The human lymphoblastoid cell, TK6, exhibited a dose-dependent cytotoxic and apoptotic response following treatment with the food borne heterocyclic
amine, 2-amino-1-methyl-6-phenylimidazo[4,5-
b]pyridine (
PhIP). Augmentation of the p53
protein and increases in p21-WAF1 levels were also observed. Comparison of the survival by clonogenic assays and the percentage of apoptotic cells (cells containing subG1
DNA or condensed nuclei) revealed that only 10-20% of the
PhIP-induced cell death could be attributed to apoptosis that occurred in the first 24h
after treatment. MT1, a derivative of TK6 that contains mutations in both alleles of its hMSH6 gene and is mismatch repair deficient, showed a decreased apoptotic response. A significant increase (P<0.05) in apoptosis was observed in TK6 and not in MT1 following treatment with 2.5microg/ml
PhIP. A five- to six-fold increase and less than a two-fold increase in the fraction of apoptotic cells were observed in TK6 and MT1, respectively. Treatment with 5microg/ml
PhIP resulted in significant increases in apoptosis (P<0.05) in TK6 and MT1. The percentages of apoptotic cells were, however, two- to three-fold higher in TK6 than in MT1. HCT116, a hMLH1 defective mismatch repair deficient
colorectal carcinoma cell line, also exhibited lower
PhIP-induced apoptosis than its mismatch repair proficient chromosome transfer cell line (HCT116+chr3) following
PhIP treatment. These results show that
PhIP-induced apoptosis is mediated through a mismatch repair dependent pathway. Accumulation of p53 in TK6 and MT1 were evident in samples taken 24h after
PhIP treatment. Increases in p21-WAF1 were also observed in both cell lines confirming that the p53 was functional. The lower apoptotic response of MT1 but similar p53 accumulation in TK6 and MT1 suggest that the mismatch repair
protein(s) are involved downstream of p53 or that
PhIP-induced apoptosis is p53-independent.