초록/요약

Apoptosis is a programmed cell death which can be induced by several agents and radiations. This suicidal mechanism is accompanied with the characteristic cellular changes includng DNA fragmentation(laddering), nuclear membrane degradation, and chromatin condensation. One of the most well charaterized signal pathway involves receptors such as TNFR, DR, and Fas, some adaptors, caspases, Apaf-1, and DFF/CAD-ICAD. Also this pathway appears to be regulated by Bcl familiy, protein kinase, and serpin type inhibitors. However, evidence of the existence of other apoptic pathway has been suggested. Ca is one of the well known apoptosis inducing agent but the mechanism of Ca action in the apoptosis is relatively not well characterized. In order to isolate and characterize the factors of the apoptotic signal pathway involving Ca and other metal ions, in vitro apoptosis assay was established using rat liver nuclei and cell extract. Mn, Co as well as Ca but not other metals(Cu, Zn, Fe, Ni) induced DNA fragmentation(DF) in dose-dependent manner. Mn and Ca but not Co could induce the DF in nuclei alone without cell extract, indicating that the nuclei may have whole machinery for the DF. The cell extract show DNase activities activated by Mn and Co but Ca has relatively low DNase activity at a concentration of up to 5 mM. The DNase activities are inhibitable by TPCK(a serine protease inhibitor), APF-cmk(nuclear scaffold protease inhibitor), Zinc, and ATA, a nuclease inhibitor with about 5 times more sensitivity for nucleus only group. DNase activity of cell extract were very sensitive(IC50＜100 uM). Nuclear fragmentation(NF) also were inducible by Ca, Mn but Co with less sensitivity. The effect were augmented by the inclusion of cell extract, indicating that some factor may be necessary for the completion of NF. The NF were inhibited by Zn but not by TPCK. In summary, Ca, Mn induce DF and NF in nuclei which can be augmented by the addition of cell extract. Cell extract were essential for the DF and NF induced by Co. The factors will be isolated by using conventional chromatography.