Increased CYP3A4 copy number in TONG/HCC cells but not in DNA from other humans

Abstract

Two recent screens for copy-number variations in the entire human genome found 12.4 gene copy number variations per person, including 2.5% of individuals with gains between 7q21.1 and 7q22.1, the chromosomal location of CYP3A4. CYP3A4 is involved in the metabolism of approximately 50% of all drugs, including many cancer chemotherapeutic agents. CYP3A4 gene copy was determined in DNA from 143 individuals: normal human livers, primary and secondary liver tumors, human hepatic cell lines, and immortalized cell lines representing eight ethnically diverse populations. CYP3A4 gene copy was normal in all but one sample, a primary human hepatocellular carcinoma cell line (TONG/HCC). Southern blots of TONG/HCC DNA revealed an approximate 10-fold increase in CYP3A and a corresponding increase in CYP3A mRNA expression and catalytic activity. Fluorescent in situ hybridization of TONG/HCC revealed specific amplification of the CYP3A4 gene on chromosome 7q21 but no amplification of the MDR1 gene that localizes 11.9 Mb upstream of CYP3A4. High resolution analysis of DNA copy number by comparative genomic hybridization confirmed amplification at 7q21.3–7q22. The amplicon spanned 1.7 Mb and contained 30 known genes, including the entire CYP3A locus. To determine whether CYP3A4 expression affected chemotherapeutic toxicity, LLC-PK1 cells were transduced with adenoviruses expressing CYP3A4 and P450 reductase. CYP3A4 conferred resistance to taxol, vinblastine and topotecan. These studies demonstrate that CYP3A4 copy number differences do not contribute to the normal variation in CYP3A4 expression. Tumors with increased CYP3A copy number (via amplification or increased chromosome 7q) would be expected to show reduced cytotoxicity to some chemotherapeutic drugs and potentially an increase in the outgrowth of drug resistant tumors.