Association Between MDR1 Polymorphisms in Exon 26 and Mitomycin C Sensitivity in Cultured Human Fibroblasts

Abstract

Abstract: : Purpose: To determine whether single nucleotide polymorphisms (SNPs) in exon 26 of the multi-drug resistance gene (MDR1) account for the variable sensitivity of different fibroblast cells to mitomycin C (MMC). Methods: The sensitivity of primary fibroblast cell cultures and cell lines to low (0.1 mg/mL) and high (0.4 mg/mL) doses of MMC was evaluated by the MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) Cell Proliferation Assay. The genotype at exon 26 of MDR1 was determined by DNA sequencing. Cells were exposed to either MMC or control treatments for five minutes. The effect of MMC on cell proliferation was assessed following a six and 13 day recovery period. The association between fibroblast genotype in exon 26 of MDR1 and MMC effect on proliferation was evaluated. Results: A total of six different fibroblast cultures were selected for MMC treatment, with four cultures at nucleotide 3435 in exon 26, one culture homozygous C-C, and one heterozygote C-T. After a six day recovery, all cells exposed to 0.4 mg/mL MMC showed a range of proliferation from 35-60% compared with control or vehicle treated cells. The C-C homozygous culture showed the biggest effect of MMC with only 35% proliferation. After a 13 day recovery, cell proliferation ranged from 25-55% with the exception of one homozygous C-C culture, which showed an increase in proliferation from 35% after six days to 72% after 13 days. After a six-day recovery following exposure to 0.1 mg/mL MMC, cell proliferation was not affected. Conclusions: MMC has a variable effect on fibroblast cell proliferation, which is dependent upon dose and recovery period, but does not appear to be associated with SNPs in exon 26 of MDR1. Other important considerations include the biological activity of the individual fibroblasts, including deficiency of DT-diaphorase, and increased efflux of MMC by p-glycoprotein. Studies are in progress to determine the optimal dose to test the association between specific MDR1 SNPs and MMC sensitivity.

Keywords: drug toxicity/drug effects • genetics • wound healing