Microsatellite instability at tetranucleotide repeats in sporadic colorectal cancer in Japan.

Most tumors of patients with Lynch syndrome and a fraction of sporadic colorectal cancers (CRCs) exhibit high levels of microsatellite instability (MSI) at mono- and dinucleotide repeat loci. A different type of instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has been found in non-colonic cancers. Our previous study demonstrated that EMAST is common in sporadic CRC. Here, we focused on the relationships between EMAST and other genomic instability parameters or clinicopathological features in an unselected series of 88 sporadic CRCs. Of the tumors in the sample, 4 (4.5%) were MSI-high (MSI-H), 9 (10.2%) were MSI-low (MSI-L) and 75 (85.2%) were microsatellite stable. EMAST status was determined using 7 EMAST markers. Fifty-three (60.2%) tumors without MSI-H showed instability at >or=1 EMAST loci. All 4 MSI-H tumors showed instability at several EMAST loci. Instability profiles of MSI-H tumors at EMAST loci were more complex than those of non-MSI-H tumors. A tendency of positive association was observed between MSI-L and EMAST (P=0.023). The frequency of loss of heterozygosity (LOH) for the 14 loci in EMAST-positive tumors was significantly higher than negative tumors (P=0.048). Among the clinicopathological parameters, only tumor location at the distal colon was associated with EMAST-negative tumors (P=0.0084, one-tailed). A relatively higher frequency of well-differentiated adenocarcinomas was observed in EMAST tumors as opposed to non-EMAST tumors, though the survival rate was similar. These results suggest that overlapping mechanisms that cause MSI-L, EMAST and LOH in CRCs may exist.

A single nucleotide substitution (-107C-->G) in the hMLH1 promoter found in colorectal cancer population reduces transcriptional activity.

Inactivation of the DNA mismatch repair gene hMLH1 predisposes one to colorectal cancer. We have identified a C to G nucleotide substitution at position -107 relative to the hMLH1 gene translation initiation site in three of 163 colorectal cancer patients with an allele frequency of 0.0092 (3/326). One of the three -107G alleles occurred in one patient out of five with reduced hMLH1 expression in the tumor tissue. The -107G was not found in 63 healthy individuals. This substitution reduced transcriptional activity by 51% compared with -107C (P<0.01) and impeded the promoter-binding capacity of nuclear proteins. Although the small number of identified -107G alleles is insufficient to evaluate the contribution to the carcinogenesis and clinicopathological properties of the tumors, the effects of -107G on hMLH1 gene transcription and nuclear protein binding to the promoter sequence implicate the site, including -107C, as a crucial element interacting with the activator that maintains hMLH1 gene expression.

A novel dysfunctional p53 mutation in the human neuroblastoma cell line TGW.

Mutations of p53 are rare in primary and advanced neuroblastomas. The p53 gene was studied in a TGW cell line established from a TNB1 xenograft, derived from metastasized neuroblastoma. The p53 protein level in TGW was elevated at baseline. Treatment with doxorubicin to induce genotoxic stress neither altered the p53 protein level nor induced p21 protein within 24 hours. DNA sequencing analysis revealed a novel triplet deletion mutation at codon 282 (R282del) of the p53 gene, a mutation also found in TNB1, indicating that the mutation occurred in the relapsed tumor. The mutant was incapable of transactivation and had no effect on the transactivational activity of the wild-type p53 gene product in reporter assays using a plasmid possessing a p53 responsive element of p21, bax or mdm2. These results suggest that the mutant p53R282del found in TGW is a non-functional mutant and has no dominant negative nature.

Establishment of cisplatin-resistant variants of human neuroblastoma cell lines, TGW and GOTO, and their drug cross-resistance profiles.

PURPOSE: The emergence of multidrug resistance (MDR) in neuroblastoma is a critical issue for chemotherapy. In order to study low-level MDR, we developed variants derived from two neuroblastoma cell lines, TGW and GOTO, by exposure to low doses of cisplatin (CDDP). The cross-resistance to other cytotoxic agents and expression of MDR-related proteins in the variants and their clones were examined. MATERIALS AND METHODS: Cells were exposed to 3 or 10 micro M CDDP and three variants were obtained from each cell line, TGW and GOTO. Clones TR1 and TR2, derived from the TGW variants, were also established. Cytotoxicity was determined using a dye-staining method. Expression of MDR-related proteins was detected by immunoblotting. RESULTS: Resistant variants exhibited 1.1- to 2.5-fold increased resistance to CDDP, N-methyl- N'-nitro- N-nitrosoguanidine (MNNG), doxorubicin and vincristine. The cytotoxicity of these agents varied between clones of resistant variants. The microsatellite profiles of the TR1 clones differed, indicating that the TR1 variant comprised a heterogeneous cell population. The cytotoxicities of cytosine beta- D-arabinofuranoside (Ara-C) and chlorambucil in these variants and clones were similar to those in the parent cells. No significant changes in the cellular levels of MDR1, MRP, hMLH1 and hMSH2 were detected in the TGW variants. Cyclosporin A increased the sensitivity of both parental cell lines and the variants to doxorubicin and vincristine, but not to CDDP or MNNG. CONCLUSIONS: Ara-C and chlorambucil may be useful for the treatment of neuroblastoma exhibiting an MDR phenotype. These CDDP-resistant variants and clones may be useful for studying the mechanisms of low-level drug resistance in neuroblastoma.