DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors.
DNA strand breaks occurring during apoptosis - their early insitu detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors.
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DOI：
10.3892/ijo.1.6.639
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发表时间：
1992-11
影响因子：
5.2
通讯作者：
Wojciech Gorczyca;S. Bruno;Rj Darzynkiewicz;Jp Gong;Zbigniew Darzynkiewicz
中科院分区：
文献类型：
--
作者：
Wojciech Gorczyca;S. Bruno;Rj Darzynkiewicz;Jp Gong;Zbigniew Darzynkiewicz
通过使用外源性末端转移酶或DNA聚合酶基于生物素化DUTP的标记，基于将3'-OH末端标记的新测定，在单个细胞中，在单个细胞中检测到DNA链断裂的出现。 DNA拓扑异构酶I和II抑制剂在HL-60细胞中诱导了凋亡，并通过泼尼松龙诱导大鼠胸腺细胞。丝氨酸蛋白酶不可逆抑制剂二异丙磷酸（DFP），L-1-甲基酰胺-2-苯基乙基氯甲基酮（TPCK），N-P-苯基-l-赖氨基氯甲基酮（TLCK）预防链断裂的形成。 -Alpha-Tosyl-L-精氨酸甲酯（驯服）和N-苯甲酰l-L-酪氨酸乙酯（BTEE）。数据表明，凋亡过程中DNA降解的开始是蛋白水解步骤的接头DNA与凋亡相关的内切酶。
The appearance of DNA strand breaks during apoptosis was detected in individual cells, in relation to the cell cycle phase, by a novel assay based on labeling 3'-OH termini with biotinylated dUTP using exogenous terminal transferase or DNA polymerase. Apoptosis was induced in HL-60 cells by the DNA topoisomerase I and II inhibitors, and in rat thymocytes by prednisolone. Formation of strand breaks was prevented by the serine protease irreversible inhibitors diisopropyl fluorophosphate (DFP), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and by the substrates N-alpha-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-tyrosine ethyl ester (BTEE). The data indicate that initiation of DNA degradation during apoptosis is preceded by a proteolytic step and suggest that apoptosis starts with activation (e.g. by DNA lesions) of a serine protease which hydrolyses protein(s) associated with the internucleosomal linker DNA sections, thus increasing accessibility of linker DNA to the apoptosis-associated endonuclease.