Inhibition of CYP1B1 in MCF-7
and V79 H1B1 Cells in Culture.
Tuan M. Nguyen
Major: General Science/Pre-Optometry
Mentors: Dr. William M. Baird & Dr. Brinda Mahadevan
Overview
• Introduction
• Methods
• Results
• Summary
Introduction
• Polycyclic aromatic hydrocarbons (PAH)
are environmental carcinogens
• Cytochrome P450 (CYP) enzymes such
as CYP1B1 have been identified to be
involved in the activation of
dibenzo[a,l]pyrene (DBP)
Introduction
• DBP is one of PAH forms
• DBP commonly found in cigarette smoke,
diesel exhaust, urban dust and other
environmental carcinogens.
3
2
fjord re gion
DNA adduct
formation on
exposure to DBP
6
7
13
12
8
11
10
dibenzo[a,l]pyrene
( DB[a,l]P)
9
CYP 1A1
CYP 1B1
O
O
+
HO
HO
OH
OH
(+)-syn-DB[a,l]P11,12-dihydrodiol 13,14-epoxide
HO
DNA adducts
5
14
DBP diol-epoxides
DBPDE
4
1
(–)-anti-DB[a,l]P11,12-dihydrodiol 13,14-epoxide
HO
HO
HO
NH
OH
N
N
N
NH
OH
N
N

OH
O
OH
(+)-syn-DB[a,l]P11,12-dihydrodiol 13,14-epoxidedA adduct
N

OH
O
OH
(–)-anti-DB[a,l]P11,12-dihydrodiol 13,14-epoxidedA adduct
Objective
• To investigate the importance of CYP1B1
as key enzyme in metabolizing DBP to its
metabolites.
Aspects of Study
• DNA adducts
• CYP1B1 enzyme activity
• CYP1B1 gene expression
Experimental Design
DNA Adducts
MCF-7 Cells
V79 H1B1 Cells
•
•
•
•
•
•
•
•
TMS (- control)
TMS+DBP
DBP
DBPDE (+ control)
TMS (- control)
TMS+DBP
DBP
DMSO (solvent ctrl)
Methods
Add fresh media to cells
24 hrs prior to treatment
V79 H1B1
MCF-7
TMS (-)
TMS+DBP
DBP
DBPDE (+)
20 ml media
TMS (-)
TMS+DBP
DBP
DMSO (solvent ctrl)
20 ml media
24hr
DNA
isolation
Harvest
RNA &
isolation
Microsome
RT-PCR
Postlabeling &
HPLC
P450 Glo Assay
Measurement
of DNA
Adducts
•Postlabeling
•Sep-pak
•HPLC
Adducted
dinucleotide
monophosphates
Dinucleotide
adducts
Nucleoside
5’ phosphate
HPLC Profiles
6000
12000
(+)-anti-B[a]PDE-dG
(+)-anti-DB[a,l]PDE-dA
Radioactivity
5000
dG
10000
dA
4000
dA
•DBPDE + control
8000
dG
3000
6000
2000
4000
1000
2000
0
0
0
20
40
60
80
100
120
0
20
Retention Time [min]
40
60
80
100
120
P450 Glo Assay
Cytochrome P450 Glo Assay enable the measurement of the activity of
CYP1B1.
CYP1B1
Luciferin CEE
luciferin firefly luciferase
light
(P450 Glo
substrate)
Luminescence
reading
P450-Glo Assay
Luminescence (RLU)
P450-Glo Assay: S9 Microsomes
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
Room Temp
37ЎC
1mg/ml
10mg/ml
20mg/ml
Concentration
30mg/ml
41.6mg/ml
How does RNAi work?
•Antler, C. ‘Antisense RNA’, http://www.bioteach.ubc.ca/MolecularBiology/AntisenseRNA/.
RNAi
Count Cells
Untreated RNAi
Ctrl
V79 H1B1
NCsiRNA NC
Ctrl
Plate Cells
V79 H1B1
+ RNAi
Transfect
Isolate RNA
V79 H1B1
+ RNAi
MCF-7
Ctrl
MCF-7 +
RNAi
RNAi
Untreated
Ctrl
Reverse Transcription Reaction
Polymerase Chain Reaction
RNAi
Electrophoresis
- V79 H1B1 cells - MCF-7 cells
Isolated RNA
100 bp Ld
V79H NC
V79H1B1
Untreated
V79H1B1+RNAi
MCF-7 Ctrl
MCF-7+V79H1B1
100 bp Ld
RT-PCR
RT-PCR and amplification of CYP1B1 cDNA
Total RNA
Random primers
Superscript RT
RNase inhibitor
RP
First strand cDNA
RP
SPP
Amplify cDNA
SPP
PCR
Amplified product
RP – Random Primer
SPP – Specific Pair Primer for CYP1B1(18-25 nt)
Amplified CYP1B1 Gene
Ld siRNA NC V79H1B1 Ctrl
V79H1B1+RNAi
MCF-7 Ctrl
MCF-7+RNAi
Summary
• Familiarized with postlabeling technique
• P450-Glo Assay
• RNAi
• Amplified CYP1B1 gene.
Future Work
Predictions
DBP alone
TMS alone
TMS+DBP
RNAi alone
RNAi+DBP
DNA Adducts
Activity of
CYP1B1
enzyme
Expression of
CYP1B1 gene
+
-
+
-
+
N/A
N/A
-
Acknowledgements
• William M. Baird
• Brinda Mahadevan
• Kevin Ahern
• Jennifer Atkin
• Howard Hughes Medical Institute