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Inhibition of CYP1B1 in MCF-7 and V79 H1B1 Cells in Culture.

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.

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Inhibition of CYP1B1 in MCF-7 and V79 H1B1 Cells in Culture.

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  1. 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

  2. Overview • Introduction • Methods • Results • Summary

  3. 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)

  4. Introduction • DBP is one of PAH forms • DBP commonly found in cigarette smoke, diesel exhaust, urban dust and other environmental carcinogens.

  5. DNA adduct formation on exposure to DBP CYP 1A1 CYP 1B1 DBP diol-epoxides DBPDE DNA adducts

  6. Objective • To investigate the importance of CYP1B1 as key enzyme in metabolizing DBP to its metabolites.

  7. Aspects of Study • DNA adducts • CYP1B1 enzyme activity • CYP1B1 gene expression

  8. Experimental DesignDNA Adducts MCF-7 Cells • TMS (- control) • TMS+DBP • DBP • DBPDE (+ control) V79 H1B1 Cells • TMS (- control) • TMS+DBP • DBP • DMSO (solvent ctrl)

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

  10. Measurement of DNA Adducts Adducteddinucleotide monophosphates Dinucleotideadducts Postlabeling Sep-pak HPLC Nucleoside 5’ phosphate

  11. 6000 5000 4000 3000 2000 1000 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 HPLC Profiles 12000 (+)-anti-B[a]PDE-dG (+)-anti-DB[a,l]PDE-dA dG dA 10000 dA DBPDE + control 8000 dG Radioactivity 6000 4000 2000 0 Retention Time [min]

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

  13. P450-Glo Assay

  14. How does RNAi work? Antler, C. ‘Antisense RNA’, http://www.bioteach.ubc.ca/MolecularBiology/AntisenseRNA/.

  15. RNAi Count Cells Untreated Ctrl RNAi Plate Cells V79 H1B1 Ctrl V79 H1B1 + RNAi siRNA NC NC Transfect Isolate RNA V79 H1B1 + RNAi MCF-7 Ctrl MCF-7 + RNAi Reverse Transcription Reaction RNAi Untreated Ctrl Polymerase Chain Reaction RNAi Electrophoresis - V79 H1B1 cells - MCF-7 cells

  16. Isolated RNA 100 bp Ld 100 bp Ld V79H NC V79H1B1 Untreated V79H1B1+RNAi MCF-7 Ctrl MCF-7+V79H1B1

  17. 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)

  18. Amplified CYP1B1 Gene Ld siRNA NC V79H1B1 Ctrl V79H1B1+RNAi MCF-7 Ctrl MCF-7+RNAi

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

  20. Future Work Predictions

  21. Acknowledgements • William M. Baird • Brinda Mahadevan • Kevin Ahern • Jennifer Atkin • Howard Hughes Medical Institute

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