No abstract

The mismatch repair (MMR) gene hMLH1 is mutated in ∼50% of hereditary non‐polyposis colon cancers and transcriptionally silenced in ∼25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR‐proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR‐proficient cells, activation of the MNNG‐induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited cancer predisposition syndrome caused by germ line mutations in DNA mismatch repair genes, predominantly MLH1 and MSH2, with large genomic rearrangements accounting for 5% to 20% of all mutations. Although crucial to the understanding of cancer initiation, little is known about the second, somatic hit in HNPCC tumorigenesis, commonly referred to as loss of heterozygosity.Here, we applied a recently developed method, multiplex ligation-dependent probe amplification, to study MLH1/MSH2 copy number changes in 16 unrelated Swiss HNPCC patients, whose cancers displayed microsatellite instability and loss of MLH1 or MSH2 expression, but in whom no germ line mutation could be detected by conventional screening. The aims of the study were (a) to determine the proportion of large genomic rearrangements among Swiss MLH1/MSH2 mutation carriers and (b) to investigate the frequency and nature of loss of heterozygosity as a second, somatic event, in tumors from MLH1/MSH2 germ line deletion carriers. Large genomic deletions were found to account for 4.3% and 10.7% of MLH1 and MSH2 mutations, respectively. Multiplex ligation-dependent probe amplification analysis of 18 cancer specimens from two independent sets of Swiss and Finnish MLH1/MSH2 deletion carriers revealed that somatic mutations identical to the ones in the germ line occur frequently in colorectal cancers (6 of 11; 55%) and are also present in extracolonic HNPCC-associated tumors. Chromosome-specific marker analysis implies that loss of the wild-type allele predominantly occurs through locus-restricted recombinational events, i.e., gene conversion, rather than mitotic recombination or deletion of the respective gene locus. (Cancer Res 2006; 66(2): 659-64)

In 10-30% of patients with classical familial adenomatous polyposis (FAP) and up to 90% of those with attenuated (<100 colorectal adenomas; AFAP) polyposis, no pathogenic germline mutation in the adenomatous polyposis coli (APC) gene can be identified (APC mutation-negative). Recently, biallelic mutations in the base excision repair gene MYH have been shown to predispose to a multiple adenoma and carcinoma phenotype. This study aimed to (i) assess the MYH mutation carrier frequency among Swiss APC mutationnegative patients and (ii) identify phenotypic differences between MYH mutation carriers and APC/MYH mutation-negative polyposis patients. Seventy-nine unrelated APC mutation-negative Swiss patients with either classical (n 5 18) or attenuated (n 5 61) polyposis were screened for germline mutations in MYH by dHPLC and direct genomic DNA sequencing. Overall, 7 (8.9%) biallelic and 9 (11.4%) monoallelic MYH germline mutation carriers were identified. Among patients with a family history compatible with autosomal recessive inheritance (n 5 45), 1 (10.0%) out of 10 classical polyposis and 6 (17.1%) out of 35 attenuated polyposis patients carried biallelic MYH alterations, 2 of which represent novel gene variants (p.R171Q and p.R231H). Colorectal cancer was significantly (p < 0.007) more frequent in biallelic mutation carriers (71.4%) compared with that of monoallelic and MYH mutation-negative polyposis patients (0 and 13.8%, respectively). On the basis of our findings and earlier reports, MYH mutation screening should be considered if all of the following criteria are fulfilled: (i) presence of classical or attenuated polyposis coli, (ii) absence of a pathogenic APC mutation, and (iii) a family history compatible with an autosomal recessive mode of inheritance. ' 2005 Wiley-Liss, Inc.