Tuan M. Nguyen Major: General Science/Pre-Optometry Mentors: Dr. William M. Baird & Dr. Brinda...
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Transcript of Tuan M. Nguyen Major: General Science/Pre-Optometry Mentors: Dr. William M. Baird & Dr. Brinda...
Tuan M. Nguyen
Major: General Science/Pre-Optometry
Mentors: Dr. William M. Baird & Dr. Brinda Mahadevan
Inhibition of CYP1B1 in MCF-7 and V79 H1B1 Cells in Culture.
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.
DNA adduct formation on
exposure to DBPO
7
812
910
13
11
fjord region1
4
6
14
OHHO
OHHO
N
N
Ν
N
O
2
5
3
+
(–)-anti- [DB ,a l] -P11,12- 13,14-dihydrodiol epoxide
O
(+)-syn- [DB ,a l] -P11,12- 13,14-dihydrodiol epoxide
[dibenzo ,a l]pyrene( [DB ,a l] )P
OHHO
HO
OH
OH
NH
N
N
Ν
N
O
NHHO
HO
OH
OH
OH
(+)-syn- [DB ,a l] -P11,12- 13,14- -dihydrodiol epoxide
dA adduct
(–)-anti- [DB ,a l] -P11,12- 13,14- -dihydrodiol epoxide
dA adduct
DBP diol-epoxides
DBPDE
DNA adducts
CYP 1A1
CYP 1B1
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• TMS (- control)• TMS+DBP• DBP• DBPDE (+ control)
V79 H1B1 Cells• TMS (- control)• TMS+DBP• DBP• DMSO (solvent ctrl)
MethodsAdd fresh media to cells 24 hrs prior to treatment
20 ml media
TMS (-)TMS+DBPDBPDMSO (solvent ctrl)
DNA RNA & Microsome isolation isolation
Postlabeling & HPLC
20 ml media
P450 Glo Assay
24hr
RT-PCR
TMS (-)TMS+DBPDBPDBPDE (+)
MCF-7 V79 H1B1
Harvest
Measurement of DNA Adducts
•Postlabeling
•Sep-pak
•HPLC
Dinucleotideadducts
Adducteddinucleotide monophosphates
Nucleoside 5’ phosphate
HPLC Profiles
Retention Time [min]
0
1000
2000
3000
4000
5000
6000
0 20 40 60 80 100 120
(+)-anti-B[a]PDE-dG
0
2000
4000
6000
8000
10000
12000
0 20 40 60 80 100 120
(+)-anti-DB[a,l]PDE-dA
Rad
ioac
tivi
ty
dGdA
dG
dA•DBPDE + control
P450 Glo Assay
Luciferin CEE
(P450 Glo substrate)
CYP1B1
luciferin lightfirefly luciferase
Cytochrome P450 Glo Assay enable the measurement of the activity of CYP1B1.
Luminescence reading
P450-Glo Assay
P450-Glo Assay: S9 Microsomes
0100002000030000400005000060000700008000090000
1mg/ml 10mg/ml 20mg/ml 30mg/ml 41.6mg/ml
Concentration
Luminescence (RLU)
Room Temp
37°C
How does RNAi work?
•Antler, C. ‘Antisense RNA’, http://www.bioteach.ubc.ca/MolecularBiology/AntisenseRNA/.
RNAi
siRNA NC V79 H1B1 Ctrl
V79 H1B1 + RNAi
V79 H1B1 + RNAi
MCF-7 Ctrl
MCF-7 + RNAi
Plate Cells
Transfect
Isolate RNA
Reverse Transcription Reaction
Polymerase Chain Reaction
Electrophoresis
Count Cells
NC
Untreated Ctrl
RNAi
RNAi
Untreated Ctrl
RNAi
- V79 H1B1 cells - MCF-7 cells
Isolated RNA
100 bp Ld V79H NC V79H1B1+RNAi MCF-7 CtrlV79H1B1 Untreated
MCF-7+V79H1B1 100 bp Ld
Total RNA
Random primersSuperscript RT
RNase inhibitorRP
RPFirst strand cDNA
Amplify cDNASPP
SPP
PCR
Amplified product
RT-PCR and amplification of CYP1B1 cDNA
RT-PCR
RP – Random Primer SPP – Specific Pair Primer for CYP1B1(18-25 nt)
Amplified CYP1B1 Gene
Ld siRNA NC V79H1B1+RNAi MCF-7 CtrlV79H1B1 Ctrl MCF-7+RNAi
Summary
• Familiarized with postlabeling technique
• P450-Glo Assay
• RNAi
• Amplified CYP1B1 gene.
Future Work
DNA Adducts Activity of CYP1B1 enzyme
Expression of CYP1B1 gene
DBP alone + + +
TMS alone - - N/A
TMS+DBP - - N/A
RNAi alone - - -
RNAi+DBP - - -
Predictions
Acknowledgements
• William M. Baird• Brinda Mahadevan
• Kevin Ahern• Jennifer Atkin
• Howard Hughes Medical Institute