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.

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Presentation transcript:

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 DBP 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)

Methods Add fresh media to cells 24 hrs prior to treatment 20 ml media TMS (-) TMS+DBP DBP DMSO (solvent ctrl) DNA RNA & Microsome isolation isolation Postlabeling & HPLC 20 ml media P450 Glo Assay 24hr RT-PCR TMS (-) TMS+DBP DBP DBPDE (+) MCF-7 V79 H1B1 Harvest

Measurement of DNA Adducts Postlabeling Sep-pak HPLC Dinucleotide adducts Adducted dinucleotide monophosphates Nucleoside 5’ phosphate

HPLC Profiles Retention Time [min] (+)-anti-B[a]PDE-dG (+)-anti-DB[a,l]PDE-dA Radioactivity dG dA dG dA DBPDE + control

P450 Glo Assay Luciferin CEE (P450 Glo substrate) CYP1B1 luciferinlight firefly luciferase Cytochrome P450 Glo Assay enable the measurement of the activity of CYP1B1. Luminescence reading

P450-Glo Assay

How does RNAi work? Antler, C. ‘Antisense RNA’,

RNAi siRNA NC V79 H1B1 Ctrl 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 Untreated Ctrl RNAi - V79 H1B1 cells - MCF-7 cells

Isolated RNA 100 bp LdV79H NCV79H1B1+RNAiMCF-7 CtrlV79H1B1 Untreated MCF-7+V79H1B1 100 bp Ld

Total RNA Random primers Superscript RT RNase inhibitor RP First strand cDNA Amplify cDNA 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 LdsiRNA NCV79H1B1+RNAiMCF-7 CtrlV79H1B1 CtrlMCF-7+RNAi

Summary Familiarized with postlabeling technique P450-Glo Assay RNAi Amplified CYP1B1 gene.

Future Work DNA AdductsActivity 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