Masahiko Nezu

Centromere Protein H Is Up-regulated in Primary Human Colorectal Cancer and Its Overexpression Induces Aneuploidy

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Several centromere proteins such as CENP-A and CENP-H are the fundamental components of the human active kinetochore, and inappropriate expression of the centromere proteins could be a major cause of CIN. We have previously shown that CENP-A was overexpressed in primary human colorectal cancer. In this study, we show that CENP-H was also up-regulated in all of 15 primary human colorectal cancer tissues as well as in CIN tumor cell lines. Surprisingly, transient transfection of CENP-H expression plasmid into the diploid cell line HCT116 remarkably induced aneupoidy. Moreover, CENP-H stable transfectant of mouse embryonic fibroblast/3T3 cell lines showed aberrant interphase micronuclei, characteristic of chromosome missegregation. In these CENP-H overexpressed cells, CENP-H completely disappeared from the centromere of mitotic chromosomes, which might be the cause of the chromosome segregation defect. These results suggest that the aberrant expression and localization of a kinetochore protein CENP-H plays an important role in the aneuploidy frequently observed in colorectal cancers.

Comparative analyses of four different methods of genotyping ALDH2.

BACKGROUND A number of methods of genotyping single nucleotide polymorphisms (SNPs) are currently available, ranging from the traditional restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) to more sophisticated technologies. We determined the utility of three novel methods by genotyping aldehyde dehydrogenase-2 (ALDH2). METHODS DNA was isolated from blood samples of 241 control subjects and 74 patients with esophageal cancer. The utility of three novel genotyping methods-melting curve analysis using a LightCycler, SNaPshot analysis using an ABI PRISM 310 genetic analyzer, and denaturing high-performance liquid chromatography using a WAVE DNA fragment analysis system-were compared with that of conventional fluorescent-based polymerase chain reaction (PCR)-SSCP using an ALF express DNA sequencer. RESULTS The frequency of the mutant ALDH2*2 allele was significantly higher in patients with esophageal cancer (27.7%) than in control subjects (16.2%; p < 0.01; habitual alcohol drinkers). The melting curve analysis was accurate, more rapid, and easier to use than the SNaPshot analysis or denaturing high-performance liquid chromatography analysis. Fluorescent-based PCR-SSCP proved useful for analyzing a large number of samples. CONCLUSION Melting curve analysis using the LightCycler is suitable for the genotyping of small numbers of samples in a routine clinical setting; fluorescent-based PCR-SSCP analysis using the ALF express DNA sequencer can be used for large-scale genotyping in epidemiologic studies.

Identification of the CAB2/hCOS16 Gene Required for the Repair of DNA Double-strand Breaks on a Core Amplified Region of the 17q12 Locus in Breast and Gastric Cancers

We previously reported that CAB1 and c‐ERBB‐2 genes were found to be located in a core amplified region of the 17q12 locus, which is frequently amplified in various cancers. During identification of this core region, CAB2, a human homologue of the yeast COS16 required for the repair of DNA double‐strand breaks was cloned. Autofluorescence analysis of cells transfected with its GFP fusion protein demonstrated that CAB2 translocates into vesicles, suggesting that overexpression of CAB2 may decrease intercellular Mn2+ by accumulating it in the vesicles, in the same way as yeast COS16. This is the first report identifying all of the genes on the core amplified region of the 17q12 locus in breast and gastric cancers.