Kimiko Nishizawa

Mismatch repair and microsatellite instability in esophageal cancer cells

Using in vitro mismatch repair (MMR) assay, we have identified 3 of 22 esophageal cancer cell lines exhibiting reduced MMR activity. By means of gel‐shift assay, decreased binding ability to GT mismatch and CA loop was observed in these 3 cell lines. However, we could not find any mutations in the hMSH2, hMSH3 and hMSH6 genes, the protein products of which exhibit mismatch binding activity in human cells. In addition, when using antibodies against 5 MMR‐related proteins (hMSH2, hMSH3, hMSH6, hPMS2 and hMLH1), no aberrant expression was detected in any of them. When we examined 9 microsatellite loci in endogenous genomic DNA, these 3 esophageal cancer cell lines, deficient in MMR, did not exhibit microsatellite instability. However, when we examined the repetitious sequence on exogenous plasmid DNA which was introduced into these 3 esophageal cancer cells, the results suggested that MMR deficiency in esophageal cancer cells could result in moderate instability of the exogenous sequence.

Different susceptibility of each L-myc genotype to esophageal cancer risk factors.

To understand the relationship between the L‐myc genotypes and esophageal cancer risk, a polynierase chain reaction‐based restriction fragment length polymorphism analysis was performed on 91 Japanese patients with esophageal cancer and 241 non‐cancer outpatients. No significant difference in the distribution of genotypes was observed between patients and controls; 18.7% LL genotype, 56.0% LS and 25.3% SS among patients, and 24.5%, 55.6% and 19.9%, respectively, among controls. Frequency of the s‐allele in patients (0.533) was slightly higher than in controls (0.477), but the difference was not statistically significant. However, the odds ratios (ORs) for smoking or heavy drinking were markedly higher in SS and LS genotypes than in LL genotype; age‐sex‐adjusted ORs for smoking was 7.57 in the SS genotype, 6.40 in the LS genotype and 1.77 in the LL genotype. Age‐sex‐adjusted ORs for heavy drinking were 19.78, 18.20 and 7.40, respectively. The age‐sex‐adjusted ORs for both factors combined were 12.77, 18.45 and 1.44, respectively. These results suggested that the L‐myc polymorphism might modify the effects of lifestyle factors on esophageal cancer risk.

UV-induced mutations of supF gene on a shuttle vector plasmid in p53-deficient mouse cells are qualitatively different from those in wild-type cells

To verify the genetic instability of p53-deficient cells, UV-induced mutation of the supF gene on a shuttle vector was analyzed. UV-irradiated or non-irradiated shuttle vector plasmid carrying the supF gene as a target of mutation (pYZ289) was introduced into p53-deficient and p53-proficient mouse embryonic fibroblasts, and then the plasmid DNA replicated in mouse cells was recovered. Survival of UV-irradiated plasmid was almost equivalent in both p53-deficient and p53-proficient cells. The frequencies of UV-induced mutation of the supF gene were also the same in both types of cells. However, the distributions of base change mutations in the supF sequence were different between p53-deficient and p53-proficient cells; especially the locations of tandem CpC to TpT changes exhibited a marked difference. Since DNA repair activities of these two types of cell were almost the same, these qualitative differences in UV-induced mutations were probably caused by as yet unidentified differences in other than DNA repair activity.

A genetic effect of altered gravity : mutations induced by simulated hypogravity and hypergravity in microsatellite sequences of human tumor cells

To determine the possible genetic effects of gravity alterations, we analyzed mutation induction in microsatellite sequences of human tumor cells treated with simulated hypogravity provided by a clinostat or hypergravity by a centrifuge. Microsatellite mutations were detected as changes in the size of polymerase chain reaction (PCR)-amplified allelic markers. The frequencies of mutant clones in cultures treated with simulated hypogravity for 24 or 48 h were almost the same as those of controls, but after 72 h of treatment, the mutant frequencies had increased significantly in all three microsatellite loci examined. Significantly higher mutant frequencies were similarly detected in cultures treated for 72 h with a hypergravity condition as low as 18xg, but not detected in 24 or 48 h treated cultures. These findings clearly show that gravity alterations that last for 3 days can induce microsatellite mutations in human cells. A genetic effect of gravity change, therefore, is established for the first time. Moreover, high frequencies of microsatellite mutations were induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates protein kinase C-mediated signal transduction pathways and causes genetic instability. These findings suggest that gravity change induces microsatellite mutations by modulating the pattern of gene expression involved in signal transduction pathways.