Evaluation of a real-time polymerase chain reaction method for the quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells.
BACKGROUND
Cytochrome P450 1B1 (CYP1B1) catalyzes the bioactivation of numerous procarcinogens and it is expressed in tumor cells, including human breast cancer cells. To study CYP1B1 gene expression, it is important to have an accurate, precise, reproducible, specific, and quantitative method.


METHODS
MCF-7 human breast carcinoma cells were treated with beta-naphthoflavone (BNF; 50 microM), emodin (0.1-3 microM), trans-resveratrol (2.5-20 microM), or 0.1% dimethylsulfoxide (DMSO; vehicle control). Total cellular RNA was isolated and reverse transcribed. cDNA samples were quantified by a fluorescence assay and a constant amount (1 ng) was amplified in a real-time DNA thermal cycler (LightCycler).


RESULTS
Melting curve analysis and agarose gel electrophoresis of the amplicons resulted in a single peak and a single band, respectively. The identity of the amplicon was confirmed to be CYP1B1 by sequencing analysis. The standard curve for the real-time PCR amplification of CYP1B1 cDNA was log-linear for at least four orders of magnitude. The limit of quantitation (LOQ) of the assay was 100 copies. At the LOQ, the assay had an accuracy of 8% and a precision of 10%. The intraday (n=4) variability (expressed as percent coefficient of variation) was 9% for a sample with low CYP1B1 mRNA expression (cells treated with 0.1% DMSO; i.e., Sample A) and 3% for a sample with elevated CYP1B1 mRNA expression (cells treated with BNF; i.e., Sample B). The interday (n=4) variability was 16% for Sample A and 15% for Sample B. Emodin increased CYP1B1 mRNA expression in cultured MCF-7 cells (maximal effect of ninefold induction achieved at 1 microM), whereas trans-resveratrol suppressed it (IC(50)=6.6+/-1.0 microM, mean+/-S.E.M., n=3).


CONCLUSIONS
An accurate, precise, reproducible, and specific method is described for the real-time PCR quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells.
Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada V6T 1Z3.
Catherine Y S
CY
Cheung
Jie
J
Chen
Thomas K H
TK
Chang
D000230:Adenocarcinoma / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D001189:Aryl Hydrocarbon Hydroxylases; D001943:Breast Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000235:genetics; D045744:Cell Line, Tumor; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004642:Emodin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis; D015203:Reproducibility of Results; D000077185:Resveratrol; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013267:Stilbenes / Q000494:pharmacology; D019324:beta-Naphthoflavone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013267:Stilbenes; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D004642:Emodin; D000077185:Resveratrol