[Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines].
Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.
Tronov
V A
VA
Kramarenko
I I
II
Smirnova
T D
TD
Terekhov
S M
SM
D000477:Alkylating Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D020137:Base Pair Mismatch / Q000502:physiology*; D045744:Cell Line, Tumor / Q000187:drug effects; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D008770:Methylnitrosourea / Q000494:pharmacology*; D013997:Time Factors
D000477:Alkylating Agents; D000972:Antineoplastic Agents, Phytogenic; D008770:Methylnitrosourea; D005047:Etoposide