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Molecular Mechanism of Induction and Suppression of Apoptosis by Adenovirus E1A and E1B Gene Products

腺病毒E1A和E1B基因产物诱导和抑制细胞凋亡的分子机制

基本信息

批准号：
06680699
负责人：
NAKAJIMA Takuma
金额：
$ 1.41万
依托单位：
Science University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

The human epithermoid carcinoma derived cell line MA1, established by introduction of the adenovirus E1A 12S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone (dex). The level of topoisomerase IIalpha begin to decreases steeply within 36 h preceding the onset of DNA fragmentation, while its mRNA level unchanges. Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dex for 42 h (the 42 h extract) than in the extract from unreated MA1 cells (the 0 h extract) in an ATP and ubiquitin dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and much reduced in the 42 h extract prepared from MA1-derivative cell lines expressing E1B19k or Bc1-2. The proteolytic activity was lost in the S70 and P70 fractions prepared from the 42h extract by centrifugation at 70,000 x g for 6 h, but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42 h extract was several fold higher than that prepared from the 0 h extract. Ubiquitination could be detected with the N-terminal 198 amino acid fragment, but not with the central and C-terminal fragments. These results suggest that a component (s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha is activated or induced during the latent phase of E1A-induced apoptosis.

通过导入与小鼠乳腺肿瘤病毒长末端重复序列连接的腺病毒E1 A12 S cDNA建立的人表皮热样癌细胞系MA 1，在地塞米松（dex）诱导E1 A12 S后诱导细胞凋亡。拓扑异构酶II α的水平开始急剧下降，在36小时内发生的DNA片段化，而其mRNA水平不变。拓扑异构酶Ⅱ α制备的免疫沉淀或提取的核基质中降解更有效地从MA 1细胞处理dex 42小时（42小时提取物）比在提取物从unreated MA 1细胞（0小时提取物）在ATP和泛素依赖的方式制备的S10提取物。拓扑异构酶II α的降解的蛋白水解活性被抑制剂的蛋白酶体和42小时的提取物中制备的MA 1-衍生细胞系表达E1 B19 K或Bc 1 -2大大减少，特别是抑制。通过以70，000 x g离心6 h从42 h提取物制备的S70和P70级分中蛋白水解活性丧失，但当合并这些级分时部分恢复。拓扑异构酶II α的多泛素化形式可以通过将其在缺乏大部分蛋白酶体活性的S70或S100提取物中孵育来检测。从42 h提取物制备的S70中的泛素化活性比从0 h提取物制备的高数倍。N-末端198个氨基酸片段可检测到泛素化，但中心和C-末端片段不能检测到泛素化。这些结果表明，在泛素蛋白水解途径中的一个组件（S），负责泛素化和拓扑异构酶II α的降解过程中激活或诱导E1 A诱导的细胞凋亡的潜伏期。