXRlAabf2
IXR1 abf2::TRP1
JM43
MATa, his4-580, trpl-289, leu2-3,112, ura3-52
JM45
JM43 MATa
JM43Aixrl
JM43 ixrl::LEU2
JM43Aroxl
JM43 rox::LEU2
JM43Acox5a
JM43 cox5a::URA3
Ixrl-1
JM43 cox5a::URA3, ixrl-l
JM43Acox5aAixrl
JM43 cox5a::URA3, ixrl::LEU2
F::5
JM43 cox5a::URA3,ixrl::LEU2,grows quickly on YPGE
BMH200
MATa, ade2, ura3-52
BMH281
BHM200 Acox5a
Ixrl-9
BMH281 ixrl-9
BWG
BWG1-7A ura3, leu2, his4, adel
BWGAixrl
BWG ixrl::LEU2
120
Table II
Plasmids used in cytotoxicity experiments
Plasmid
Description
pYes2
yeast GAL expression plasmid with no protein after the
promoter, 2g origin
Often used as a control plasmid
pCD43
yeast GAL expression plasmid with no protein after the
promoter, CEN origin
Often used as a control plasmid
pYY
pYes2-Ixrl, plasmid pYes2 with IXR1 after the promoter
pCY
pCD43-Ixrl, plasmid pCD43 with IXR1 after the promoter
pSL301
E. coli plasmid with a large polylinker
pSL301-Ixrl
IXR1 in the pSL301 polylinker
pMM1
yeast expression plasmid with Met promoter linked to IXR1
pCox5a
yeast Cox5a expression plasmid, uses the Cox5a genomic
promoter
121
Table III
Cytotoxicity assay results
WT Strain
Slope (k)
Mutant
k(WT)/k(mutant)
range of ratios
JM43
43 + 1.0
Aixrl
3.9 + 2
2.0 - 6.5
Aroxl
1.1 + 0.2
0.78 - 1.2
Acox5a
2.0 ± 0.3
1.7 - 2.3
Ixrl-1
0.76 ± 0.1
0.66 - 0.84
Acox5aAixrl
1.4 ± 0.01
1.4
F::5
1.0 + 0.08
1.0 - 1.1
BWG
27.9 + 9.5
Aixrl
1.65 + 0.22
1.5 - 2.0
IXR1
7.9 + 1.6
Aixrl
1.5 + 0.2
1.3 - 2.1
Table IV
Ixrl overexpression cytotoxicity assay
Strain
Slope (k)
JM43+pYes2
11.4 ± 1.5
JM43Aixrl+pYes2
k(J+pYes2)/k(other)
range of ratios
4.2 + 0.4
2.7 + 0.3
2.4 - 2.9
JM43+pMM
11.5 ± 2.0
1.0 + 0.09
0.9 - 1.1
JM43Aixrl+pMM
8.0 ± 1.2
1.4 + 0.05
1.4 - 1.5
122
Table V
Ixrl-1 cytotoxicity assay
Strain
Slope (k)
JM43+pCD43
11.7 + 0.9
JM43Aixrl+pCD43
4.0 + 0.9
Ixrl-l+pCD43
10.9 ± 1.0
Ixrl-l+pCox5a
10.8 + 1.2
Table VI
Different growth conditions
Condition
WT strain
Slope (k)
k(WT)/k(Aixrl)
range of ratios
Anaerobic
JM43
9.1
2.4
0.8
1.7 - 3.2
YPGE saturation
IXR1
2.0 + 0.1
3.2 + 1.1
2.2 - 4.4
2.4
123
124
Figure 2.1. Ixrl domains. A. Domains of Ixrl and the mutant protein Ixrl-l. B.
Comparison of Roxl HMG domain to an ordinary HMG domain.
125
A.
HMG domains
Ixrl
Ixrl-1
ig- - :
.....
i I i...
:"
::':
-- ..
.........-
I
-----1..
....-,
B.
HMG domain
Roxl
.......:
go~.
Normal
lg
::....
....
''...
"":.:.:........
i
..
.ii
Ixxxxx7:
4
5 amino acid spacer
i
126
Figure 2.2. Natural functions of Ixrl and related proteins. Ixrl represses the
transcription of CoxVb. CoxVa and CoxVb are both regulated by oxygen and
glucose levels. Expression of CoxVa is activated by oxygen through heme and
HAP2/3/4 and repressed by glucose through SSN6, TUP1 and HAP2/3/4.
Expression of CoxVb is repressed by oxygen through heme, SSN6, TUP1, and
Roxl, and repressed by glucose through MIG1, SSN6 and TUP1.
127
*- heme
/
SSN6&TUP1
SSN6&TUP1
/
HAP2/3/4
A ..
...
:ion
:ose
Roxl
.pression
CoxVa
02 repression
CoxVb
glucose
rep
repression
MIGI&SSN6&TUP1
Ixrl
Glucose
128
Figure 2.3. Ixrl expression plasmids.
129
BamHI
/
EcoRV
SacI
IXR1
I
I
I
I
'I
XhoI
I
I
I
I
I
I '
pSL301-Ixrl
BamHI
I
Gal
pmtr
IXR1
I
-
I
*pCY
I
-SacI
pCY
V
CEN originBamHI
I Gal pmtr
II
I
I
If
g
m
I
URA3
IXR1
I
XhoI
pYY
L I
k
I
I
I
I
I
origin
URA3
2g origin
URA3
I
Met pmtr
IXR1
-
I
I
~L
XhoI
pMM1
ii
·
I
!
Im
origin
2g origin
URA3
I
·
~~~~~
130
Figure 2.4. Cytotoxicity assays performed on JM43 and JM43Aixrl using
standard conditions.
131
.9.
100
10
cl
?i
0.1
0
0
JM43
JM43Aixrl
-
I error
0.1 -
II
0
0.2
0.4
I
I
I
0.6
0.8
1
[Cisplatin] mM
I
1.2
132
Figure 2.5. Confirmation of construction of BWGAixrl strain. A) Southwestern
blot on BWG (IXR1) and putative BWGAixrl (ixrl) strain. B) Southern blot of
genomic DNA from those strains digested with SacI and probed with a fragment
from Ixrl present in the normal and interrupted genes.
133
I ,--1
ITVD1I
E-Ixrl
Degraded
}
P.
-_
r
Ixrl
x .
-0
=
,.,
--'
A. Southwestern Blot
==
*-ixrl
4.6--Abf2
sf
t
.
-- IXR1
B. Southern Blot
135
136
Figure 2.6. Cytotoxicity assays performed on BWG and BWGAixrl using
standard conditions.
137
100
10
._'
cf
0.1
0.01
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
0.6
0.7
138
Figure 2.7. Ixrl binds cisplatin damaged DNA but not other forms of damaged
DNA. Bandshift assay using Ixrl. Pairs of lanes contain DNA without protein
and DNA with Ixrl protein. From left to right, the pairs of lanes contain
undamaged DNA, cisplatin damaged DNA, trans-DDPdamaged DNA,
ultraviolet light damaged DNA, and Pt(dien)Cl+ damaged DNA.
139
141
142
Figure 2.8. Cytotoxicity assays performed on JM43Aroxl, JM43, and JM43Aixrl
using standard conditions.
143
100
10
C
·,
1
0.1
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
144
Figure 2.9. Southwestern blot of abf2strains.
145
ABF2
interruption
30
I
V
Abf2, 20 kD-
21.5
rp
jim.
m~
____
14.3
147
148
Figure 2.10. Southwestern blots of overexpression of Ixrl by using the GALl
promoter. A. Induction profile of BWG+pCYcells. 2% galactose was added to
the cells at time 0 and aliquots were collected at the indicated times. The same
weight of cells were used in each lane. B. Induction of Ixrl expression when the
cells were grown to saturation. Lane 1 contains BWG cells with the control
plasmid pCD43. Lane 2 contains BWG with the Ixrl expression plasmid pCY.
The cells were induced for 2 hours with 2% galactose.
149
B.
A.
Induction
Induction in log phase
Induction time (hrs)
at
saturation
BWG
BWG
IpCDI
Ixrl
pCY
I
151
152
Figure 2.11. Cytotoxicity assays performed on BWG and BWGAixrl cells with
the control plasmid pCD43 and the Ixrl overexpression plasmid pCY. Cells
treated with cisplatin in exponential phase after 1 hour of induction by 2%
galactose.
153
100
10
;>
1
cn
8o
0.1
0.01
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
0.6
0.7
154
Figure 2.12. Ixrl overexpression from the Met promoter. Southwestern showing
the levels of overexpression obtained from the pMM Ixrl overexpression
plasmid.
155
JM43
JM43
JM43 JM43
Aixrl
Aixrl +pMM +pMM
<-- Ixrl
Degraded
Ixrl
<- Abf2
157
158
Figure 2.13. Cytotoxicity assays performed on JM43 and JM43Aixrl with the
control plasmid pYes2 and the Ixrl overexpression plasmid pMM. The cells
were treated with cisplatin after being grown to saturation in SCAUraAMet
media.
159
100
10
.>
cn
0--
1
0.1
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
0.6
0.7
160
Figure 2.14. Cytotoxicity assays performed on mutant Ixrl and cox5astrains. A.
Assay on JM43, JM43Aixrl, JM43Acox5a,and Ixrl-l using standard conditions. B.
Assay on BMH200, BMH200Acox5a,and Ixrl-9 using standard conditions.
161
A.
a
1UU
10
.c
d
1
0.1
0.01
0
0.2
0.4
0.6
0.8
1.2
1
[Cisplatin] mM
12
100
.
10
(>
0.
1
0.1
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
162
Figure 2.15. Cytotoxicity assays performed on Ixrl-1 strains. Assay was
performed on JM43,JM43Aixrl, and Ixrl-1 with the control plasmid pCD43 and
on Ixrl-1 with the CoxVa expression plasmid pCox5a. Cells were grown to
saturation on SCAUramedia.
163
100
10
C1
1
Qn
0.1
0.01
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
0.6
0.7
164
Figure 2.16. Cytotoxicity assays performed on JM45, F::5,JM43Acox5a,and
JM43Acox5aAixrlusing standard conditions.
165
100
10
cQ
0
1
0.1
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
166
Figure 2.17. Cytotoxicity assays performed on JM43 and JM43Aixrl using
anaerobic conditions. Cells were grown on YPD without oxygen, and the
cytotoxicity assay was performed using anaerobic conditions.
167
100
10
-4
1
cr
cn
0.1
0.01
0.001
0)
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
168
Figure 2.18. Cytotoxicity assays performed on IXR1and ixrl grown on YPD and
YPD with 10% dextrose.
169
100
--- 4
mb
10
::I
V)
1
0.1
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
170
Figure 2.19. Cytotoxicity assays performed on JM43 and JM43Aixrl grown on
YPD with 10% dextrose. The cell in B. were grown longer than those in A.
171
A.
100
4
10
1c
0.1
0
0.2
0.4
0.6
0.8
1
1.2
0.6
0.7
[Cisplatin] mM
B.
~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~
AA
100
10
>
1
cn
0.1
0.01
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
172
Figure 2.20. Cytotoxicity assays performed on IXR1and ixrl grown in YPGE. A.
Cells were treated with cisplatin in exponential phase. B. Cells were treated
with cisplatin after they were grown to saturation.
173
A.
100
10
(c
oe
1
0.1
0
0.2
0.4
0.6
0.8
1
1.2
[Cisplatin] mM
B.
100
10
-4
;-
80
1
0.1
0
0.5
1
1.5
2
[Cisplatin] mM
2.5
3
3.5
174
175
CHAPTER 3
The HMG domain protein Ixrl shields cisplatin-DNA
adducts from excision repair in vivo
176
INTRODUCTION
Ixrl is an HMG domain protein from Saccliaromyces
cerevisiae(Brown et al.,
1993). Also known as Ordl, it regulates the expression of cytochrome c oxidase
subunit 5b, which is used in aerobic respiration when oxygen levels are low
(Lambert et al., 1994). A yeast strain in which the Ixrl gene had been deleted was
less sensitive to cisplatin than the corresponding wild-type strain (Brown et al.,
1993). This result showed that Ixrl can modulate cisplatin cytotoxicity. Since
there was no significant difference between the IXR1and ixrl strains when they
were treated with ultraviolet light (Brown et al., 1993),the difference in cisplatin
cytotoxicity was not due to some intrinsic alteration in the cells. This result is
supported by the results detailed in the preceding chapter. It was also
demonstrated in chapter 2 that the difference in cisplatin cytotoxicity was due to
the HMG domains of Ixrl, not the natural function of Ixrl. The cause of the
difference in cisplatin cytotoxicity is investigated further in this chapter.
There are several ways Ixrl could alter cisplatin cytotoxicity. Ixrl could
affect the formation of cisplatin-DNA adducts, either by assisting the binding
reaction or by regulating the uptake, export, or availability of the drug. Ixrl
could also be titrated or "hijacked" away from its natural binding site by
cisplatin-DNA adducts, a possibility demonstrated by in vitro studies of another
HMG domain protein, hUBF (Treiber et al., 1994). Such behavior could either
cause faulty regulation of transcription at the natural binding site or result in
improper transcription at the site of the cisplatin-DNA adduct. A third
possibility is that Ixrl might affect the repair of cisplatin-DNA adducts. Ixrl is
not a repair protein, since removing it increased the resistance of the yeast cells to
cisplatin. It probably does not regulate the transcription of repair genes, since
deleting Ixrl had no effect on the sensitivity of yeast cells to ultraviolet light
(Brown et al., 1993). Ixrl could block the repair of the cisplatin-DNA adducts by
177
preventing the excision complex from recognizing the cisplatin-DNA adduct
(Fig. 3.1, or, in less detail, Fig. 1.8). Relatively high levels of HMG domain
proteins block the excision repair of cisplatin d(GpG) cross-links in mammalian
cell extracts (Huang et al., 1994). In this mechanism, removing Ixrl would cause
the 1,2-intrastrand cross-links to be repaired more readily, which would increase
the resistance of the yeast cells to cisplatin.
If repair shielding could be demonstrated in yeast, it would have
important implications for humans, since the excision repair system of S.
cerevisiaeis highly homologous to that of human cells (Friedberg, 1991a; Cleaver,
1994). Changes in the level of HMG domain proteins may account for the
acquired resistance found in many ovarian carcinoma patients. Many of the
yeast repair proteins (Rad) have mammalian counterparts in the xeroderma
pigmentosum (XP) cell complementing proteins or in the excision repair crosscomplementing (ERCC)proteins (Cleaver, 1994). Cisplatin-DNA adducts are
repaired by the excision repair system in both human and yeast cell extracts
(Hansson and Wood, 1989;Wang et al., 1993). Most laboratories study excision
repair by using UV-irradiated DNA. Very little work has been carried out to
examine the sensitivity of yeast excision repair mutants to cisplatin (Brendel and
Ruhland, 1984;Hannan et al., 1984;Wilborn and Brendel, 1989).
Currently, functions are being assigned to both yeast and mammalian
repair gene products and specific interactions among repair proteins have been
identified (Fig. 3.1). The human protein corresponding to Radl4 (Bankmann et
al., 1992),XP-A complementing factor, binds to DNA damaged with cisplatin
and with UV light (Jones and Wood, 1993). Radl4 itself binds to UV-damaged
DNA (Guzder et al., 1993). XP-A interacts with the rodent counterparts of the
Radl-RadlO complex, ERCC1 and ERCC4 (XP-F)(Li et al., 1994;Park and Sancar,
1994). Another large complex identified contains Rad2, Rad4, Rad3, Rad25, Ssll,
178
Tfbl, and possibly Rad23 (Feaver et al., 1993;Bardwell et al., 1994a; Bardwell et
al., 1994c). Rad3, Ssll, and Tfbl are part of RNA polymerase II transcription
initiation factor b, the yeast homologue of TFIIH (Gileadi et al., 1992; Feaver et
al., 1993; Wang et al., 1994). Rad2 is a structure-specific endonuclease (Habraken
et al., 1993; Harrington and Lieber, 1994), which is capable of degrading circular
single-stranded, but not double-stranded, DNA (dsDNA) (Habraken et al., 1993).
It specifically cleaves a branched DNA structure and is thought to cut on the 3'
side of the adduct (Harrington and Lieber, 1994),within a few nucleotides of the
Ixrl binding site (Huang et al., 1992). XP-G (Rad2) is required for the 3' incision
whereas ERCC-1 (RadlO) is not (O'Donovan et al., 1994). Cell extracts lacking
XP-G do not have 5' incision activity either, suggesting that the 3' cut is made
first (O'Donovan et al., 1994). Radl and RadlO form a complex that acts as a
ssDNA endonuclease; it binds ssDNA preferentially but shows no preference for
damaged DNA (Sung et al., 1993;Tomkinson et al., 1993;Tomkinson et al., 1994).
Thus, Radl and RadlO cleave DNA, but other proteins are involved in adduct
recognition and duplex unwinding to form the single-stranded substrate. The
Radl-RadlO complex is responsible for the excision at the 5' side of the adduct
(Bardwell et al., 1994b),which is approximately two turns of helix upstream of
the cisplatin binding site (Huang et al., 1992).
In the present study we have evaluated the repair blockage hypothesis by
constructing yeast strains in which both IXR1 and an excision repair gene are
interrupted. If repair shielding occurs, the differential cisplatin sensitivity of the
IXR1 and ixrl strains should substantially diminish when a component of the
excision complex involved in damage recognition is removed (Fig. 3.1).
Specifically, we constructed double mutants of the IXR1 gene and the excision
repair genes RADI, RAD2, RAD4, RAD10, and RAD14. These genes are
homologous to the mammalian genes encoding XP-F/ERCC4, XP-G/ERCC5, XP-
179
C, ERCC1, and XP-A, respectively. Other repair-related genes were also
interrupted, including Rad6, which is involved in error-prone repair, Rad9,
which regulates DNA-damage induced cell-cycle arrest, and Rad52, which is
involved in double-strand break repair.
MATERIALS AND METHODS
Yeast Strains and plasmids
Yeast strains used to construct the rad mutants were IXR1: MATa, ura3-52,
ade2, trpl-1, lys2A201, his3-200, leu2-3,112 and ixrl: MATa, ura3-52, ade2, trpl-1,
lys2A201, his3-200, ixrl::LEU2 (Brown et al., 1993). The Cumsky laboratory kindly
supplied their IXR1 (JM43) and ixrl (JM43Aixrl)strains (Lambert et al., 1994);
JM43: Matoc, his4-580, trpl-289, leu2-3,112, ura3-52. Plasmids used for gene
interruption
are in Table I. The following plasmids were used for RAD
expression: pG12-RAD1 1 , pWS502 (Rad2) 2 , pR67 (Rad6) 2 , pG12-RAD10 2 , pR14.9
(Rad14)3 , and pTB301 (Rad52) 3 .
Constructionofrad strains
The various RAD genes were disrupted by transforming both the IXR1
and the ixrl strains with plasmid fragments containing the RAD gene interrupted
by a selectable marker (Table I) (Ausubel et al., 1994). Cells with the rad gene
disruptions were selected by growing the transformed cells on synthetic media
lacking a necessary component such that only the presence of the interrupting
gene permitted cell growth. The sensitivity of the resultant colonies to ultraviolet
light was examined in a streak test to confirm interruption of the RAD gene
(Naumovski and Friedberg, 1984). rad9 strains are not as sensitive to ultraviolet
light as excision repair mutations, so a higher UV dose was required to see the
difference between rad9 and RAD9. Since interruption of the RAD52 gene does
1 Plasmids obtained from E. Friedberg.
2 Plasmids obtained from L. Prakash.
180
not sensitize the cells to UV light, the putative rad52strains were tested by
examining their sensitivity to methyl methanesulfonate (MMS) (Prakash and
Prakash, 1977). Expression or lack of expression of Ixrl was monitored by
Southwestern blot analysis (Brown et al., 1993), and several of the strains were
also checked by expressing the deleted gene from a plasmid. In particular, the
rad2, rad6, rad14, rad52, IXR1 radl, and IXR1 randO strains were no longer UV-
sensitive following expression of the appropriate RAD gene from a plasmid.
Cytotoxicity assays
Yeast cells were grown to saturation in 20 mL of YPD media (Ausubel et
al., 1994) for approximately 64 hours at 30°C. All strains grew to similar densities
except for the rad6and rad52strains, which saturated at approximately one half
of the density of the other strains. Portions containing 2x107 cells of each strain
were centrifuged to remove the media and resuspended in 1 mL of SD media, pH
6.5 (Ausubel et al., 1994)plus 0 to 1 mM cisplatin. The cells were incubated in an
Eppendorf tube with gentle rocking for 2 hours at 30°C. The medium containing
cisplatin was removed by pelleting the cells, which were resuspended in SD
medium, diluted, and plated at 20 to 200 cells per plate. Three identical YPD
plates were made for each level of platination. The plates were grown for 3 days
in a 30°C incubator. Colonies on the plates were counted and the percent
survival at the various concentrations was calculated relative to controls treated
identically except for the lack of cisplatin. The IXR1 RAD and ixrl RAD strains
were treated with only three different cisplatin concentrations because their
differential sensitivity has already been investigated (Brown et al., 1993). Each
strain was assayed in three to five separate experiments and results from a
representative experiment are shown in the figures. The error bars in the percent
survival plots show a 2a level derived from colony counts on the three identical
plates and do not represent multiple experiments. The data were evaluated by
181
fitting a line to the log of the percent survival. The log(% survival) was used
because, when a least squares fit was attempted to percent survival data, points
below 1% survival were insufficiently weighted. The slope of the line
corresponds to k in the expression
survival=exp(-k[cisplatin]) used to fit the
data. The reported values for k are an average of three to five separate
experiments, except for k for the RAD strains, which corresponds to the average
of 18 experiments. All experiments were performed with RAD IXR1 and RAD
ixrl strains grown and treated identically to the rad strains.
RESULTS
Construction of rad IXR1 and rad ixrl yeast strains
The genes for Radl, Rad2, Rad4, Rad6, Rad9, RadlO, Radl4, and Rad52
were interrupted by transforming IXR1and ixrl strains with plasmids containing
an interrupted copy of the RAD gene in question. The removal of repair activity
was checked by assaying the sensitivity of the cells to DNA damaging agents
(Fig. 3.2). The rad strains were significantly more sensitive to ultraviolet light or
MMS. Several of the strains were also checked by expressing the interrupted
gene from a plasmid.
Cisplatincytotoxicity in IXR1 and ixrl strains
The cytotoxicity of cisplatin in various yeast strains was measured by
treating cells with varying amounts of the drug for 2 hours. As found previously
(Brown et al., 1993), removing the Ixrl protein by interrupting its gene caused the
yeast cells to be less sensitive to cisplatin (Fig. 3.3). This result was repeated in
two other pairs of IXR1 and ixrl strains with different backgrounds. In one pair
of strains, constructed in a different laboratory (Lambert et al., 1994), the ixrl
strain was 6 times more resistant to cisplatin treatment (Fig. 2.4), demonstrating
that the differential cisplatin cytotoxicity between IXR1 and ixrl strains is not
dependent on a specific strain of yeast.
182
Excision repair mlrtants
All excision repair mutants were substantially more sensitive to cisplatin
than either IXR1 or ixrl (Figs. 3.3, 3.4, and 3.5). The rad IXR1 and rad ixrl strains
were compared in order to determine whether the differential sensitivity of IXR1
and ixrl strains was related to excision repair. Interrupting the RAD2, RAD4, or
RAD14 genes caused ixrl strains to be nearly as sensitive to cisplatin as IXR1
strains (Fig. 3.3). Thus, the difference in sensitivity between the RAD IXR1 and
RAD ixrl strains results from modulation of the actions of the Rad2, Rad4, and
Radl4 proteins by Ixrl, indicating that Ixrl blocks the access of Rad2, Rad4, and
Radl4 to cisplatin adducts.
When the cisplatin cytotoxicity of rad IXR1 and rad ixrl strains was
compared for radl and radiO,the ixrl strains were still less sensitive to cisplatin
than the IXR1 strains (Fig. 3.5). This result suggests that Radl and RadlO are
functionally distinct from Rad2, Rad4, and Radl4, as measured by this assay. As
discussed below, this finding is in accord with recent studies performed in vitro,
which assign specific functions to some of these proteins.
Differences in cytotoxicity were quantitated by fitting % survival =
exp(-k[cisplatin]) to the data and examining the slopes (k-values) (Table II). The
results indicate that all 5 excision repair mutants were approximately 5-8 times
more sensitive to cisplatin than wild type cells. The ratio of slopes for the IXR1
compared to the ixrl strains was 1.1 for the rad4 and radl4 strains, and 1.2 for the
rad2 strain, significantly less than the average value of 1.5 for the repair proficient
cells. The ratio is greater than 1.0 because excision repair is not the only repair
process which removes cisplatin adducts. Deletion of genes involved in either
double-strand break repair (Fig. 3.6) or error-prone repair (Fig. 3.7) also caused
the cells to be more sensitive to cisplatin. Comparison of the range of k-value
ratios obtained in the various experiments performed clearly indicates that the
183
difference between RAD and rad2,rad4,and radl4 strains is significant because
they do not overlap. When the average ratios of the various strains are
compared, that for RAD is greater than that for any of the excision repair strains.
The Student t-test, applied by using the Fisher-Behrens problem, demonstrated
the IXRl/ixrl ratios for rad2,rad4,and radl4 to be significantly different from the
corresponding ratio in the RAD lines at the 95% confidence level. The radl and
radiOratios were not significantly different from the ratio in wild type cells.
Double-strandbreak repairmutant
Rad52 is involved in homologous recombination and the repair of doublestrand breaks, MMS-DNA adducts, and ionizing-radiation-DNA adducts
(Prakash and Prakash, 1977). The rad52strain was three-fold more sensitive to
cisplatin treatment than the RAD52 strain (Fig. 3.6 and Table II). This result is
reasonable since cisplatin forms interstrand cross-links that would have to be
repaired by a system that can treat both strands. The sensitivity of the rad52
strain to cisplatin fell in between those of the excision repair mutants and that of
the wild-type strain, consistent with the lower frequency of interstrand compared
to intrastrand cross-link formation (Bruhn et al., 1990). The rad52ixrl strain was
liesssensitive to cisplatin than the rad52IXR1 strain, indicating that the difference
between IXR1 and ixrl does not involve Rad52. The k-value ratio of the IXR1 to
the ixrl strains is slightly higher for the rad52strains, probably because the ability
to remove interstrand adducts has been diminished. Under these circumstances,
intrastrand adduct repair will be more important to cell survival, and the effect of
Ixrl on cisplatin cytotoxicity correspondingly greater. Ixrl apparently had no
effect on double-strand break repair, which was expected since this protein
probably does not bind specifically to interstrand adducts.
184
Error-pronerepairmutant
Rad6 is part of the error-prone repair pathway, which is distinct from the
excision repair pathway (Friedberg, 1991b). Cisplatin cytotoxicity was
determined for rad6 IXR1 and rad6 ixrl strains (Fig. 3.7, Table II). Both rad6
strains are very sensitive to cisplatin, more so than the excision repair deficient
strains. The rad6 IXR1 strain is more sensitive to cisplatin than the rad6 ixrl
strain, indicating that Rad6 does not effect the underlying differential
cytotoxicity of RAD IXR1 and RAD ixrl.
Checkpointmutant
Treatment of yeast with DNA damaging agents arrests the cell cycle in
either the G1/S or the G2/M transition, but not in rad9strains (Schiestl et al.,
1989; Siede and Friedberg, 1990; Weinert et al., 1994). When Rad9 is removed,
the cells can no longer check to see whether the DNA is damaged before
proceeding to the next stage in the cell cycle. rad9mutants are less sensitive to
UV light than excision repair mutants (Siede et al., 1993). The rad9 IXR1 and rad9
ixrl mutants were studied in order to determine whether Rad9 had any affect on
cisplatin cytotoxicity or could mediate the difference between IXR1 and ixrl
strains. Cisplatin cytotoxicity assays showed no difference between the RAD9
and rad9 strains (Fig. 3.8 and Table II). On the other hand, rad9 ixrl was less
sensitive to cisplatin than rad9IXR. These results indicate that the difference
between IXR1 and ixr strains is unaffected by deletion of Rad9 and that this
protein does not mediate the response of yeast cells to cisplatin.
DISCUSSION
Ixrl affects excision repair
The antitumor activity of cisplatin might be mediated by HMG domain
proteins, which bind specifically to cisplatin-DNA 1,2-intrastrand cross-links.
Deletion of Ixrl, a yeast HMG domain protein with similar binding
185
characteristics,
3
caused the cells to be as much as 2.1-fold less sensitive to
cisplatin (Brown et al., 1993 and Table II). In the present work we have measured
this effect in a different yeast background where the difference was six-fold (Fig.
3.4). One problem with cisplatin is that a number of cancers have either natural
or acquired resistance to cisplatin treatment. A 2.3-fold difference in repair
capability can lead to clinically relevant cisplatin resistance in human tumors
(Ali-Osman et al., 1994). In this context, the differences observed in the yeast
cells are sufficiently significant to warrant further investigation into their origins.
Since less platinum was bound to DNA isolated from the ixrl strain following
cisplatin treatment, it was suggested that the difference in sensitivity was related
to the repair of the cisplatin-DNA adduct (Brown et al., 1993). There were
several alternative possibilities, however; for example, reduced uptake of
cisplatin into the ixrl cells would also have produced the observed results.
Here we demonstrate that the difference in cisplatin cytotoxicity between
the IXR1 and ixrl strains involves Rad2, Rad4, and Radl4, since interrupting the
genes encoding any of these three proteins diminishes the difference between
IXR1 and ixrl strains. All three proteins are required for excision repair, so it is
likely that Ixrl blocks the excision repair of cisplatin-DNA adducts. Increased
excision repair of cisplatin-DNA adducts in the RAD ixrl strain thus accounts for
:itsgreater survival. Although the differences in cisplatin sensitivity that
distinguish the rad2,rad4,and radl4 strains from the RAD strain are not large,
they are quite reproducible and statistically significant at the 95% confidence
level (Table II; note that the ranges are not overlapping). This finding is
supported by recent in vitro excision repair assays using human cell extracts.
Addition of relatively high levels of HMG1 to the extracts blocked repair of the
cisplatin 1,2-d(GpG) intrastrand cross-link by the human excinuclease, but did
3 Whitehead, JP and SJL, manuscript in preparation.
186
not affect the repair of adducts to which HMG1 does not bind, specifically, the
cisplatin 1,3-d(GpTpG) intrastrand cross-link (Huang et al., 1994). The present
results show that HMG domain proteins can block removal of cisplatin-DNA
adducts by the excision repair proteins in vivo.
The results for the rad6and rad52strains demonstrate that the increased
sensitivity to cisplatin of the radstrains is not masking differences between IXR1
and ixrl strains. The rad6 strains are substantially more sensitive than the
excision repair deficient strains, yet the differences between the rad6 IXR1 and
rad6 ixrl strains is still evident. Thus, the results for the rad2,rad4,and radl4
strains cannot be explained solely by theorizing that the difference between the
IXR1 and ixrl strains disappears because these strains are more sensitive to
cisplatin than the RAD strains.
Although Radl and RadlO are also required for excision repair,
interrupting their genes does not affect the difference between IXR1 and ixrl
strains. The radl ixrl and radiOixrl strains are more resistant to cisplatin than
their IXR1 counterparts; there is a measurably smaller difference between the
IXR1 and ixrl strains in rad2,rad4,or radl4 strains (Table II). Even though all five
of these Rad proteins are involved in excision repair (Cleaver, 1994), they are not
equally capable of mediating the difference between the IXR1 and ixrl strains.
We suggest that the difference in the results for the radl and radiOstrains,
compared with those for the rad2,rad4,and rad14strains, arises because they act
at different stages in the repair pathway (Fig. 3.1). The functions of the Rad
proteins, elucidated by experiments carried out in vitro, and described in the
introduction, support the present in vivo data. To elaborate, excision of cisplatin
1,2-intrastrand cross-links occurs in at least three steps, which currently appear
to be performed by different proteins. The initial step involves adduct
recognition and is the step that Ixrl would block by binding to the adduct.
187
Subsequently, the DNA is unwound and cleaved at both the 5' and 3' ends, and
the damaged strand is removed. The proteins deleted in the present study
function at different stages in this process. Radl4 recognizes DNA damage,
whereas Rad2 and RadI-RadlO cleave DNA but have no specific affinity for
damaged DNA. Rad4 has not been sufficiently studied to determine its activity.
As discussed above, in vitro data indicate that Rad2 cleaves the DNA first. Until
the DNA is cleaved, all steps in the excision repair pathway are reversible (Fig.
3.1). Since the RadI-RadlO complex acts after the first cut is made by Rad2, any
disruption by Ixrl in Radl4 binding to the adduct will not affect the ability of
Radl-RadlO to make the second incision.
The ability of the cells to respond differentially to cisplatin when different
members of the excision repair family of proteins are deleted indicates that, in
vivo, there must be some factor that partially compensates for the loss of a
particular protein. Such an activity is absent in cell extracts used to study
excision repair, where deletion of a gene product results in complete inability to
remove cisplatin 1,2-intrastrand cross-links (Huang et al., 1994). Such a
hypothesis is supported by studies performed in mammalian cells having
severely reduced excision repair (Venema et al., 1991;Evans et al., 1993;Zhen et
al., 1993). A reduced level of excision repair would probably not be apparent in
cell extract studies even if it were present, because the assays are inefficient and
unable to detect repair that is 10-fold diminished.
Comparisonto previouscisplatinyeaststudies
Very little work has been done with cisplatin and yeast cells. CisplatinDNA adduct repair was defective in yeast cell extracts lacking Radl, Rad2, or
RadlO (Wang et al., 1993). Only a few studies have been performed where whole
yeast cells were treated with cisplatin. radl, rad2,rad3,rad52,and double mutant
radl rad2 strains were more sensitive to cisplatin than wild-type yeast (Brendel
188
and Ruhland, 1984;Hannan et al., 1984;Wilborn and Brendel, 1989). The present
results are generally in agreement with these previous experiments, but the
conditions used by other workers were sufficiently different such that
quantitative comparisons are not possible. The cisplatin sensitivities of the
excision repair mutant strains compare well with those for cells treated with UV
light. The sensitivity of rad9 strains to ultraviolet light falls midway between the
sensitivity of RAD and radl strains (Siede and Friedberg, 1990);in the cisplatin
cytotoxicity assay, the rad9 and RAD strains were equally sensitive to cisplatin.
Double-strandbreakrepairmutant
The results with the double-strand break repair protein, Rad52, provide
further evidence that excision repair is responsible for the difference between the
IXR1 and ixrl strains. Although rad52strains are more sensitive to cisplatin than
RAD52 strains, rad52 ixrl remains less sensitive than rad52IXR1. Presumably,
Rad52 helps repair interstrand cross-links, which are not recognized by HMG
domain proteins, such as Ixrl. rad52is approximately three-fold more sensitive
to cisplatin treatment than RAD52. Interstrand cross-links comprise ~ 4% of the
total cisplatin adducts, whereas 1,2-intrastrand cross-links represent - 90% of the
population (Lepre and Lippard, 1990). When the sensitivities of the different
yeast strains are corrected for the number and types of adducts formed, it is
apparent that interstrand cisplatin cross-links are more toxic than intrastrand
cross-links. Our data indicate that Ixrl does not block repair of all types of
cisplatin-DNA adducts, probably because Ixrl does not bind interstrand crosslinks.
Error-pronerepairmutant
Rad6 is involved in several pathways, including error-prone DNA repair
(Friedberg, 1991b). It is a ubiquitin-conjugating enzyme, an activity required for
its ability to function in repair (Sung et al., 1990). Although the rad6 strains are
189
20-fold more sensitive to cisplatin, the difference between the rad6 IXR1 and rad6
ixrl strains is not significantly different from that of the RAD strains. Ixrl does
not seem to block the action of Rad6, which seems reasonable since it should only
interfere with proteins which act at or after the damage recognition step. Rad6
may effect error-prone repair by modifying other proteins that actually perform
the repair steps.
Cell-cycle checkpoint mutant
Rad9 induces cell cycle arrest at the G1/S border when cells are damaged
with ultraviolet light and at the G2/M border when cells are damaged with
ionizing radiation (Siede and Friedberg, 1990;Siede et al., 1993;Weinert et al.,
1994). Cisplatin arrests the cell cycle at the G2/M border or in the S phase in
mammalian cells (Sorenson and Eastman, 1988b;Sorenson and Eastman, 1988a;
Sorenson et al., 1990;Inoue et al., 1992). Since there is not a large difference
between the rad9 and RAD strains, the cytotoxicity assay makes it seem unlikely
that Rad9 affects cisplatin cytotoxicity significantly. Thus, Rad9 is probably not
involved in cisplatin-induced cell cycle arrest.
CONCLUSIONS
The ability of the HMG domain protein Ixrl to sensitize yeast to cisplatin
has been linked to excision repair. The Ixrl protein probably shields the
cisplatin-DNA 1,2-intrastrand cross-links from the repair complex. This
hypothesis is explored further in the next chapter by examining the level of
cisplatin-DNA adducts on the DNA of the various strains. If HMG domain
proteins are blocking repair in tumor cells, then some tumors might adjust the
activity of the HMG domain proteins to become more resistant to cisplatin
therapy. It should be possible to design cisplatin analogs to circumvent this
resistance; if the analog-DNA adduct bound HMG domain proteins more tightly
than the cisplatin-DNA adduct, then the analog-DNA adduct would not be
190
repaired as quickly. The analog would be a more effective drug against the
resistant tumors, and possibly against normal tumors of other origin.
191
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Jones, C.J. and R.D. Wood (1993)Preferential binding of the Xeroderma
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Lambert, J.R., V.W. Bilanchone and M.G. Cumsky (1994)The ORD1 gene encodes
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Lepre, C.A. and S.J.Lippard (1990).Interaction of platinum antitumor
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Naumovski, L. and E.C. Friedberg (1984) Saccharomyces cerevisiaeRAD2 gene:
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Park, C.H. and A. Sancar (1994)Formation of a ternary complex by human XPA,
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Siede, W. and E.C. Friedberg (1990)Influence of DNA repair deficiencies on the
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196
Table I
Plasmids used in construction of radstains
Gene
Plasmid
Interrupting
gene
Restriction enzymes used
to cut plasmid
RAD1
pWS15101
URA3
PvuII
RAD2
pWS5201
TRP1
SalI
RAD4
pNF416 2
URA3
SphI, XmnI, isolate 4.2 kB
RAD6
pR6.71 3
URA3
BamHI
RAD9
pTWO39-URA3 4
URA3
BglII, SalI
RAD10
pBRWS-RAD10 5
URA3
BglII, partial SalI
RAD14
pBRWS-RAD14 1
HIS3
SpeI, EcoRV
RAD52
pSM22 6
URA3
BamHI
1 E. Friedberg,
4
unpublished.
(Siede et al., 1993).
5
2
(Fleer et al., 1987). 3 L. Prakash,
(Weiss and Friedberg, 1985).
6 D.
unpublished.
Schild, unpublished.
197
Table II
Quantitation of cisplatin cytotoxicity in yeasta
Strain
IXR1
IXR1 / ixrl
range
RAD
7.9+1.6
1.5±0.2
1.3-2.1
ad2
49+7
1.2+0.06
1.09-1.24
rad4
42+12
1.1+0.09
0.93-1.16
43+14
1.1+0.13
0.94-1.26
radil
66+7
1.3+0.02
1.30-1.34
ladlO
53+5
1.5+0.16
1.3-1.7
rad6
202+50
1.4+0.05
1.40-1.50
rad9
9.4+0.5
1.5+0.06
1.48-1.60
rad52
20+6
2.2+0.3
1.9-2.6
ad14
a Values reported in the table are the slopes (k) of cytotoxicity plots such as
those in Figs. 3-8, where % survival=exp(-k[Pt]) and [Pt] is the concentration
of cisplatin in the medium. The Student t-test, applied using the FisherBehrens problem, demonstrated that the IXR1 /ixrl ratios for rad2,rad4, and
radl4 were significantly different at the 95% confidence level than that for
RAD. The ratios for radl and radi0 were not significantly different.
198
Figure 3.1. Model for excision repair of cisplatin-DNA adducts. The numbered
proteins are the Rad proteins. S1 stands for Ssll, and T1 stands for Tfbl. The
details of the figure are explained in the text.
199
-
-
ad14 recognize:
Ixrl binds
-
-
I
I
adduct
Le
-
3'
ad3
*Rad2 cle
.Ll-- 11f - : A - -. C J.'L-- -1
ULC O IUC UI LI1C
I
DNA
polymerase
& ligase
Radl/10
cuts at the
5' side
t
adduct
*Rad3 may help
unwind the duplex/
* RT,"1
/1 n hind"
/
"
"""'f L '"
"" .
'
201
202
Figure 3.2. A. Ultraviolet light sensitivity of various yeast strains. Strains were
streaked onto YPD agar and 80% of the plate was exposed to ultraviolet light.
Strains which were rad did not survive this operation. B. Sensitivity of rad52
strains to MMS. The plate on the left was YPD only, the plate on the right was
YPD + 0.017%MMS. rad52strains are highly sensitive to MMS damage.
203
A
ixrl
rat
rad2
rad
rad
B
IXI
RA
d52
IXIr
I
IX
:rl
152
rad
0d52
IXI
ad52
ntrol RA
rad,
no MMS
ontrol
MMS treated
205
206
Figure 3.3. Cisplatin cytotoxicity assays performed on the excision repair
deficient strains RAD IXR1, RAD ixrl, rad2 IXR1 and rad2 ixrl. RAD IXR1 and
RAD ixrl strains were also treated in the same experiment; those lines were left
out since they were similar to those in Fig. 2A.
207
100
10
(d
1
0
0.1
0.01
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
208
Figure 3.4. Cisplatin cytotoxicity assays performed on the excision repair
deficient strains rad4 IXR1, radl4 IXR1, rad4 ixrl, and radl4 ixrl. RAD IXR1 and
RAD ixrl strains were also treated in the same experiment; those lines were left
out since they were similar to those in Fig. 2A.
209
100
10
(n
1
0---
0.1
0.01
0
0.05
0.1
0.15
[Cisplatin] mM
0.2
0.25
0.3
210
Figure 3.5. Cisplatin cytotoxicity assays performed on the excision repair
deficient strains radl IXR1, radIOIXR1, radl ixrl and radi0 ixrl . RAD IXR1 and
RAD ixrl strains were also treated in the same experiment; those lines were left
out since they were similar to those in Fig. 2A.
211
100
10
1
0.1
0.01
0.001
0
0.05
0.1
[Cisplatin] mM
0.15
0.2
212
Figure 3.6. Cisplatin cytotoxicity assays performed on RAD IXR1 and the double
strand break repair deficient lines rad52IXR1 and rad52ixrl. The RAD ixrl strain
was also treated in the same experiment; that line was left out of the figure since
it was similar to that in Fig. 2A.
213
100
10
1
.
0.1
0.01
0.001
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1
1.2
214
Figure 3.7. Cisplatin cytotoxicity assays performed on the error-prone repair
deficient strains rad6 IXR1, and rad6 ixrl. RAD IXR1 and RAD ixrl strains were
also treated in the same experiment; those lines were left out since they were
similar to those in Fig. 2A.
215
100
10
cfn
C>
1
0.1
0
0.01
0.02
0.03
0.04
0.05
[Cisplatin] mM
0.06
0.07
0.08
216
Figure 3.8. Cisplatin cytotoxicity assays performed on RAD IXR1, RAD ixrl, and
the cell cycle checkpoint deficient lines rad9IXR1 and rad9 ixrl.
217
1)0
10
c
~>
1
01
0.01
0.001
0
0.2
0.4
0.6
0.8
[Cisplatin] mM
1.2
218
219
CHAPTER 4
Repair of cisplatin-DNA adducts
220
INTRODUCTION
The processing of cisplatin-DNA adducts by repair proteins affects the
cytotoxicity of the drug. In the previous chapter, the hypothesis that Ixrl
partially blocks excision repair proteins from repairing cisplatin-DNA adducts
was supported by cytotoxicity assays involving various rad mutants. If this
hypothesis is correct, there should be fewer cisplatin-DNA adducts in the ixrl
cells because more DNA repair occurs in those cells. In this chapter, the level of
cisplatin-DNA adducts was determined both for genomic DNA and for a
transcribed gene.
Other experiments were performed to investigate the processing of
cisplatin-DNA adducts in various systems. The cleavage of cisplatin and
trans-DDP DNA adducts by bacterial restriction enzymes was investigated. The
repair of cisplatin and trans-DDPadducts by mammalian cell extracts was
studied. These experiments support the theory that trans-DDP-DNA adducts are
repaired more readily than cisplatin-DNA adducts.
The level of platinum adducts was measured to determine whether the
difference between IXR1 and ixrl strains was really due to differential repair.
Platinum levels were checked on the genomic DNA by atomic absorption. These
experiments give the level of platinum on global DNA. Excision repair of
cisplatin adducts often occurs faster on genes that are being transcribed (May et
al., 1993;Zhen et al., 1993). Therefore, the platinum adduct levels were also
investigated on a highly transcribed gene, specifically, phosphoglycerate kinase
(PGK) (Hitzeman et al., 1982). The platinum levels were determined by using
quantitative polymerase chain reaction (PCR) (Grimaldi et al., 1994). CisplatinDNA adducts block Taq polymerase (Comess et al., 1992),which means that
DNA with cisplatin adducts is not amplified. The level of adducts can be
221
estimated from the amount of product obtained from cisplatin-treated DNA
compared to control DNA.
Restriction enzyme digestion can be used as a simple model for the ability
of platinum adducts to inhibit protein function. The activity of restriction
enzymes is easy to measure, and restriction enzymes bind to small regions of
DNA. BamHI activity, for example, is blocked by cisplatin-DNA adducts (Ushay
et al., 1981), as are other restriction enzymes such as EcoRI and SalI (Balcarova et
al., 1992; Brabec and Balcarova, 1993). The studies presented here investigate
BamHI and EcoRI, as well as several other restriction enzymes. Further
experiments were done on a site-specifically platinated construct that contains a
trans-DDP-DNA adduct within a XhoI restriction site.
The presence of cisplatin- and trans-DDP-DNA adducts was monitored by
restriction enzyme digestion. Since adducts block some restriction enzymes well,
it is possible to watch the removal of the adducts in mammalian cell extracts by
measuring enzymatic cleavage of the DNA. This assay was developed to
confirm that trans-DDP-DNA adducts are repaired more readily than cisplatinDNA adducts. Two different sorts of repair assays indicated that such was the
case (Ciccarelli et al., 1985;Heiger-Bernays et al., 1990). It was hoped that this
new assay would be more sensitive than the other assays.
MATERIALS AND METHODS
Determiningcisplatin-DNA adduct levels
Saccharomycescerevisiaestrains and growth media are defined in chapters 2
and 3.
Atomic Absorption experiments to measure platinum adduct levels in cells
Yeast cells were grown to saturation in 100 mL of synthetic complete
media at 30°C for 4-5 days. SC was used instead of YPD media because, when
the cells were grown in the latter media, they turned red due to the absence of
222
the Ade2 protein. This red-brown color would have been carried with the DNA
through most if not all of the isolation procedure; high levels of impurities
prevent the furnace from effectively vaporizing the platinum and thus lead to
anomalous platinum levels. One mL portions of IXR1and ixrl cells were used in
a cisplatin cytotoxicity assay. The difference in cisplatin sensitivity between IXR1
and ixrl was not as pronounced as for cells grown in YPD, but was still
reproducibly present. Cells were pelleted in sterile flasks and resuspended in
cisplatin-containing SC media. Fresh media were used since the pH value
decreased while the cells were growing. The cells were shaken in media
containing cisplatin for 4 hours. The amount of cisplatin used was adjusted to
correspond to approximately the same percentage of cells killed for each of the
strains. Thus, the radl, rad2,rad4,radlO,and rad14strains were treated with 33
pRMcisplatin whereas the rad52strains were treated with 83 gM cisplatin and the
rad9 and RAD strains with 167 gM cisplatin. The DNA was isolated by making
spheroplasts, breaking them open, precipitating most of the protein with KOAc,
and subsequently precipitating the DNA (Philippsen et al., 1991). The pellet was
resuspended
in 10 mL of TE (10 mM Tris pH 8.0, 1 mM EDTA) buffer. The RNA
present in the sample was digested by treating the sample with RNAce-itTM
(Stratagene) for 16-24 hours at 37°C. The DNA was then treated with Proteinase
K (Sigma) for 16 hours at 37°C to digest any remaining protein. The sample was
extracted once with phenol/chloroform/isoamyl
alcohol (25:24:1).The DNA
was precipitated with NH4OAc and ethanol. The pellet was resuspended in 1
mL of TE buffer and a 10 L portion was run on an agarose gel to confirm that
the RNA was completely digested. The sample volume was reduced to
approximately 500 jtL in vacuo and then extracted with phenol/chloroform/
isoamyl alcohol until no further precipitate was visible at the interface of the
phases. The DNA was again precipitated with NH4OAc and ethanol; the pellet
223
was redissolved in 500 gL of TE buffer. Samples were dialyzed against several
changes of 0.1 M NH4OAc over a 48 hour period. Excess NH4OAc was removed
by dialysis into distilled, deionized H20 with several changes over a 24 hour
period. The concentration of DNA was quantitated by measuring the A260.
Equal amounts of DNA for the IXR1and ixrl strains were dried down and
resuspended in 70 [tL of water. The DNA was digested with DNAse I
(Boehringer Mannheim). The digestion solution was made 1 mM in MgCl2 since
the DNAse I requires magnesium ion; the level of magnesium was kept low since
higher quantities of magnesium caused false platinum readings. The total
reaction volume was 100 AL. A 1 L portion of DNAse I (10 mg/mL) was added
three times during the course of a 3 day incubation at 37°C. A 10 gL portion of
the sample was run on an agarose gel to confirm that the digestion was complete.
Atomic absorption analysis was performed on these samples with a Varian AA1475 and a GTA95 graphite tube atomizer, as described previously (Ciccarelli et
al., 1985). The bound drug/nucleotide
ratios, (D/N)b, were the average of
values from three to four experiments.
Polymerase chain reaction basedassay
Cisplatin-DNA adducts block polymerases, including Taq polymerase, so
the extent of cisplatin damage can be measured by monitoring the quantity of
product from a polymerase chain reaction (PCR) assay. Cells were grown and
treated with cisplatin for these assays as they were for cytotoxicity assays
(chapters 2, 3), except that a 5 mL volume was employed. Cells were pelleted,
washed once with water, repelleted in an Eppendorf tube, and stored at -80°C
until the DNA was extracted. The cells were resuspended in 0.2 mL of buffer (1%
SDS, 2% Triton X-100, 100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA). The cells
were broken by 3 minutes of vortexing after 0.2 mL phenol/CHC13/isoamyl
alcohol, and 0.3 g acid-washed beads (Sigma, 425-600microns) were added. The
224
aqueous layer was increased by adding 0.2 mL TE. The tubes were centrifuged
for 5 minutes and the aqueous layer was transferred to a new tube. The DNA
was precipitated with 1 mL of EtOH. The pellet was resuspended in 0.4 mL of
TE plus 1 gL of RNAce ItTM (Stratagene) and was incubated overnight at 37°C.
Proteinase K was then added and the DNA was incubated for 2-16 hrs at 37°C.
The DNA was extracted three times with phenol/CHC13/isoamyl alcohol and
ethanol precipitated. The pellet was again resuspended in TE and RNAce It, and
incubated overnight. The DNA was extracted two times with phenol/CHC13/
isoamyl alcohol and once with CHC13/isoamyl alcohol to remove all remaining
protein. The DNA was ethanol precipitated and resuspended in 0.6 mL H 20.
The concentration of the DNA was determined by measuring the A260 value of
an 0.5 mL aliquot of that solution. The remaining solution was diluted if
necessary to permit pipetting 10 ng of DNA for use in the PCR reactions.
Cells for investigating the time dependence of the removal of cisplatinDNA adducts were grown in YPD media with 1 gCi of tritiated adenine or uracil
per flask (20 mL) of YPD media. The cells were treated with cisplatin as
described above. The cells were then pelleted and resuspended in five times as
much YPD media, which sufficiently diluted the cells so that they would
eventually grow. Aliquots of cells were taken at the times indicated in the
individual experiments. Cells were pelleted and stored in the -80 until DNA was
prepared as detailed above. The level of tritium in the DNA was measured with
a Beckman scintillation counter where 0.5 mL of DNA was added to 5 mL of
Hydrofluor scintillation fluid. This procedure yields disintegrations per minute
(dpm)/gg of DNA.
The method for carrying out the PCR reactions was derived from Grimaldi
et al. (1994). PCR reactions were performed in 100 gL with 10 ng DNA, 1.2 mM
each dNTP, 0.5 mM MgCl2, 0.5 M KCl, 0.1 M Tris pH8.3, 50 pmol of each oligo, 2
225
~tCi dCTP (NEN), and 2 units Taq polymerase (GIBCO). The oligos used were 5'CACGAATT(GAGCTCTTTGGCTGAT-3' and 5'-CCCTTGCCAGCCAGCTGGAATACC-3' which amplify a 432 bp segment to give a 480 bp piece of DNA. The
reactions were carried out in a 96 well dish with oil overlays on each sample.
The samples were incubated for 2 minutes at 94°C, cycled through 20 cycles of 1
min. at 94°C, 1 min. at 60°C, 1 min. at 72'C, and finally incubated for 4 min. at
72°C. Reactions with 5 ng and 10 ng of control DNA were always performed to
be sure that the PCR was still solely dependent on DNA. A 30 gL aliquot of the
reaction was mixed with formamide loading dye and run on a 4.5%
polyacrylamide gel. The gel was dried and analyzed using a phosphorimager
(Molecular Dynamics). The lesions/strand ratio was calculated by assuming a
Poisson distribution of adducts, so lesions/strand = -ln (PCR product/PCR
product of undamaged DNA).
PCR experiments were also performed on plasmid DNA damaged with
cisplatin. The plasmid used, pDEC (pDEC006),and the oligos skpl3 and skpl4
were the gift of Dave Coufal and Sonja Komar-Panicucci. These oligos amplify a
473 bp section of DNA to make a 513 bp piece of DNA. The plasmid was treated
with cisplatin and the adduct levels were determined by atomic absorption
analysis. The 1.00gL PCR reactions were performed with 3 pg DNA, 10 gL
10OxPCR
buffer (GIBCO),0.273 gg primers, 0.2 mM each dNTP, 1.5 mM MgCl2, 2
iuCidCTP (NEN), and 2 units Taq polymerase (GIBCO). The samples were
incubated for 3 min. at 94°C, 28 cycles of 45 sec. at 94°C, 30 sec. at 57°C, and 1.5
min. at 72°C, and finally 45 sec. at 94°C, 30 sec at 57°C, and 10 min. at 72°C.
]Products were analyzed as described above.
226
Restriction enzyme cleavage of cisplatin and trans-DDP treated DNA.
Platinationof DNA
The virus M13mpl8 DNA was treated with trans-DDPat 0.075
Pt/nucleotide
37'C (Ushay et al., 1981). Aliquots were taken at 30 min, 1, 2, 4, 6,
10, and 24 hrs, and the reaction was stopped by adding NaCl to 0.2 M. M13mp18
DNA was also treated with trans-DDPat 1.5 Pt/nucleotide. pUC19 plasmid
DNA was treated similarly with cisplatin and trans-DDPat (D/N)formal=l
Pt/nucleotide and at 0.075 Pt/nucleotide. Excess platinum was removed from
the DNA by applying the DNA to G-50 sephadex quick-spin columns
(Boehringer Mannheim) or by repeated ethanol precipitation of the DNA.
Restriction enzyme digestion
Restriction enzyme digestion was performed such that unplatinated
samples were completely cleaved. All restriction enzymes were supplied by
New England BioLabs, and the digestions were performed in their suggested
buffer. After digestion, samples were run on an 0.8% agarose plus ethidium
bromide gel (Sambrook et al., 1989).
Constructionof a circularpieceof DNA containinga site-specifictrans-DDPd(GAG)
adduct
The construction was done using the gapped-duplex protocol (Naser et al.,
1988). First, a 12 base pair sequence, CCTCGAGTCTCC,was inserted into the
HindII restriction site of M13mp18, and the construct was confirmed by
sequencing. This construct was called M13mpl8-Xhol2C because the inserted
oligonucleotide had a site for the XhoI restriction enzyme. Single stranded DNA
was made from M13mpl8-Xhol2C. Double stranded M13mp18 was made and
cleaved with HindII. When the linear M13mpl8 DNA was denatured and slowly
renatured in the presence of a great excess of the single-stranded M13mp18Xhol2C DNA, gapped duplexes were formed. Unadducted CCTCGAGTCTCC
227
and trans-DDP treated CCTCGAGTCTCCwere ligated into the gap. Any
remaining M13mpl8 was linearized by digesting with HindII; the closed-circular
DNA was isolated by agarose gel electrophoresis and subsequent electroelution
in an Amicon centrilutor.
The trans-DDP treated CCTCGAGTCTCColigonucleotide was obtained
from Chris Lepre. It was examined by atomic absorption analysis and by
complete enzymatic degradation of the oligonucleotide (Bellon and Lippard,
1990).
RepairAssay
Preparationof substrate
pUC19 plasmid was treated with cisplatin or trans-DDPto an (D/N)b of
approximately, 0.006, and then digested with Sma I for 1 hour, which cut about
one half of the pUC19 circles. The remaining closed-circular DNA was purified
by 2 successive 5% to 20% sucrose gradient centrifugation steps which were each
spun at 2°C in the ultracentrifuge in a SW27 rotor for 27 hrs at 24000 rpm. The
fractions from the gradient were analyzed by agarose gel electrophoresis.
Fractions containing closed-circular DNA were pooled and ethanol precipitated.
Restriction enzyme based repair assay in cell extracts
Repair reactions were run with 0.2 gg pUC19 DNA, 200 gg HeLa cell
extract, 45 mM Hepes (pH 7.8), 60 mM KCl, 7.5 mM MgCl2, 0.9 mM DTT, 0.4 mM
EDTA, 2 mM ATP, 20 jiM each dGTP, dCTP, dTTP, and dATP, 40 mM creatine
phosphate, 1 gg creatine phosphokinase, 3.4% glycerol, and 18 gg BSA for 6-24
hrs. at 30°C. The pUC19 was recovered by digestion of the above mixture with
RNAse and proteinase K in the presence of 0.5% SDS. The mixture was then
phenol extracted to remove proteins and ethanol precipitated. The DNA was
resuspended in TE, digested with Nde I and Sma I, and radiolabelled by filling in
the Nde I produced overhanging ends with the Klenow fragment of DNA
228
polymerase I. Sucrose loading buffer (40%) was then added and the DNA was
loaded onto a 1.8% agarose gel. The gel was run at 100 volts, dried, and put onto
the phosphorimager for quantitation of the bands.
RESULTS
Levels of cisplatin-DNA adducts on yeast genomic DNA
The amount of cisplatin covalently bound to the yeast genomic DNA was
measured. For these experiments, cells were grown to saturation in synthetic
complete media and treated with cisplatin for four hours. After platinum
treatment, the cells were harvested and the DNA was isolated. The experimental
conditions differed from those in the cytotoxicity assays because the cells never
grew in fresh media following cisplatin treatment.
There was a significant difference between the IXR1and ixrl strains (Table
I). This result supports the repair blockage hypothesis. The difference in
cisplatin-DNA adduct level between IXR1 and ixrl strains is not present in the
radl, rad2,rad4,or rad10strains, further supporting the hypothesis (Table I). It
was not clear if a difference was present in the radl4 strains. The difference was
present in the rad52 strains indicating that, as found in the cytotoxicity assay,
Rad52 is not part of the cause of the difference. The rad9 strains showed no
particular difference, which is not in agreement with previous cytotoxicity
results. Overall, the results examining the level cisplatin-DNA adducts on
globally platinated DNA in excision repair deficient strains indicate that the
repair shielding hypothesis is valid.
Levels of cisplatin-DNA adducts on a transcriptionally active yeast gene
Cisplatin-DNA adduct levels on part of a gene that is highly expressed
were investigated. Actively transcribed genes are thought to be repaired more
readily than other types of DNA (May et al., 1993;Zhen et al., 1993). The gene
for phosphoglycerate kinase (PGK) was chosen since it is well studied (Hitzeman
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et al., 1982). Quantitative PCR was exploited to give information about the
adducts. Taq polymerase is blocked by cisplatin-DNA adducts, so if a piece of
DNA has an adduct, it will not be amplified by PCR. The adduct levels can be
obtained by comparing the amount of product obtained in control, unplatinated
DNA PCR reactions to the product for the cisplatin treated DNA reactions.
Adducts are assumed have a Poisson distribution, which means that the lesions
per strand = -I:n(Product(damaged)/Product(undamaged)).
(Drug/nucleotide)bound
((D/N)b) ratios can be calculated by dividing the
lesions per strand by the length of the product, 480 bases. This calculation
assumes that product cannot be produced from DNA when there is an adduct
between the furthest ends of the primer hybridization sites.
Initial experiments were carried out with a plasmid, pDEC, which has a
513 bp PCR product. This plasmid was treated with three different levels of
cisplatin. The resulting PCR experiment gave (D/N)b ratios that were between
1.2 and 2.5 fold higher than (D/N)b ratios obtained from atomic absorption
readings. More extensive studies of the accuracy of PCR based (D/N)b ratios are
present in the literature(Jennerwein and Eastman, 1991). The results confirm that
quantitative PCR is a reasonably accurate technique for determining the amount
of platinum bound per nucleotide.
Cells were grown and treated with cisplatin exactly as for the cytotoxicity
assays, except that the volume and number of cells were increased 5 fold. DNA
was extracted from these cells and the proteins and RNA were removed from the
DNA preparation. The PCR conditions were established on unplatinated DNA
by varying [DNA], [MgC12], [dNTP], and the number of cycles. The conditions
were adjusted so that the PCR reaction was always dependent solely on the
substrate DNA. This aspect was examined in every PCR reaction by amplifying
5 ng of unplatinated DNA and comparing the amount of product to that of the 10
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ng sample. The PCR products were analyzed on a 4.5% polyacrylamide gel,
which was dried and the bands were quantified on a phosphorimager. The 480
bp product was the only product of the reaction (Fig. 4.1). The lower diffuse
band was due to the primers themselves becoming radiolabelled.
The time dependence of cisplatin-DNA adduct removal was investigated
by treating cells with the drug and then letting them recover from treatment by
growing in fresh media for various times. The level of DNA synthesis that is not
related to repair was calculated by using cells grown in media containing
tritiated uracil or adenine. The recovery was performed in normal media, where
new DNA synthesis diluted out the amount of tritium per glgof DNA. The
calculated level of cisplatin-DNA adducts was adjusted to take into account only
the tritiated DNA present in the original cells.
Cisplatin adduct level and removal studies were performed on the IXR1,
ixrl, radl4 IXR1, radl4 ixrl, JM43, and JM43Aixrl strains. The cisplatin-DNA
adduct levels rose with the level of cisplatin treatment in both the IXR1 and ixrl
cells (Fig. 4.2). There was no significant difference in cisplatin adduct level
between the IXR1 and ixrl strains. The (D/N)b values for the strains at the same
platination levels used in the atomic absorption experiments was 1.6x10-3
adducts/nucleotide, and hence the level determined by the PCR technique for
the transcribed gnee was an order of magnitude less than the level for bulk DNA
from the atomic absorption experiment. This result indicates that the PGK gene
is repaired more rapidly than the rest of the DNA. The rate of cisplatin adduct
removal experiment also showed no significant difference between the IXR1 and
ixrl strains (Fig. 4.3). The small difference observed in Fig. 4.3 was not
reproducible; also, since the slopes are essentially the same, the rate of repair is
also the same. The IXR1and ixrl cells did not grow significantly during the 6
hour experiment, according to the level of tritium in the DNA. The cells did
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grow significantly when the cells were grown for 24 hours; the DNA in these
samples was almost entirely newly synthesized. Repair was nearly complete at 6
hours for both strains. These results indicate that removing Ixrl does not cause
any difference in the repair of cisplatin-DNA adducts in the highly transcribed
PGK gene.
The radl4 strains were much more sensitive to cisplatin in the cytotoxicity
assays (Chapter 3) and the cisplatin-DNA adduct levels are correspondingly
higher at the same platination levels. At 33 gM cisplatin, the (D/N)b was
1.1x10-3 adducts/nucleotide, again an order of magnitude lower than that from
the atomic absorption experiment (Fig. 4.4). Again, this result is consistent with
the possibility that the PGK gene is repaired more readily than the rest of the
DNA. One interesting and reproducible aspect of this experiment was that the
amount of PCR product increased at low platination levels in the ixrl strain. This
increase caused the apparent number of adducts per strand to become negative
(Fig..4.4). This phenomenon was sometimes encountered with the IXR1 strain as
well. Cisplatin adducts are known to unwind DNA which may make it easier for
the primers to anneal to the genomic DNA. When the primers can anneal more
readily, more product can be formed from the same amount of substrate. The
repair rate in the radl4 strains was also examined (Fig. 4.5). This experiment
showed that the radl4 strains do indeed remove cisplatin-DNA adducts. The
rate at which this occurred was not reproducible; two experiments are shown in
Fig. 4.5 to show the range of results obtained. Full repair of the adducts took
eight hours for these strains. The strains did not grow significantly during those
eight hours. There was no significant difference in the repair of cisplatin-DNA
adducts in the radl4 ixrl strain. Interestingly, repair was observed; the radl4
strains should be deficient in global excision repair, so some other form of repair
must be occurring.
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The results with the JM43 and JM43Aixrl strains were similar to those for
the IXR1 and ixrl strains. No difference in adduct level was noted between JM43
and JM43Aixrl (Fig. 4.6). This result again implies that the lack of ixrl does not
affect the repair of the PGK gene. The cisplatin repair rates in these strains were
similar when DNA replication was not accounted for (Fig. 4.7a). These cells grew
substantially during the experiment, however, as can be seen in Fig. 4.7b. This
growth level was high enough to account for all of the increase in PCR product
(Fig. 4.7c). The adduct levels on the original DNA appear to actually increase
significantly with time. It is possible that repair of the cisplatin-DNA adducts in
these strains involved large pieces of DNA, which would mean that a significant
part of the new DNA synthesis was part of the repair event. If such were the
case, not all the decrease in tritium signal should be used to adjust the adduct
levels because the majority of the tritium signal is no longer due to cell growth.
Alternatively, this result would also be explained if repair occurred in the time
between platination and recovery. Repair that occurs before the time 0 point is
naturally not seen in this experiment; if a significant amount of the repair occurs
when the DNA is treated with cisplatin, then that would explain the low rate of
repair in this experiment. Since the JM43 strains both grow significantly faster
than any of the other strains tested, it is quite likely that they are repairing their
DNA more quickly.
Restrictionenzyme cleavageof cisplatin-and trans-DDP-DNA adducts
There are several publications which discuss how cisplatin and trans-DDP
form adducts on DNA which block restriction enzyme cleavage (for example,
Ushay et al., 1981;Balcarova et al., 1992). A series of experiments was performed
to define how well some restriction enzymes were blocked by such damage on
the DNA. DNA was treated with the same concentration of cisplatin or transDDP for varying times. The cleavage of several restriction enzymes was checked
233
for the minimum times needed to cleave fully unplatinated DNA, since if DNA is
over-digested, nonspecific or star activity can occur, which also cleaves the DNA.
Digestion was measured by running digested DNA on agarose gels and
monitoring the conversion of the closed-circular plasmid band to nicked and
linear forms.
Both pUC'19and M13mp18 DNA were used in these experiments.
M13mp18 treated with trans-DDPwas cleaved with SacI. SacI digestion is fully
inhibited by DNA damaged at a (D/N)b between 0.12 and 0.17 (Fig. 4.8).
Cisplatin and trans-DDP damaged pUC19 were cleaved with BamHI, SmaI, PstI,
and EcoRI. Two of these experiments are shown in Figs. 4.9 and 4.10, and the
data are summarized in Table II. Higher levels of cisplatin cause all three of the
plasmid bands to migrate faster on the gel. The nicked and linear bands merge,
as has been shown separately (data not shown); this phenomena has also been
noted in the literature (Ushay et al., 1981). This result is probably due to the
adducts bending the DNA and altering its mobility in the gel. It is clear from
Table II that different restriction enzymes cleave damaged DNA to different
extents.
The different extents of cleavage may be because, at lower levels of
platination, the damage is not actually within the restriction enzyme site. A sitespecifically platinated construct was made as shown in Fig. 4.11. This construct
contains a trans-DDP-DNA adduct in the XhoI site, CTCGAG. The
oligonucleotide was characterized in several ways. It was shown to be free of
detectable impurities by running HPLC columns that separate unplatinated and
platinated oligonucleotides. The oligo itself was digested to completion to
demonstrate that all of the G bases are bound to the platinum; no G peak was
visible in the HPLC trace, whereas a peak for Pt-d(G)2 was present. The
platination level of the oligonucleotide was also checked by atomic absorption
234
analysis, and the oligo was found to be fully platinated. The presence of the
adduct on the final construct was also determined. Replication blockage
experiments demonstrate that polymerases are blocked by platinum adducts; it
has been found that different adducts are blocked to different extents. If one
assumes complete blockage of the polymerase, then one can determine how
many adducts are present on the DNA. The trans-DDP-M13-Xhol2C construct
was characterized in a replication-blockage experiment (Comess, 1991). The
polymerase was blocked in at least 75% of the strands at the site of platination,
indicating that at least 75% of the DNA circles had platinum attached. The actual
number of circles with platinum attached is probably higher than this since in
similar experiments, the cisplatin d(GCG) 1,3-intrastrand adduct only blocked
replication by about 75% (Comess, 1991). The adduct probably does not
rearrange into interstrand adducts since it was never heated (Locker et al., 1995).
These experiments demonstrate that the trans-DDPd(GAG) adduct was present
both on the oligonucleotide and on the final trans-DDP-M13-Xhol2C construct.
trans-DDP-M13-Xhol2C was cleaved fairly efficiently by XhoI, even
though the adduct was in the XhoI site. This result was observed for several
different preparations of trans-DDP-M13-Xhol2C (data not shown). In Fig. 4.12,
it is clear that the closed circular trans-DDP-M13-Xhol2C construct was
converted to nicked and linear pieces. The XhoI enzyme did not convert this
construct to linear DNA as readily as it linearized the unplatinated construct
(lanes 4 and 5). The trans-DDP-M13-Xhol2Cconstruct was treated with cyanide
to remove the platinum adduct. The resulting unplatinated construct was
cleaved more readily than trans-DDP-M13-Xhol2C (lanes 6 and 7); this particular
preparation did not have much closed circular DNA, however, other experiments
showed complete cleavage of such DNA when far more was present. There was
more linear product in both of the constructs without a platinum adduct. XhoI
235
cleaved the DNA with the platinum adduct, but it could not make a doublestranded cut as easily. The platinum adduct does not block the binding and
nicking activity of the XhoI enzyme, even when the adduct is present in the
binding site.
Restriction enzyme based DNA adduct repair assay
A repair assay can be used that takes advantage of the ability of platinumDNA adducts to block cleavage by some restriction enzymes. Certain restriction
enzymes, such as SmaI, are blocked by relatively low quantities of platinum
adducts. The repair of the adducts can be examined with the restriction enzymes
since when adducts are removed, the restriction enzyme can cut the DNA. A
substrate for such a repair assay was created by treating pUC19 DNA with
cisplatin or trans-DDP. The treated plasmid was cut with SmaI to linearize any
DNA that SmaI could cut naturally, and the closed circular portion was isolated.
The DNA was run on two sucrose gradients to purify further the closed circular
form so that the assay only measured actual repair events. Any nicks present in
the DNA can act as initiation points for polymerases which might remove the
adducts by displacing the damaged strand. The presence or absence of the
adducts was verified by digesting the plasmids with SmaI and NdeI (Fig. 4.13).
NdeI does not have a potential platination site in its recognition site and is not
particularly sensitive to platinum adducts, so it can be used to help generate a
small, readily visible piece of DNA. The SmaI+NdeI digest releases a small piece
of DNA in unplatinated constructs (Fig. 4.13, lane 4). Cisplatin and trans-DDP
treated plasmids do not form this small piece of DNA (Fig. 4.13, lanes 2 and 3).
The untreated, cisplatin treated, and trans-DDP treated plasmids were
used in a repair assay, where they were treated with HeLa cell extract and then
assayed for release of the small piece of DNA (Fig. 4.14). The presence of the
small piece of DNA indicates that there were an insufficient number of adducts
236
present on the DNA to block the SmaI restriction enzyme. The small piece of
DNA is present in the untreated plasmid lane (Fig. 4.14, lane 1, at product
arrow). There is no significant amount of product in the cisplatin treated
plasmid lane (Fig. 4.14, lane 2), which implies that the cisplatin-DNA adducts are
not readily repaired in this assay. The product band is present in the trans-DDP
treated plasmid lane (Fig. 4.14, lane 3), which means that the trans-DDPadducts
are repaired enough to permit SmaI cleavage of the DNA. The trans-DDP
damaged plasmid was repaired more strongly than the cisplatin damaged
plasmid.
DISCUSSION
Platinum levels on yeast DNA--generalDNA
The repair shielding hypothesis was supported by experiments examining
the amount of platinum adducts on yeast genomic DNA. The level of platinum
adducts on the DNA was quantified in various IXR1 and ixrl strains by atomic
absorption analysis. The repair shielding hypothesis predicts that the adduct
levels will be lower in the ixrl strains because the adducts are removed more
readily by the excision repair system. When the adduct levels were measured in
IXR1 and ixrl by atomic absorption, the ixrl DNA clearly had less platinum
attached (Table I, RAD strain). This result was previously reported by S. Brown
(Brown et al., 1993). This result was further investigated by examining the
excision repair deficient strains radl, rad2,rad4,radlO,and radl4. If the decrease
in cisplatin adduct levels in the ixrl strain is due to the shielding of excision
repair, then inactivating the excision repair pathway should remove the
difference in adduct levels between ixrl and IXR1 strains. The repair deficient
strains were treated with much lower levels of cisplatin so that the number of
dead cells was not significantly greater in the rad strains. Dead cells can lead to
abnormally high levels of adducts since they no longer repair their DNA. At
237
these lower levels of platination, rad strains had about the same number of
adducts as RAD strains (Table I). Since cisplatin-DNA adducts are removed by
excision repair, the rad strains cannot repair the cisplatin-DNA adducts as readily
so more adducts were accumulated, for example, compare rad2 to RAD (Table I),
taking into account the higher levels of cisplatin used to treat the RAD strains.
The excision repair deficient strains did not show any difference in
platinum adduct levels between rad ixrl and radIXR1 strains, which supports the
repair shielding hypothesis. The difference between RAD IXR1 and ixrl strains
present in the atomic absorption experiment disappeared in the radl, rad2,rad4,
and radlOstrains. Therefore, the decreased level of adducts in the ixrl strain was
dependent upon active excision repair, indicating that Ixrl shields the adduct
from excision repair in the IXR1strain. In the cytotoxicity assays of radl and
radlO,the rad ixrl strains were still less sensitive than the rad IXR1 strains
(Chapter 3), although the strains were not different in the atomic absorption
assay. The cytotoxicity assay was more sensitive and more reproducible than the
platinum level experiments, so small differences are more apparent. The results
of the atomic absorption experiments with the rad14strain are unclear; while the
average (D/N)b of the two strains were significantly different, the standard
deviation on those numbers was large enough to cause them to overlap. It is
likely that further experiments would demonstrate that there is no significant
difference between these two strains. Therefore, no real conclusion can be made
about the presence of a difference between the radl4 IXR1 and rad14ixrl strains.
Overall, these results support the repair shielding hypothesis because the
difference in platination levels between the IXR1and ixrl cells was dependent
upon excision repair.
238
Platinum levels on actively transcribed DNA in yeast
DNA repair occurs more rapidly in the actively transcribed gene PGK
than in the rest of the DNA. Adduct levels were measured in the highly
transcribed PGK gene by quantitative PCR. In all four strains tested using both
PCR and atomic absorption analysis, the adduct levels were an order of
magnitude lower in the PCR experiments. There were fewer adducts present on
the PGK gene than on the remainder of the DNA. This result could be due to
either increased repair or decreased adduct formation. The adduct formation is
unlikely to be decreased selectively in the PGK gene, since cisplatin-DNA
adducts should form more readily in actively transcribed regions of DNA since
they are not wound up as chromatin. Increased repair of cisplatin-DNA adducts
in actively transcribed genes has been demonstrated in mammalian cells (May et
al., 1993). This phenomenon is also present in mammalian and yeast cells for UV
adducts (Bohr, 1991;Verhage et al., 1994). These results are the first
demonstration that gene-specific repair occurs in yeast cells with cisplatin
adducts.
Ixrl apparently does not block repair of cisplatin adducts in the actively
transcribed PGK gene. There was no difference in adduct levels between IXR1
and ixrl strains in the PGK gene. This result was observed in both the IXR1and
JM43 strains. Interrupting RAD14 caused the adduct levels to increase on the
PGK gene because excision repair was disabled. It takes about 5 fold less
cisplatin to give the radl4 strains the same amount of adducts as the IXR1 strains.
This result implies that the PGK gene was repaired at least in part by excision
repair in the IXR1 strain. Since the adduct levels in the IXR1 and ixrl strains
were the same, Ixrl cannot be affecting the repair of this gene. Therefore, Ixrl
does not block cisplatin-DNA adduct repair in the PGK gene, probably because it
is highly repaired due to active transcription.
239
The rate of cisplatin-DNA adduct repair was investigated in the highly
-transcribed gene PGK. There was no significant difference in the rate of the
repair of this gene between IXR1 and ixrl cells in two different yeast
backgrounds, which supports the above conclusion that Ixrl does not block
cisplatin-DNA adduct repair in the PGK gene. There is a significant amount of
repair of this gene occurring during the platination step since it has much lower
levels of adducts than the rest of the DNA. The JM43, IXR1,and rad14 strains all
had different repair rates. In the IXR1cells, the repair of the cisplatin adducts
was 50% complete in 1-2 hours, and finished within 6 hours of removal of the
cisplatin from the media. The rate of cisplatin repair in the JM43cells was not
clear since they grew so rapidly that, when the repair levels were adjusted for
DNA synthesis, no repair was apparent. The difference between the JM43result
and that for the IXR1cells was probably because JM43 cells grow significantly
faster than the IXR1cells. Since radl4 cells are deficient in excision repair, one
would expect the repair to occur more slowly than in the wild-type IXR1cells.,
As monitored by PCR, the rate of repair in the radl4 cells was not reproducible
from experiment to experiment, but in some cases, it was about 2 times slower
than that for the IXR1cells (Fig. 4.5). It is possible that there are other forms of
repair that are used in the actively transcribed regions which repair the adducts
in the rad14cells. These repair mechanisms are not as efficient as excision repair,
since more adducts build up in radl4 cells. They are, however, sufficient to help
repair the gene. This result is reasonable because other forms of repair do exist,
excision repair is simply the most studied. These results indicate that repair
occurs in the PGK.gene in both wild-type and radl4 strains.
Platinum levels on yeast DNA--Other types of repair
The platinum adduct levels in the rad52and rad9 strains were also
measured by atomic absorption spectroscopy. The results with the double-
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strand break repair deficient rad52strains are similar to those for the same strains
in the cytotoxicity assay. Interruption of RAD52 caused the cells both to become
more sensitive to cisplatin (Chapter 3) and to accumulate higher levels of adducts
( Table I). Therefore, Rad52 is involved in the cellular response to cisplatin. It
participates in double-strand break repair and recombination, and is probably
involved in the repair of cisplatin interstrand cross-links (Chapter 3). The rad52
ixrl strain was less sensitive to cisplatin and fewer adducts were present on its
DNA than for the rad52 IXR1 strain (Chapter 3; Table I). Rad52 is therefore not
part of the mechanism by which Ixrl affects cisplatin cytotoxicity. Ixrl does not
block the type of repair that Rad52 assists. This result is reasonable since Ixrl
does not bind to interstrand cross-links. Rad52 repairs cisplatin-DNA adducts,
probably interstrand adducts, but is not blocked by Ixrl.
The results with the checkpoint mutant, rad9,are substantially different
from those obtained in the cytotoxicity assay experiments. There was very little
difference between the rad9and RAD strains in the cytotoxicity assay, which
made it seem unlikely that Rad9 affected cisplatin cytotoxicity significantly. The
level of cisplatin bound to the DNA in the rad9 resting cells, however, was
substantially higher than that for RAD cells (Table I). This result may indicate
that Rad9 does have some role in signaling the cell to remove cisplatin adducts
from DNA. It is possible that, in resting cells, Rad9 is necessary for cisplatinDNA adduct repair whereas it is not required for cells that proceed more swiftly
through the GO phase of the cell cycle.
Comparison to mammalian and E. coli cell adduct levels
The levels of cisplatin bound to wild-type yeast DNA were about two
orders of magnitude higher than equally toxic levels in human or Escherichiacoli
cells (Razaka et al., 1986; Bouayandi et al., 1993; Mamenta et al., 1994).
Apparently, yeast can survive higher levels of cisplatin adducts, at least in their
241
resting state. The dose of cisplatin needed to kill yeast cells was also higher than
that for human or E. coli cells (Razaka et al., 1986; Bouayandi et al., 1993;
Mamenta et al., 1994). Presumably, yeast repair many of these adducts when
they start replicating. The adduct levels were an order of magnitude lower in
genes that are actively transcribed, which indicates that many of the extra
adducts are present in regions of the DNA where they will not block RNA
polymerases.
RestrictionEnzyme digestionof platinum-treatedDNA
Restriction endonucleases bind DNA prior to cleaving and thus model
other DNA binding proteins. If a platinum-DNA adduct can disrupt cleavage by
a restriction enzyme, it might also disrupt the activity of a transcription factor or
polymerase. Experiments were performed to investigate whether platinumDNA adducts affect all restriction enzymes. Cisplatin and trans-DDPDNA
adducts have been shown to block restriction enzyme digestion in the literature
(Ushay et al., 1981; Balcarova et al., 1992; Brabec and Balcarova, 1993, and
references therein). Contrary to these results, a trans-DDP-d(GAG) adduct in the
XhoI recognition site did not block XhoI from nicking and cleaving the DNA.
The activity of XhoI was not completely blocked by the trans-DDPadduct, even
though other restriction enzymes were blocked by other site-specific platinum
adducts (Naser et al., 1988;Comess et al., 1992). These results indicate that
different restriction enzymes are affected differently by platinum-DNA adducts.
This result was not found in previous publications.
Several other restriction enzymes were also investigated. BamHI, SmaI,
and PstI digestions were all blocked at approximately the same relatively low
adduct levels following treatment by cisplatin or trans-DDP(Table II). SacI,
which recognizes the inverse of the XhoI site, required many more trans-DDP
adducts to block its cleavage than were required for repression of BamHI
242
activity. Inhibition of EcoRI required more cisplatin or trans-DDPadducts than
BamHI or SmaI or PstI. These differences may be because the adducts present in
and around the recognition sites were different types. The quantity of adducts
blocking BamHI, SmaI and PstI was sufficiently low that there is only an adduct
in the recognition site of about one in six plasmids. This result means that the
distortions caused by adducts in the surrounding DNA must affect the cleavage
of these enzymes. The difference cannot be solely due to the presence of d(GG)
sites in some sites and not in others, because trans-DDPdoes not bind to d(GG)
sites, yet the same difference was observed. The higher levels of platinum
required to inhibit SacI and EcoRI reflect approximately one adduct within the
recognition site in about 75% of the plasmids. Apparently, the adducts must be
much closer to the recognition site to disturb cleavage by these enzymes. The
different restriction enzymes probably contact the DNA in different manners and
with different specificities. EcoRI is known to make 12 hydrogen bonds with its
recognition sequence, including one to the N7 of the guanine in its recognition
sequence, which is where any adducts in the recognition sequence would bind
(McClarin et al., 1986). BamHI contacts the DNA in both the major and minor
grooves, and has a similar crystal structure to that of EcoRI (Newman et al., 1994;
Kang et al., 1995). SmaI contacts not only both the N7 of the guanines and the
phosphate backbone within the site but also the three adjacent bases on each side
(Withers and Dunbar, 1995). Clearly, the additional contacts of SmaI will make it
less tolerant to the distortion of the DNA duplex caused by platinum adducts.
These experiments show that different restriction enzymes are affected
differently by adducts, implying that generalizations about adducts blocking the
binding of whole groups of proteins are not valid.
Several other publications measured the inhibition of restriction
enzymes by platinum adducts. The data for BamHI presented here is similar to
243
that from one such publication (Ushay et al., 1981). A different group has
published two papers studying EcoRI, BamHI, and SalI (Balcarova et al., 1992;
Brabec and Balcarova, 1993). Both studies were performed on the same piece of
DNA and it was platinated in the same manner; in all respects, the experiments
were identical. Yet the results were significantly different, since the second
paper reported that the inhibition of BamHI, EcoRI and Sall required cisplatin
adduct levels that were an order of magnitude lower than in the first paper.
Even though the two papers have several of the same authors, there is no
reference to the first paper in the second. Both papers report that BamHI and
EcoRI were inhibited by similar levels of cisplatin, which does not agree with the
present results. The papers do not indicate the flanking sequences of the
restriction enzyme sites, nor do they use a standard plasmid, so comparisons of
the flanking sequence are not possible. The difference between the current data
and these past results cannot be explained since they give no explanation for
their extremely different results, making it unclear which adduct levels should be
used in comparisons to the present data.
Restriction enzyme based repair assay
A platinum adduct repair assay was developed and used to demonstrate
that trans-DDP DNA adducts were repaired more readily than cisplatin-DNA
adducts in HeLa cell extracts. The inhibition of restriction enzyme cleavage by
platinum-DNA adducts can be used as a probe for the presence of adducts. An
assay for the repair of those adducts was designed around platinum treated
probes that are not cleaved by SmaI. If repair occurs, the SmaI cleaves the DNA,
giving rise to a small fragment of DNA. The trans-DDPtreated probe was
partially repaired when it was incubated in HeLa cell extracts. The cisplatin
treated probe was not noticeably repaired. Therefore, the trans-DDPadducts
were repaired more easily than the cisplatin adducts in cell extracts. trans-DDP
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adducts do not seem to be repaired more readily than cisplatin adducts by
excision repair in mammalian cells as measured by transcription reactivation
experiments (Mello et al., 1995). Increased repair in mammalian cell extracts has
been seen with two other assays as well; the reason for the discrepancy between
these experiments is unknown (Heiger-Bernays et al., 1990; Mello et al., 1995).
The more proficient repair of the trans-DDP-DNA adducts may be because HMG
domain proteins do not bind to these adducts, and thus do not block their repair.
The HMG domain proteins present in the HeLa cell extract may block the repair
of the cisplatin adducts but not the repair of the trans-DDPadducts. Another
possible explanation is that the trans-DDPadducts may be more easily
recognized by the repair proteins, since trans-DDPadducts distort the DNA in a
different manner from cisplatin adducts. Whatever the correct explanation is,
trans-DDP adducts are repaired more readily than cisplatin adducts in cell
extracts.
CONCLUSIONS
The data in this chapter support the hypothesis that Ixrl shields cisplatinDNA adducts from excision repair. They also demonstrate that cisplatin-DNA
adducts are repaired more readily from actively transcribed genes in S. cerevisiae.
In another set of experiments, it was shown that a restriction enzyme, XhoI,
could still cleave DNA even with a trans-DDPadduct in its recognition site. This
result and the results with the other restriction enzymes indicate that the
inhibition of cleavage by platinum adducts is not a universal phenomenon. It is
thus likely that other DNA processing proteins exist which platinum adducts do
not inhibit. It is important that generalizations about inhibition of proteins are
not made too broadly, since proteins seem to be able to adapt to DNA damage.
Finally, it was demonstrated that trans-DDP-DNA adducts are repaired more
readily than cisplatin-DNA adducts in mammalian cell extracts. Overall, these
245
data support the conclusion that the repair of cisplatin-DNA adducts is a key
element of its cytotoxicity.
246
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Bellon, S.F. and S.J. Lippard (1990)Bending studies of DNA site-specifically
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Bohr, V. (1991) Gene specific DNA repair. Carcinogenesis 12, 1983-1992.
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Brabec, V. and Z. Balcarova (1993)Restriction-enzyme cleavage of DNA modified
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Brown, S.J.,P.J. Kellett and S.J. Lippard (1993)Ixrl, a yeast protein that binds to
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Ciccarelli, R.B., M.J. Solomon, A. Varshavsky and S.J. Lippard (1985) In vivo
effects of cis- and trans-diamminedichloroplatinum(II) on SV40chromosomes:
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Biochemistry 24, 7533-7540.
Comess, K.M. (1991) M.I.T. Thesis
Comess, K.M., J.N. Burstyn, J.M. Essigmann and S.J. Lippard (1992)Replication
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Jennerwein, M.M. and A. Eastman (1991)A polymerase chain reaction-based
method to detect cisplatin adducts in specific genes. Nucleic Acids Res. 19, 62096214.
Kang, Y.K., H.B. Lee, M.J. Noh, N.Y. Cho and O.J. Yoo (1995) Different effects of
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Locker, D., M.. Decoville, J.C. Maurizot, M.E. Bianchi and M. Leng (1995)
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Mamenta, E.L., E.E. Poma, W.K. Kaufmann, D.A. Delmastro, H.L. Grady and S.G.
Chaney (1994)Enhanced replicative bypass of platinum-DNA adducts in
cisplatin-resistant human ovarian carcinoma cell lines. Cancer Res. 54, 3500-3505.
May, A., R.S. Nairn, D.S. Okumoto, K. Wassermann, T. Stevnsner, J.C. Jones and
V.A. Bohr (1993)Repair of individual DNA strands in the hamster dihydrofolate
reductase gene after treatment with ultraviolet light, alkylating agents, and
cisplatin. J. Biol. Chem. 268, 1650-1657.
McClarin, J.A., C.A. Frederick, B.C. Wang, P. Greene, H.W. Boyer, J. Grable and
J.M. Rosenberg (1986)Structure of the DNA-EcoRI endonuclease recognition
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Mello, J.A., S.J. Lippard and J.M. Essigmann (1995) DNA adducts of cis-
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Naser, L.J., A.L. Pinto, S.J. Lippard and J.M. Essigmann (1988)Chemical and
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Newman, M., T. Strzelecka, L.F. Dorner, I. Schildkraut and A.K. Aggarwal (1994)
Structure of restriction endonuclease BamHI and its relationship to EcoRI.
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Philippsen, P., A. Stotz and C. Scherf (1991) DNA of Saccharomyces cerevisiae.
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248
Sambrook, J., E.F. Fritsch and T. Maniatis (1989). Molecular cloning: a laboratory
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Ushay, H.M., T.D. Tullius and S.J.Lippard (1981)Inhibition of the BamHI
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Biochemistry 20, 3744-3748.
Verhage, R., A.-M. Zeeman, N. de Groot, F. Gleig, D.D. Bang, P. van de Putte and
J. Brouwer (1994) The RAD7 and RAD16 genes, which are essential for
pyrimidine dimer removal from the silent mating type loci, are also required for
repair of the nontranscribed strand of an active gene in Saccharomyces cerevisiae.
Mol. Cell. Biol. 14, 6135-6142.
Withers, B.E. and J.C. Dunbar (1995)Sequence-specific DNA recognition by SmaI
endonuclease. J. Biol. Chem. 270, 6496-6504.
Zhen, W., M.K. Evans, C.M. Haggerty and V.A. Bohr (1993)Deficient gene
specific repair of cisplatin-induced lesions in xeroderma pigmentosum and
Fanconi's anemia cell lines. Carcinogenesis 14, 919-924.
249
Table I
(D/'N)b levels measured by atomic absorption
Strain
[Cisplatin] mMa
IXR1I (x103 )
ixrl (x103 )
RAD
167
36 + 11
16 + 0
rad2
33
12 + 8
9 +3
rad4
33
13 + 3
15+ 3
radl4
33
13 + 3
6+3
radl
33
13 + 5
11 + 2
radiO
33'
15 + 5
16 + 3
rad9
167
64 + 23
85 + 23
rad52
83
58 + 24
20 + 1
a The different levels were used to treate the cells in order to equalize the number
of killed cells.
250
Table II
Platinum-DNA adducts block restriction enzyme activity
Restriction
Enzyme
Cleavage site
Reaction
conditions
(D/N)b values between which
restriction enzymes were
completely inhibited
cis
trans
SacI
ttcGAGCTCggt
aagCTCGAGcca
37°C, 60 min.
--
.12
.17
BamHI
cggGGATCCtct
37°C, 40 min.
.020
.028
.026
.038
gccCCTAGGaga
SmaI
gtaCCCGGGgat
catGGGCCCcta
25°C, 10 min.
.012
.020
.023
.028
PstI
gacCTGCAGgca
ctgGACGTCcgt
37°C, 10 min.
.020
.026
.028
.038
EcoRI
agtGAATTCgag
tcaCTTAAGctc
37°C, 45 min.
.089
.13
.19
.21
251
252
Figure 4.1. Gel of PCR product from PGK gene from yeast genomic DNA treated
with various levels of cisplatin. The first two lanes contain unplatinated DNA;
the first lane contains one half of the DNA of the second. This lane shows that
the PCR reaction is dependent solely on the level of substrate DNA.
253
Cisplatin
DNA 51
0*404* & to*4_S
480 bp
primers
10
*
iiiii
255
256
Figure 4.2. Analysis of PCR products from DNA extracted from IXR1and ixrl
cells treated with increasing levels of cisplatin.
257
1
0.8
r.
c7
U
.4
Vx
cn
4-A
0.6
0.4
Cbl
0.2
0
0
0.05
0.1
[Cisplatin] mM
0.15
0.2
258
Figure 4.3. Analysis of PCR products from DNA extracted from IXR1and ixrl
cells treated with cisplatin and permitted to recover in fresh media for various
times.
259
1.2
1
to
(J
u(J
0.8
0.6
0.4
0.2
0
0
1
2
3
4
Time (hrs)
5
6
7
260
Figure 4.4. Analysis of PCR products from DNA extracted from radl4 IXR1 and
radl4 ixrl cells treated with increasing levels of cisplatin.
261
I,nu.u
0.5
0.4
$a
0.3
cn
0.2
0.1
O
-0.1
-0.2
0
0.005
0.01
0.015
0.02
[Cisplatin] mM
0.025
0.03
0.035
262
Figure 4.5. Analysis of PCR products from DNA extracted from rad14IXR1 and
radl4 ixrl cells treated with cisplatin and permitted to recover in fresh media for
various times. The two graphs represent two different experiments.
263
1 .J
:
1
rj
u
7;
0.5
0
0
2
4
6
8
10
Time (hrs)
.L
1
-'
c¢/
-
0.8
0.6
cjI
0.4
0.2
0
0
2
4
6
Time (hrs)
8
10
264
Figure 4.6. Analysis of PCR products from DNA extracted from JM43 and
JM43Aixrl cells treated with increasing levels of cisplatin.
265
11
2.5
2
1.5
1
0.5
TO
0
0.1
0.2
0.3
0.4
[Cisplatin] mM
0.5
0.6
0.7
266
Figure 4.7. Analysis of PCR products from DNA extracted from JM43 and
JM43Aixrl cells treated with cisplatin and permitted to recover in fresh media for
various times. A. Adduct level calculated without accounting for cell growth. B.
Level of tritium in DNA shows tritium level decreasing as cells grow. C. Adduct
level adjusted for cell growth.
267
A
1 .
1
1
0.8
c
0.6
u 0.4
0.2
0
0
1
2
3
4
5
6
7
8
Time (hrs)
R2
Li.
1
ID
10
8
cr.
4
2
0
0
2
4
6
Time (hrs)
8
10
268
C.
4
"d
: 3
"0
2
1
;-4
0
2
4
6
Time (hrs)
8
10
269
270
Figure 4.8. SacI restriction enzyme digestion of trans-DDPtreated M13mp18
virus DNA. A. Lower levels of platinum adducts. Lane 0: untreated M13mp18
and digested M13mpl8 mixed to give marker lane. Lanes 1-8:M13mp18 treated
with various levels of trans-DDPand digested with SacI. (D/N)b levels are
0.0012, 0.0049, 0.0094, 0.015, 0.027, 0.032, and 0.054 Pt/nucleotide
for lanes 1-8,
respectively. B. Higher levels of platinum adducts. Lanes 0-8 are the same
except that lanes 1-8 contain trans-DDPtreated DNA with 0.023, 0.061, 0.085,
0.098, 0.13, 0.12, 0.17 Pt/nucleotide,
respectively.
271
trans-DDP
A.
0
1
2
3
4
5
6
7
8
partially digested
nicked
I
linear
completely
digested
\_
\
closed
circular
B.
partially
digested
completely
digested
undigested
undigested
trans-DDP
0
1
2
3
4
5
6
7
8
273
274
Figure 4.9. SmaI restriction enzyme digestion of cisplatin and trans-DDPtreated
pUC19 plasmid DNA. A. Treated pUC19 plasmid DNA run an agarose gel with
no digestion. The "m" lane contains the X-HindIII DNA size marker. The "p"
lane contains undamaged pUC19 plasmid DNA. The small band visible below
the main closed-circular plasmid band is a marker plasmid that helps show the
change in electrophoretic mobility between the various levels of platinum-treated
DNA. The (D/N)b in the cisplatin treated samples are 0.0026, .00050,0.012,
0.020, 0.026, 0.034, 0.040 Pt/nucleotide
for lanes 1-7, respectively.
The (D/N)b in
the trans-DDP treated samples are 0.0030, 0.0057, 0.011, 0.017, 0.023, 0.028, 0.038
Pt/nucleotide, for lanesl-7, respectively. B. SmaI digested pUC19 plasmid
DNA. Lane markings are the same as for (A).
275
A.
I
trans-DDPdamaged
Cisplatin damaged
1 2 3
4
5
6
7 1 2
3 4
nickedclosed circular
marker plasmid"
B.
my
0 1 2 3 4 5
partially digested
completely digested
undigested.
Cisplatin damaged
0
12
3 4 5 6 7
partially digested
completely digested
undigested
trans-DDPdamaged
6
7
5 6
7
277
278
Figure 4.10. EcoRI restriction enzyme digestion of cisplatin and trans-DDP
treated pUC19 plasmid DNA. A. Treated pUC19 plasmid DNA run an agarose
gel with no digestion. The "m" lane contains the X-HindIIIDNA size marker.
The "p" lane contains undamaged pUC19 plasmid DNA. The small band visible
below the main closed-circular plasmid band is a marker plasmid that helps
show the change in electrophoretic mobility between the various levels of
platinum-treated DNA. The (D/N)b in the cisplatin treated samples are 0.032,
0.053, 0.089, 0.13, 0.17, 0.20, 0.26 Pt/nucleotide
for lanes 1-7, respectively.
The
(D/N)b in the trans-DDP treated samples are 0.062, 0.092, 0.19, 0.21, 0.20, 0.26,
0.34 Pt/nucleotide, for lanesl-7, respectively. B. SmaI digested pUC19 plasmid
DNA. Lane 0 contains undamaged pUC19 DNA digested with EcoRI. Other
lane markings are the same as for (A).
279
A.
m
7 6
5
4 3
2
1
/
nicked
- closed circular
% marker plasmid
Cisplatin damaged
m p
1 2
3
4
5
6
7
nicked
closed circular
marker plasmid
trans-DDPdamaged
B.
partial.
m vO
1 2 34
5
6 7
digestE
Cisplatin damaged
completely
digested
undigested
1 2 3 4
partially digested
completely digested
undigested
5
6
7
trans-DDPdamaged
281
282
Figure 4.11. Construction of trans-DDP-M13-Xhol2C. Double stranded
M13mp18 was linearized with HindII (step 1). This DNA was mixed with an
excess of single-stranded M13-Xho12CDNA and denatured in formamide (step
2). The mix was then slowly renatured to give M13-Xhol2C gapped duplexes,
one strand of M13mpl8, and the excess M13-Xho12Ccircles (step 3). The gapped
duplexes were purified away from the single-stranded material by
hydroxyapatite chromatography (step 4). The trans-DDPadducted
oligonucleotide was ligated into the gapped duplex, yielding trans-DDP-M13Xhol2C (step 5).
283
GGAGCTCAGAGG
(+)
M13-Xhol2C
single strand
1) linearize
Dialysis
(+)
(+)
2) denature in formamide
3) renature in TE
IF
GGA(CTCAGAGG4) remove
M13-Xho12C
gapped duplex (+) ( )
_.
single strand s
&
(-)
Pure gapped
duplex
Ligate
&
'GAGTCTCC
pped duplex
(+)
(+)
'rrr-fArrr
284
Figure 4.12. Digestion of trans-DDP-M13-Xhol2C by XhoI. Lane 1 contains k-
HindIII DNA size marker. Lanes 2 and 3 contain trans-DDP-M13-Xhol2C DNA.
Lanes 4 and 5 contain un-M13-Xhol2C, which is made in the same way, except
the oligonucleotide used in step 5 was not treated with trans-DDP. Lanes 6 and 7
contain trans-DDP-M13-Xhol2C DNA that was treated with cyanide to remove
the platinum adduct. DNA in lanes 3, 5, and 7 were treated with XhoI restriction
enzyme.
285
DNA
M
trans
Unpt
CN-
XhoI
digestion
nicked
linear
closed
circular
1
2
3
4
5
6
7
287
288
Figure 4.13. Test of repair assay substrates. Digestion of untreated, cisplatin
treated, and trans-DDPtreated pUC19 with the restriction enzymes SmaI and
NdeI and with NdeI alone. Lane 1 contains X-HindIII DNA size marker. Lane 8
contains the pBR322-MspDNA size marker. Lanes 2 and 5 contain trans-DDP
treated pUC19 (T lanes). Lanes 3 and 6 contain cisplatin treated pUC19 (C lanes),
and lanes 4 and 7 contain pUC19 alone (U lanes). The DNA in lanes 5-7 were
treated with NdeI alone, whereas the DNA in lanes 2-4 were treated with both
SmaI and NdeI.
289
digestion
DNA M1
SmaI+NdeI
NdeI
T C U
TCU
M2
1
E.
1
2
3 4
small piece of
DNA from
SmaI+NdeI
cleavage
5
6
7
8
291
292
Figure 4.14. Restriction enzyme based repair assay performed in HeLa cell
extracts. Lane 1 contains untreated DNA, lane 2 contains cisplatin treated DNA,
and lane 3 contains trans-DDP treated pUC19 DNA. The DNA in all three lanes
was incubated with HeLa cell extracts, purified, and digested with SmaI and
NdeI. Absence of platinum adducts was observed by watching for the band
marked product. This gel is equivalent to the gel in Fig. 4.13, except that the
DNA was treated with the HeLa cell extracts.
293
UCT
plasmid-_
product--
1
2
3
295
CHAPTER 5
HMG domain proteins binding to cisplatin-DNA
adducts in vivo
296
INTRODUCTION
Titration Hypothesis
HMG domain proteins could affect cisplatin cytotoxicity in other ways
than by shielding repair of cisplatin-DNA adducts. Many of the HMG domain
proteins are transcription factors, and cisplatin treatment may disturb the
transcription of the genes they regulate (Fig. 5.1) (Donahue et al., 1990). When
HMG domain proteins bind to cisplatin-DNA adducts, they are no longer
available to bind to their natural target sites. The affinity of HMG domain
proteins for cisplatin-DNA adducts is the same as or greater than their affinity
for their natural binding sites. In cells undergoing chemotherapy, there are more
cisplatin-DNA adduct binding sites than natural binding sites, so the HMG
domain proteins may be titrated or "hijacked" away from their natural binding
sites, thus disrupting the expression of the gene(s) that the proteins regulate.
This theory is supported by work with hUBF, the human upstream
binding factor (Treiber et al., 1994). hUBF is a transcription factor for ribosomal
RNA, which are critical for cell proliferation and survival. hUBF binds to a site-
specific cisplatin-d(GpG) adduct more readily than its natural binding site.
hUBF was removed from its natural binding site by physiological levels of
cisplatin-DNA adducts. These studies were performed in vitro, and could not
take into account any response the cell might make to the removal of hUBF from
its natural binding site. For instance, the cell might raise the level of hUBF to
prevent cisplatin-DNA adducts from hijacking enough hUBF to kill the cell.
In the work described in this chapter, the titration or "hijacking"
hypothesis was tested in vivo using Ixrl. Ixrl represses transcription of the
COX5b gene (Lambert et al., 1994). Two kinds of experiments were devised.
First, the COX5b promoter was inserted before the Bt-galactosidasegene on a
yeast plasmid. S. cerevisiaecontaining the plasmid were treated with cisplatin
297
and the level of B-galactosidase activity was determined. If Ixrl were removed
from the COX5b promoter by cisplatin-DNA adducts, then the expression of iBgalactosidase should increase. Experiments with ixrl cells containing the
plasmid were run to control for inhibition of transcription by cisplatin that bound
to the plasmid gene itself. ixrl cells expressed the iB-galactosidasegene well,
since there was no Ixrl to inhibit transcription. The titration theory was also
-testedby examining transcription from the COX5b promoter in genomic DNA.
Yeast cells were treated with cisplatin, and the levels of Cox5b mRNA were
analyzed by northern analysis.
.HMG1 binds cisplatin-DNA adducts in vivo
HMG domain proteins bind to cisplatin-DNA adducts in vitro. The
binding of HMG1, Ixrl, and hUBF have all been studied in detail, and several
other HMG domain proteins bind to globally platinated DNA (Pil and Lippard,
1992;Chow et al., 1994;Treiber et al., 1994;Whitehead and Lippard, 1995). DNA
in vivo is quite different from that used for in vitro studies. Genomic DNA is
longer and has proteins bound to it, such as histones that wind it up into smaller
packages. Therefore, it is necessary to establish whether HMG1 binds to cisplatin
l:reated genomic DNA. This question was first addressed by experiments
performed in the Scovell laboratory (Scovell et al., 1987;Hayes and Scovell,
1991a; Hayes and Scovell, 1991b),which were repeated and expanded here. It
was originally reported that HMG1 was cross-linked to the DNA by cisplatin.
Since this work was performed before HMG1 was known to bind to cisplatinDNA adducts, it was of interest to determine whether the original experiments
could be interpreted differently.
298
MATERIALS AND METHODS
Yeast strains and plasmids
The yeast strains used were JM43,JM43Aixrl, IXR1and ixrl. Their
genotypes are given in Chapter 2. Plasmid pYep contains the COX5b promoter
inserted before the i-galactosidase gene on a yeast plasmid (obtained from the
Cumsky laboratory). All media types are described elsewhere (Ausubel et al.,
1994).
Testing the titration hypothesis
Plasmidbasedtest
A plasmid, pYep, with the COX5b promoter linked directly to the 3galactosidase gene was transformed into yeast. Freshly transformed cells were
used in the assays since the P-galactosidase activity drops off after about a week.
JM43 and JM43Aixrl cells containing the plasmid were inoculated into SCAUra
(2% galactose) media and the cells were grown into log phase. The cells were
then pelleted and resuspended in 5 mL of fresh media containing varying levels
of cisplatin. The cells were incubated for 6 hours at 30°Cin culture tubes on a
rotating wheel. The cells were then frozen at -80°C. The assay was adapted from
that in Current Protocols in Molecular Biology (Ausubel et al., 1994). When the
assay was performed, the cells were thawed, pelleted in the clinical centrifuge,
and resuspended in 5 mL of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10
mM KCl, 1 mM MgSO4, 50 mM -mercaptoethanol,
pH 7.0). The OD600 (optical
density) was determined. The assay was performed by bringing 10,50, 100, or
500 gl of cells to 1 mL with Z buffer; two different dilutions were used for each
sample. A drop of 0.1% SDS and two drops of chloroform were added to each
sample with a Pasteur pipet. The samples were then vortexed for 15 seconds to
break the cells open. They were equilibrated to 30°C in a water bath for 15
minutes. The color reagent, o-nitrophenyl-p-D-galactoside (ONPG) was added to
299
each tube at 0.2 mL of a 4 mg/mL solution in 0.1 M KPO4, pH 7.0. The tubes
were mixed by vortexing for 5 seconds. The reactions were permitted to run at
30°C until the solution was a medium yellow. The reaction was stopped by
adding 0.5 mL of 1M Na2CO3; the reaction time was noted. The tube was
centrifuged for 5 minutes in an Eppendorf centrifuge to pellet the cell debris.
The OD420 of the samples was taken. The OD550 value of the samples was
consistently 0, which indicates that the cell debris was completely pelleted.
1-
Galactosidase activity was calculated according to the standard equation U =
1000*(OD420)/[(t)(V)(OD600)]where t=time the reaction ran (min), V=volume of
cell culture used (mL). Each experiment was performed 3-5 separate times; the
error bars in the figures represent the variation among these experiments at 2o.
Cox5bmRNA analysis
--Cisplatin treatmentand RNA preparation
Yeast cells were treated with cisplatin in YPGE media for 6 hours at 30°C.
The 10 ml aliquots were then pelleted and resuspended in 1 ml of cold water.
The cells were transferred to sterile, RNAse free Eppendorf tubes. The tubes
were centrifuged to pellet the cells. The cell pellets were quickly frozen by
dropping the tubes in liquid nitrogen. They were then stored in the -80°C
freezer. The next day, total RNA was prepared. The cells were thawed and
resuspended
in 400 tl TES buffer (10 mM Tris-Cl pH 7.5, 10 mM EDTA, 0.5%
SDS, room temperature). An equal volume of acid phenol (phenol equilibrated
with water, but not buffered) was added. To break open the cells, the tubes were
incubated at 65°C for 30-60 minutes and were vortexed occasionally. The tubes
were put on ice for 5 minutes, and then centrifuged for 5 minutes. The aqueous
layer was removed, placed in a fresh Eppendorf tube, and another 400 p1 of acid
phenol was added. The tubes were vortexed, left on ice for 5 minutes, and
centrifuged for 5 minutes. The aqueous layer was removed to a new Eppendorf
300
tube and extracted with 400 ptLof chloroform. The aqueous layer from that
extraction was put in another Eppendorf tube. The RNA was precipitated by
adding 40 tl of 3 M NaOAc pH 5.3 and I ml of cold EtOH, incubating in the
-80°C for 5 minutes, and centrifuging for 5 minutes. The pellet was washed once
with -20°C 70% EtOH. The RNA was redissolved in 50 gl of water and the tube
was heated to 65°C for 5-15 minutes to help dissolve the pellet. A260
(absorbance) and A280 readings were taken to confirm the purity and yield of the
sample, and the RNA was stored at -20°C. All solutions were treated with
diethyl pyrocarbonate (DEPC) and autoclaved to inactivate any contaminating
RNAse.
--NorthernBlotting
All plastic apparatus were treated with 3% hydrogen peroxide for 10
minutes and then rinsed with DEPC-treated water. Approximately 30 gg of each
sample were transferred into fresh Eppendorf tubes. The samples were dried to
reduce the volume and resuspended in 6.8 gl of water. Each tube was brought to
a volume of 30 gl by adding 3 gl of 5x formamide gel-running buffer (0.1 M
MOPS pH 7.0, 40 mM NaOAc, 5 mM EDTA), 5.25 gl of formaldehyde,
and 15 gl
of formamide.
The samples were incubated at 65°C for 15 minutes and 3 gll of
formaldehyde
gel loading buffer (50 % glycerol, 1 mM EDTA pH 8.0, 0.25%
bromphenol blue, 0.25% xylene cyanol FF) was added. A 1.8% agarose gel was
poured by dissolving 1.8 gm of agarose in 62.5 ml water and, after the solution
had cooled, adding 17.9ml of formaldehyde and 19.7ml of 5x formaldehyde gelrunning buffer. The gel was pre-run at 70 volts for 5 minutes and the samples
were loaded onto the gel. The gel was run for 3-5 hours at 50-70 volts. The RNA
was transferred to nitrocellulose using the Schleicher & Schuell Turboblotter
rapid downward transfer system. The device was set up as directed and the
RNA was transferred for 3 hours. The membrane was rinsed in 2xSSC (20xSSC:
301
3M NaCl, 300mM Na citrate, trisodium salt pH 7.0) for 15 minutes and dried in a
vacuum oven for 2 hours at 80°C. The blot was pre-hybridized and hybridized in
Stratagene's QuickhybTM. The blot was placed in a sealable bag and 9 mL of
QuickhybTMwere added. The blot was then incubated with gentle shaking for
20 minutes at 50°C. The blot was probed by adding 1 ml of QuickhybTMwith 710x106 cpm Cox5b probe to the bag and incubating for 1 hour at 50°C. The blot
was rinsed twice in 3xSSC, 0.1% SDS for 15 minutes, gently blotted dry, and
exposed to film overnight. The next day, the blot was put on the
phosphorimager cassette overnight, and then the bands were quantitated. The
blot was stripped by twice adding boiling O.1xSSC,0.1% SDSto the blot and
shaking for 15 minutes. The blot was reprobed as above using the probe for
actin. The probes used were 5'-GCACGAGGAGCGTCGTCACCGGCA-3'for
actin and a 200 bp fragment of COX5b for Cox5b mRNA (Hodge et al., 1989). The
probes were labeled with y-3 2 P-ATP and kinase, or with random priming with oa3 2 P-dCTP and
Klenow, respectively. The graphs presented in the figures
represent individual experiments. The numbers associated with the Cox5b
mRNA levels were arbitrary units, and there was no numerical correspondence
between different graphs since the numbers are based on the phosphorimager
output.
Binding of HMG-1 to DNA in platinated chicken erythrocyte nuclei
--Isolation of chicken erythrocyte (c.e.) nuclei
Chicken blood was purchased from Pel Freeze. The white blood cells
were removed by repeated centrifugation and resuspension in isolation buffer
(0.85%NaCl, 0.01% merthiolate and 6 mM Na2HPO4, pH 7.0). The blood was
stored at -20°C in isolation buffer plus 25% glycerol.
To isolate the nuclei, the cells were thawed and spun down in Corex tubes
for 10 minutes at 5000 rpm. The supernatant and the remaining white blood cells
302
were removed, and the cells were resuspended in RSB(10 mM NaCI, 3 mM
MgCl2, 0.25 M sucrose and 10 mM Tris-HCl pH 7.2) + 0.5%/o
NP40 (detergent).
The pellets were resuspended with a Dounce homogenizer and the B pestle. This
treatment broke open the red blood cells. The nuclei were pelleted for 10 min at
5000 rpm. They were washed once more (pellet resuspended by homogenizing)
in RSB + 0.5% NP40, and then 1 to 4 times in RSB alone. The washing removed
excess chromosomal proteins; the progress of the washes could be partially
followed by monitoring the disappearance of red hemoglobin. All the red color
was gone after the second RSB + NP40 wash.
--Platination of c.e. nuclei
The nuclei pellets were resuspended into RSB. The tubes were pooled and
the amount of DNA was determined by adding 0.9 mL of 5 M urea, 2 M NaCl to
0.1 ml of nuclei and measuring the A260 value. Cisplatin was dissolved into 10
mL of water, and then the necessary amount to give the desired ratio between
DNA and platinum was added to the nuclei. Incubations were done at room
temperature; unplatinated controls were also incubated at room temperature.
The nuclei were spun down as before and washed once with RSB.
--Micrococcal nuclease digestion
The pellet was resuspended into 3 mL of RSB + 1 mM CaC12. 210 units of
micrococcal nuclease were added and the tube was incubated at 37 C for 30
minutes. The nuclei were spun down as before.
--HMG-1 extraction
The final pellet was resuspended in extraction buffer (0.35 M NaCl, 5 mM
Na2HPO4 pH 7.0); this salt treatment cracked open the nuclei. The pellet was
Dounce homogenized with the B pestle 5-8 times to disrupt the DNA. The DNA
was then pelleted by centrifugation for 15 minutes at 10000rpm. The
supernatant was carefully removed and the proteins were precipitated by adding
303
0.5 volumes of 100% trichloroacetic acid. The precipitating mixture was
incubated on ice for 15-45 minutes and then spun for 15 minutes at 15000 rpm.
The sample appeared as a small film on the sides of the tubes and was washed
once in acetone-HCl and once in acetone. The pellet was then dissolved in SDSPAGE loading buffer (100 mM Tris pH 6.8, 200 mM dithiothreitol, 4 "/ SDS, 0.2 Y.
SDSbromphenol blue, and 20 % glycerol). The samples were analyzed by 12%0/
PAGE gel electrophoresis and subsequent Southwestern blotting. Southwestern
blots were probed with radiolabelled cisplatin-treated DNA and unplatinated
DNA (Pil and Lippard, 1992). Western blots were performed using anti-HMG1
antibody (Pil and Lippard, 1992).
RESULTS
Titration Hypothesisexperiments
Plasmid-based assay
The titration hypothesis was first tested by using a plasmid, pYep, with
the Cox5b promoter in front of the 9-galactosidase gene. i-Galactosidase was
easily assayed in crude cell extracts. These experiments were all performed with
cells in exponential phase growth because B-galactosidase expression only occurs
under these conditions. JM43 and JM43Aixrl cells were treated with various
levels of cisplatin and the level of B-galactosidase activity was measured (Fig.
5.2). As expected, when no cisplatin was added, the JM43Aixrl cells produced
much more B-galactosidase because there was no Ixrl to inhibit transcription
from the Cox5b promoter. As cisplatin was added, the level of B-galactosidase in
the JM43Aixrl cells dropped because the cisplatin bound to the pYep plasmid
and blocked transcription. If the titration hypothesis were correct, the level of Bgalactosidase expression in the JM43 cells should go up when the cisplatin was
added, because the adducts would titrate the Ixrl away from the Cox5b
promoter. No significant increase in B-galactosidase expression was seen in Fig.
304
5.2 or in any similar experiments. This assay was performed five times using
these conditions, and three times where cisplatin levels between 0 and 0.5 mM
were investigated.
The small peak visible in the JM43 data in Fig. 5.2 was no
greater in any other experiment, and was not visible in all experiments. These
data do not support the titration hypothesis.
Several other treatments of the cells were also investigated, to be sure that
there was no other indication of titration activity. Cells were treated with
cisplatin for two hours instead of six, which made no difference. In some
experiments, cells were incubated in fresh media after the cisplatin treatment to
see if the cells needed time to respond to the cisplatin treatment. Neither a 16
hour (Fig. 5.3) nor a 2 hour incubation showed any increase in the levels of 13galactosidase in the JM43 strain as cisplatin was added. The JM43Aixrl strain
does not lose 3-galactosidase activity with increasing cisplatin levels in the 16
hour recovery experiment, probably because the cells have repaired the cisplatin
from the plasmid (Fig. 5.3). Cells were also treated with the same amount of
cisplatin for various times to see whether there was any increase in the £galactosidase activity in the JM43 strain (Fig. 5.4). The results for this experiment
were similar to those for the other experiments. None of these experiments
supports the titration hypothesis.
mRNA based assay
Experiments investigating the titration hypothesis were also performed by
examining the level of Cox5b mRNA. These experiments do not suffer from the
drawbacks of using a plasmid based system, for example, relying on a small
circle of DNA that is not necessarily processed in the same manner as genomic
DNA. As in the g-galactosidase activity experiments, cells were treated with
cisplatin and then broken open. Total RNA was extracted and northern blots
were probed with anti-Cox5b probe. The loading was controlled in all
305
experiments by subsequently probing the blots with an actin probe. Several
experiments were performed in which exponential phase cells were treated with
various levels of cisplatin. The Cox5b bands from a typical northern blot are
shown in Fig. 5.5a. When the cells were not treated with cisplatin, ixrl produced
a higher level of Cox5b mRNA than IXR1because Ixrl was not present to inhibit
transcription (Fig. 5.5a, lanes 1 and 7). These blots were quantitated on the
phosphorimager. Low levels of cisplatin did not produce any appreciable
increase in Cox5b mRNA in the IXR1strain (Fig. 5.5b). Higher levels of cisplatin
caused the level of Cox5b mRNA to decrease in both the IXR1and ixrl strains
(Fig. 5.6); there was no significant transient increase in the IXR1strain. Both of
these experiments were repeated several times with no different results.
Different conditions of cisplatin treatment were also investigated in this
assay. In one experiment, the cells were permitted to recover for 16 hours in
fresh media after the cisplatin treatment. This experiment did not yield any
particularly different results from the experiments where recovery was not
permitted (Fig. 5.7). A similar experiment was performed in which the cells were
treated with cisplatin for 3 hours and permitted to recover for 3 hours, which did
not produce any different results. Cells grown to saturation were also
investigated by this method (Fig. 5.8). The mRNA levels in such cells are
particularly low (Werner-Washburne et al., 1993),so more mRNA was loaded
onto each blot, and the error in each lane was correspondingly greater. These
blots gave no indication that the titration hypothesis is correct. One experiment
was performed by using the JM43 and JM43Aixrl strains, which yielded no
different results. The IXR1and ixrl cells were also treated with cisplatin for
various time periods (Fig. 5.9). Again, this experiment did not show any increase
in Cox5b mRNA expression in the IXR1cells with increasing levels of cisplatin
treatment. None of these experiments support the titration hypothesis.
306
HMG1 binds to cisplatin-treated chicken erythrocyte DNA in chromatin
Experiments were performed to determine if HMG1 bound to genomic
DNA in chicken erythrocytes. Chicken blood was bought and first the red blood
cells and then their nuclei were isolated. The nuclei were broken open and
HMG1 was extracted with 0.35 M NaCl. The 0.35 M NaCl extraction separates
soluble proteins from a DNA and protein pellet. Most proteins other than
histones are present in the soluble fraction. The HMG1 band was confirmed by
Southwestern and Western blots (Fig. 5.10). The HMG1 bands appear in the
Southwestern blot only when probed with DNA that was treated with cisplatin
(Fig. 5.10c). Histone H1 bands show up faintly in both the Southwestern blots
since it does not bind specifically to cisplatin-DNA adducts (Fig. 5.10c,d). The
Western blot with the anti-HMG1 antibody confirms that the band(s) in question
belong to HMG1 and the two highly related proteins, HMG2 and HMG-E, which
run directly below HMG1 and sometimes smear into one band with HMG1 (Fig.
5.10b).
The chicken erythrocyte nuclei were treated with several concentrations of
cisplatin overnight. The higher levels of cisplatin caused the HMG1 to remain
with the DNA during the 0.35 M NaCl extraction and to disappear from the
soluble fraction which was loaded onto the gel for the Southwestern blot (Fig.
5.11, lanes 1-4). Inspection of the histone H1 bands of a Coomassie stained gel
run in parallel indicated that equal amounts of cell extracts were loaded into each
lane (Fig. 5.11b). The pure HMG1 run as a standard on this gel was substantially
degraded; the small upper band corresponded to the full length protein (Fig.
5.11, lane 6). The DNA-containing pellets from the 0.35 M NaCl extraction were
solubilized and the DNA was purified and analyzed for platinum content by
atomic absorption spectroscopy. The samples treated at 0.001, 0.01, and 0.15
(Pt/nucleotide) corresponded to actual (Drug/nucleotide)bound ratios of 1x10-4 ,
307
4x10-4 , and 19x10-4 (Pt adducts/nucleotide).
These experiments indicated that
HMG1 was bound to either the DNA or some other component of the 0.35 M
NaCl pellet. When the nuclei were treated with trans-DDP,a much smaller
amount of HMG1 remained in the DNA pellet (Fig. 5.11, lane 5).
In one experiment, the nuclei were treated with micrococcal nuclease after
cisplatin treatment (Fig. 5.11, lanes 7 and 8). Micrococcal nuclease cleaves the
DNA in the internucleosomal regions. This treatment caused the HMG1 to be
present in the soluble 0.35 M NaCl fraction in the sample treated with cisplatin.
This result indicates that the HMG1 which was in the 0.35 M NaCl pellet in the
original experiment was associated with the DNA, not any other component of
the pellet.
DISCUSSION
Titration Hypothesis
The titration hypothesis (Fig. 5.1) theorizes that cisplatin-DNA adducts
may divert HMG domain proteins such as Ixrl from their natural binding sites,
thus disturbing the transcription of the genes they regulate. Ixrl inhibits
transcription from the Cox5b promoter. The effect of cisplatin-DNA adducts on
transcription from the Cox5b promoter was measured in a plasmid-based system
and by examining Cox5b mRNA levels. Neither experiment yielded any data
that suggest that Ixrl was removed from its binding site sufficiently to affect
transcription from the promoter. Different levels and types of cisplatin treatment
were investigated. Two different sets of yeast strains were examined. Cells were
treated in exponential phase for most experiments, but some of the mRNA assays
were performed with cells grown to saturation. None of the different cisplatin
treatment protocols led to substantially increased expression from the Cox5b
promoter in the IXR1 strains. These experiments demonstrate that the titration
hypothesis is not valid for Ixrl and the Cox5b promoter in vivo. It is still possible
308
that such behavior occurs for other HMG domain proteins, like hUBF. These
experiments help define the parameters for devising new cisplatin analogs; they
indicate that only the blocking of the repair proteins may be crucial, not the
titration of the HMG domain proteins from their native sites.
HMG1 binds cisplatin-DNA addulctsin vivo
HMG1 has been shown to bind to DNA in chicken erythrocyte nuclei.
HMG1 is usually soluble in 0.35M NaCl extracts. When the nuclei were treated
with cisplatin, the HMG1 was found in the insoluble DNA pellet. If the nuclei
were treated with micrococcal nuclease, which cuts the internucleosomal region
of the DNA, the HMG1 was found in the soluble fraction. Therefore, the HMG1
was associated with the internucleosomal region. This result was originally
discovered in the Scovell laboratory (Hayes and Scovell, 1991a; Hayes and
Scovell, 1991b). They concluded that HMG1 must be covalently crosslinked to
the DNA by the cisplatin. This theory was reasonable at the time, since cisplatin
was found by several laboratories to cross-link proteins to DNA (Lippard and
Hoeschlele, 1979; Banjar et al., 1985; Gaczynki et al., 1990; Miller et al., 1991;
Ferraro et al., 1992). Cisplatin functions well as a cross-link reagent since it is less
dependent on either DNA sequence context or specific amino acids sidechains.
When it was discovered that HMG1 binds to cisplatin-DNA adducts in vivo,
these experiments were repeated to see if HMG1 is bound to the DNA covalently
or non-covalently. The latter is certainly possible since there are other proteins in
the 0.35 M NaCl DNA pellet, including the core histones (data not shown).
HMG1 binds more tightly to cisplatin-treated DNA than to unplatinated DNA
(Pil and Lippard, 1992), and this difference in binding may account for HMG1
staying bound to the DNA through the 0.35 M NaCl treatment. The lesser
binding of HMG1 to the DNA pellet when the nuclei were treated with transDDP indicates that such may be the case. HMG1 does not bind to trans-DDP-
309
DNA adducts (Pil and Lippard, 1992). An equal amount of trans-DDPcaused
much less HMG1 to be sequestered in the 0.35 M NaCl pellet. Since the
platination took place over a 24 hour period, the level of crosslinking caused by
trans-DDP and cisplatin should be very similar (Lepre and Lippard, 1990). In
other studies, no difference was noticed in non-histone chromosomal proteins
crosslinked to DNA by cisplatin and trans-DDP(Banjar et al., 1983;Banjar et al.,
1984). The significant difference between cisplatin and trans-DDPfor HMG1 is
probably caused by the binding affinity of HMG1 for cisplatin-DNA adducts.
The HMG1 in the 0.35 M NaCl pellet is most likely partly covalently bound to the
DNA, but a large part is probably non-covalently bound as well. In conclusion,
HMG1 binds to DNA when it is packaged as chromatin in chicken erythrocyte
nuclei.
CONCLUSION
The experiments in this thesis primarily examine the role of HMG domain
proteins in mediating cisplatin cytotoxicity. Much of this work focused on the S.
cerevisiaeHMG domain protein Ixrl. Interrupting the IXR1gene causes cells to
become less sensitive to cisplatin and to have lower levels of platinum adducts
on their DNA. The drop in sensitivity was shown to be related to the missing
HMG domains of Ixrl, not to its function as a transcription repressor of Cox5b.
The differences between the IXR1and ixrl strains were dependent on the
presence of several excision repair proteins. These results mean that the excision
repair proteins are involved in causing the differences between the two strains.
Since Ixrl binds to cisplatin-DNA adducts, Ixrl is probably shielding the adducts
from the repair machinery. Thus, the data supported the repair shielding
hypothesis. Another hypothesis, the titration or "hijacking" hypothesis, was also
investigated. Ixrl, however, was not titrated away from its binding site on the
Cox5b gene sufficiently to affect the transcription of Cox5b. This data showed
310
that the titration hypothesis was not valid for Ixrl binding to cisplatin-DNA
adducts. This thesis supports the premise that new cisplatin-related anticancer
treatments could be based on maximizing the interaction between the DNA
adduct and the HMG domain proteins so that the adducts are shielded as much
as possible from repair. These new anticancer drugs may be more effective
against tumors with acquired or intrinsic resistance to cisplatin chemotherapy.
311
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Hodge, M.R., G. Kim, K. Singh and M.G. Cumsky (1989) Inverse regulation of the
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313
314
Figure 5.1. Titration hypothesis. HMG domain proteins bind to their normal
binding site and activate or repress the transcription of gene X. When the cell are
treated with cisplatin, the HMG domain proteins are titrated away from their
natural binding sites and the transcription of gene X is not regulated properly.
315
HMG
domain
protein
~104 addu
per cell
Small number
of normal.
binding sites
Transcription activation or
repression of gene X
Protein not bound to
normal site so the
transcription of gene X is
not properly regulated
317
318
Figure 5.2. -galactosidase activity of JM43 and JM43Aixrl strains treated with
cisplatin. Cells were grown to exponential phase before cisplatin treatment.
319
10)0
-
o JM43
* JM43Aixrl
I
L7w
100 -
u
,
a)
(t
m
ul
uc3
t=
bC
I
'10 -
I
;
1
-
I
0
0.5
I
I
I
I
I
1
1.5
2
[Cisplatin] mM
I
I
2.5
l
I
3
320
Figure 5.3. -galactosidase activity of JM43 and JM43Aixrl strains treated with
cisplatin and permitted to recover in fresh media for 16 hours. Cells were grown
to exponential phase before cisplatin treatment.
321
1000
o JM43
* JM43Aixrl
-t~~
,
100 -
,4-
tt
!
uMd
v)
(n
Md
t~
tz
Crl
-T-
101
1
r
-
-
I
0
0.5
I
1
I
I
1.5
2
[Cisplatin] mM
I
2.5
3
322
Figure 5.4. -galactosidase activity of JM43 and JM43Aixrl strains treated with
cisplatin over time. Cells were grown to exponential phase before cisplatin
treatment.
323
--I(UU
100
.
·
10
T7
0
C
1
0.1
0
4
8
12
Time (hrs)
16
20
24
324
Figure 5.5. mRNA analysis of IXR1and ixrl strains treated with low levels of
cisplatin. A. Northern blot probed for Cox5b mRNA. B. Quantification of the
northern blot after loading levels were taken into account by probing for actin
mRNA. Cells were grown to exponential phase before cisplatin treatment.
325
A.
Cisplatin
level
I
ixrl
IXR1
V
ribosomal
RNA bands
Cox5b mRNA
two major forms
I
1 2 3
4
5 6 7 8 9 101112
327
B.
,]~
~
~
~ ~~oJR
87-
Z
C-)
6-
o IXR1
-
* ixrl
5-
X
O
432 (
1
0
I
I
0.02
0.04
I
0.06
[Cisplatin] mM
I
0.08
0.1
328
Figure 5.6. mRNA analysis of IXR1and ixrl strains treated with higher levels of
cisplatin. Cells were grown to exponential phase before cisplatin treatment.
329
3
2.5
2
s-0
1.5
X
0
©
U
1
0.5
0
0
0.5
1
1.5
[Cisplatin] mM
2
2.5
3
330
Figure 5.7. mRNA analysis of IXR1and ixrl strains treated with cisplatin and
permitted to recover in fresh media for 16 hours. Cells were grown to
exponential phase before cisplatin treatment.
331
16
14
12
10
0
u
8
6
4
2
0
0.5
1
1.5
2
[Cisplatin] mM
2.5
3
332
Figure 5.8. mRNA analysis of IXR1 and ixrl strains treated with cisplatin after
they were grown to saturation.
333
/
6
5
Z4
X
3
U
2
1
0
0
0.5
1
1.5
2
[Cisplatin] mM
2.5
3
334
Figure 5.9. mRNA analysis of IXR1and ixrl strains treated with cisplatin for
various times. Cells were grown to exponential phase before cisplatin treatment.
335
2
1.5
<
1
Lo
C
0.5
0
0
4
8
12
Time (hrs)
16
20
24
336
Figure 5.10. HMG1 is present in the 0.35 M NaCl extract of chicken erythrocyte
nuclei. A. Coomassie stained gel of 0.35 M NaCi extract and pure HMG1. B.
Western blot of (A) with anti-HMG-1 antibody. C. Southwestern blot of (A)
probed with cisplatin treated radiolabelled DNA. D. Southwestern blot of (A)
probed with untreated radiolabelled DNA. The arrows indicate the HMG1
bands.
337
A
D
C
B
&0o
,e
e·*e·e
1;
?,+, i
-
&
C>
Axis~
-
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4i
5m
46
46- is30Sif
30- ii=~
30
21.5-
14.3-
-
Coomassie
stain
Western
-
-
Cisplatin
No
cisplatin
Southwestern
339
340
Figure 5.11. HMG-1 was bound to DNA in cisplatin treated chicken erythrocyte
nuclei. A. Southwestern of 0.35M NaCl extracts of various chicken erythrocyte
nuclei. B. Coomassie stain of histone H1 bands from gel equivalent to (A); this
gel showed that equal amounts of proteins were loaded in each lane. Lane 1:
untreated nuclei. Lane 2-4: nuclei treated with cisplatin at 0.15, 0.01, and 0.001
[Pt]/[nucleotide] respectively. Lane 5: nuclei treated with trans-DDPat 0.15
[Pt] / [nucleotide]. Lane 6: pure HMG1 that has degraded, the top band is full
length HMG1. Lane 7: nuclei treated with micrococcal nuclease. Lane 8: nuclei
treated with cisplatin at 0.15 [Pt/nucleotide] and then with micrococcal nuclease.
341
A.
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343
About the Author
I grew up in Berkeley, California, and got my start in science at an early
age from my dad the chemistry professor and my mom the physicist. Sometimes
[ think I was born liking chemistry, and that liking was solidified in 7th grade
when we did experimental chemistry in a self-paced course. That was definetly
the most fun I had in my first 7 years of school. My AP Chemistry teacher, Ms.
Beck, helped turn me into a real chemist and reminded me why I liked it so
much. I graduated from Harvard with a A.B. in Chemistry and Physics, shades
of both my parents, but by my senior year it was clear to me that my real love
was molecular biology, especially DNA and the proteins which interacted with
it. The one request my parents made for all the money they poured into my
Harvard education was that I take a biology course, since I'd hated it in high
school; I begged off and took biochemistry instead. After realizing that even
biology was interesting, I went to work for Professor Greg Verdine in the
chemistry department, doing molecular biology and molecular dynamics
calculations. The experience I gained in his laboratory and on my summer job in
the laboratory of Professor Sung-Ho Kim of Berkeley was invaluable when I
joined Professor Steve Lippard's lab at M.I.T.. Without the experience in other
laboratories that taught me that there is no one true way of doing molecular
biology, the first years of my graduate school career would have been even more
frustrating. The games I've played since with yeast and antitumor drugs are
described in this thesis, and were fun and difficult while they lasted.