|Budget Amount *help
¥3,980,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥480,000)
Fiscal Year 2007: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
Applicants performed cell-biologic analysis of cell-death following abnormal mitosis, using various models to understand the machinery of the cell-death in detail, in order to induce cell-death by inhibiting the function of m-calpain. Initially, applicants performed RNA-interference (RNAi) in HeLa cells targeting mRNA of Kid that a candidate of the substrate of m-calpain in mitosis. Aberrant nuclei had already appeared in transmitted light observation after 48 hours from the start of RNAi, and the cells containing two or three nuclei had increased. Although these cells had not show apparent delay of mitosis, aberrant nuclei and enlargement of cytoplasm had investigated, indicating that they had progressed into next G1 phase after the biochemical exit from mitosis without cell division. Further investigation detected that majority cells had resulted in cell-death with fragmentation. Flowcytometry showed appearance of cells containing polyploidy in the early period of investigation, then
thinning population of cells in synthetic phase and augmentation of cell-death in the late. Although monopolar cells are investigated by addition of monastrol, a specific inhibitory agent for Eg5, at 72 hours after the treatment of RNAi, "chromosome clear zone" formed in the center of monopolarized cells had disappeared in comparison to control cells, indicating that down-regulation of polar ejection force. These results are consistent with a report of injecting a neutralizing antibody for Kid by Levesque, et. al. and that of inhibiting m-calpain by applicants.
Although these experiments had strongly suggested the relation of m-calpain and Kid in the machinery of generating polar ejection force in mitosis, it is necessary to show for clinical application that why cell-death is induced by inhibiting the functions or activities of these molecules. It was suggested that inhibitory of Kid function induced cell-death after once getting polyploid, according to the experiences of. RNAi for Kid. To investigate how the polyploid cells are lead to cell-death, applicants treated cells by various doses of monastrol that could make cells polyploidy more simply and analyzed phenotype and copies of chromosomes with time.
Ninety-six hours treatment of effective dose of monastrol had hardly induced both mitotic-arrest and cell-death at all in hTERT-MRC5 cells, the immortalized human normal fibroblast, and somewhat more mitotic-arrest and nearly equal cell-death in HEK293 cells, immortalized human fetal kidney cell. Furthermore, most HCT116 cells, a colon carcinoma cell-line containing normal p53 protein, had been induced cell-death by the same treatment, and in contrast, HCT116-p53ko containing no functional p53 protein, had showed enlargement of cytoplasm and very fewer cell-death. Flowcytometry in HEK293 and HCT116-p53ko showed appearance of polyploidized cell population containing 8N and 16N that had never appeared in the experience of other cell-lines. As already reported, these results indicate that p53 related cell-cycle checkpoint stands on the link between polyploidization and cell-death by the treatment of monastrol. However, even in p53 deleted cell-lines, a number of dell-death had been induced after becoming hexaploid, octaploid and more, suggesting that as progression of highly polyploidization, differ from below tetraploid, there is a p53-independent machinery of checkpoint or cell-death. This highly polyploidized cell population had contained many vacuole-like structures in the cytoplasm, and speculated that the machinery of autophagy plays an important role in cell-death. Proofs that these structures are identical to autophagosome and autophagy is induced biochemically could reveal that cell-death induced in the highly polyploidized cells is identical to autophagic-cell-death. Less