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

通过引入与小鼠乳腺肿瘤病毒长末端重复序列连接的腺病毒E1A12S cDNA，建立了人上皮样癌衍生细胞系MA1，该细胞系在地塞米松（dex）的诱导下诱导E1A12S凋亡。拓扑异构酶iα水平在DNA断裂发生前36 h内开始急剧下降，而其mRNA水平不变。通过免疫沉淀或提取核基质制备的拓扑异构酶iα在经dex处理42 h的MA1细胞制备的S10提取物（42 h提取物）中以ATP和泛素依赖的方式比未处理MA1细胞的提取物（0 h提取物）更有效地降解。在表达E1B19k或bb1 -2的ma1衍生物细胞系中提取42 h后，拓扑异构酶ⅱα降解的蛋白水解活性被蛋白酶体抑制剂特异性抑制。在70000 x g离心6小时后，从42h的提取物中得到的S70和P70部分失去了蛋白水解活性，但当这两部分结合在一起时部分恢复了蛋白水解活性。在缺乏大部分蛋白酶体活性的S70或S100提取物中，可以检测到多泛素化形式的拓扑异构酶iα。从42 h提取的S70的泛素化活性比0 h提取的S70的泛素化活性高数倍。n端198个氨基酸片段可以检测到泛素化，但中心和c端片段不能检测到泛素化。这些结果表明，泛素蛋白水解途径中负责泛素化和拓扑异构酶iα降解的成分在e1a诱导的细胞凋亡的潜伏期被激活或诱导。

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.