| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1993: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
At a period of programd cell death, the neonatal mouse brain generates the killer glycoprotein NBCF(62 kDa, pI 9.1), which induced cell aggregation, pycnosis, and shrinkage of cytoplasm at the initial stage as typically observed for apoptosis, and double strand scission of nuclear DNA by 180-210 bp in human neuroblastoma NB1 cells by a NBCF-treatment time of 6 hr corresponding to a point of no return as shown by agarose gel electrophoresis. Cytolysis continued to proceed at 12-24 hr after NBCF addition, whereas degrees of intracelluar DNA scission ceased to increase at 12 hr and were similar to extracellular DNA scission degrees, suggesting that a limited degree of internucleosomal DNA scission is responsible for cytolysis. The DNA scission was inhibited with Zn or TPCK added at the initial stage, but not with cycloheximide initially added or with Zn or TPCK added after 6 hr, suggesting DNA scission by activation of preexisting endonuclease (EN) precursor but not by promotion of EN transcription. NB1 cells differentiated with a human glioblastoma A172 cell-derived defferentiation factor (36 kDa, pI 5.5) exhibited outgrowth of many neurites followed by reduced susceptibility to NBCF, and concurrently secreted a proteinic NBCF-antagonizing factor (43 kDa, basic), which transiently attenuated DNA scission degrees in undifferentiated NB1 cells treated with NBCF after a slight increase in DNA scission degrees initially observed, suggesting reversibility or tolerance in spoptosis. Isolated nuclei alone or together with cell homogenate or cell membrane of NB1 cells but not whole cells were saved from DNA scission by NBCF, showing that extracellular factors built in intact cells are essential for the DNA scission.
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