Tuan M. NguyenMajor: 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

812910

13

11

fjord region 1

4

614

OHHO

OHHO

N

N

Ν

Ν

O

2

5

3

+

(–)-anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxide

O

(+)-syn-DB[a,l]P-11,12-dihydrodiol 13,14-epoxide

dibenzo[a,l]pyrene(DB[a,l]P)

OHHO

HO

OH

OH

ΝH

Ν

Ν

Ν

Ν

O

ΝHHO

HO

OH

OH

OH

(+)-syn-DB[a,l]P-11,12-dihydrodiol 13,14-epoxide-

dA adduct

(–)-anti-DB[a,l]P-11,12-dihydrodiol 13,14-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

tivity

dGdA

dG

dA•DBPDE + control

P450 Glo Assay

Luciferin CEE

(P450 Glo substrate)

CYP1B1luciferin 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 Temp37°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 RTRNase 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/ATMS+DBP - - N/ARNAi alone - - -RNAi+DBP - - -

Predictions

Acknowledgements

• William M. Baird• Brinda Mahadevan

• Kevin Ahern• Jennifer Atkin

• Howard Hughes Medical Institute