Absence or decreased levels of the hMLH1 protein in human gastric carcinoma cell lines: implication of hMLH1 in alkylation tolerance

Abstract

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage.

Involvement of the DNA mismatch repair system in antineoplastic drug resistance

“…It has been suggested that defects in the MMR system are linked via some pathway with the proapoptotic factor Bax and resistance to programmed cell death (Brimmell et al 1998). In accordance with this suggestion, there is the Friedman et al 1997N-Methyl-N¢-nitro-N-nitrosoguanidine Kat et al 1993Dosch et al 1998Shin et al 1998N-Methyl-N-nitrosourea Friedman et al 1997Procarbazine Friedman et al 1997Temozolomide Liu et al 1996 Anthracyclines Friedman et al 1997Doxorubicin Drummond et al 1996 Antimetabolites Mercaptopurine Swann et al 19966-Thioguanine Swann et al 1996Dosch et al 1998Epipodophyllotoxins Etoposide Aebi et al 1997de las Alas et al 1997Lage et al 1999Platinum compounds Carboplatin Fink et al 1996Cisplatin Aebi et al 1996Drummond et al 1996Fink et al 1996Fink et al 1997de las Alas et al 1997Lage et al 1999 observation that mutations in the Bax-encoding gene possibly arise as a consequence of microsatellite instability due to MMR de®ciency (Colella et al 1998) (overview in Fig. 3).…”

Alterations of DNA mismatch repair proteins and microsatellite instability levels in gastric cancer cell lines

“…Interestingly, the MSI-High SNU1 did not show hMLH1 promoter methylation. Thus, given previous studies indicating that SNU1 cells display a truncating mutation at codon 226, 38 our data suggest that the second hMLH1 allele is probably inactivated by a mutation or allelic loss.…”

“…The cell lines SNU1 and SNU638 show absence of hMLH1 and hPMS2 and decreased levels of hPMS1. at codon 226, 38,39 while the SNU638 cell line was shown to display methylation of the hMLH1 promoter region. 40 However, it is not known whether the loss of hMLH1 expression results from methylation of the promoter region or from mutation or allelic loss of the second hMLH1 allele.…”

Identification of a Second MutL DNA Mismatch Repair Complex (hPMS1 and hMLH1) in Human Epithelial Cells

“…The colorectal cancer cell line HCT-116 has been reported to carry a nonsense mutation in the hMLH1 gene that results in undetectable protein in Western blots and is associated with microsatellite instability (26). In addition, we examined the expression of human MutL proteins in gastric cancer cell lines, including the SNU-1 line, which also displays absent hMLH1 protein as a result of a nonsense mutation in the coding region of the gene (25). We confirmed that HCT-116 and SNU-1 cells have instability at both mononucleotide and dinucleotide repeat microsatellite loci.…”

“…The cell line SNU-1 has been reported to have a nonsense mutation at codon 226 of hMLH1 (CGA3 TGA) resulting in a premature stop codon (25). The colon cancer cell lines (HCT-116) and the cervical cancer cell line CCL-2 (HeLa) (ATCC) were also used.…”