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

人上皮样癌细胞系MA1是通过导入与小鼠乳腺肿瘤病毒长末端重复序列相连的腺病毒E1A12S基因而建立的，在地塞米松(Dex)诱导E1A12S后可诱导细胞凋亡。DNA断裂前36h内，DNA拓扑异构酶IIa水平开始急剧下降，而其mRNA水平无明显变化。免疫沉淀法或提取核基质制备的拓扑异构酶IIα在地塞米松作用42h的MA1细胞S10提取物(42h提取物)中的降解效率明显高于未缓解的MA1细胞提取物(0h提取物)，且依赖于ATP和泛素。在表达E1B19k或Bc1-2的MA1衍生细胞系制备的42h提取物中，降解拓扑异构酶IIpha的蛋白水解酶活性被蛋白酶体抑制剂特异性地抑制，并且大大降低。提取液在70,000×g下离心6h后，S70和P70组分的蛋白水解性丧失，但当这两个组分结合时部分恢复。通过将其孵育在缺乏大部分蛋白酶体活性的S70或S100提取物中，可以检测到泛素化形式的拓扑异构酶IIpha。42h提取液制备的S70泛素化活性是0h提取液制备的S70泛素化活性的数倍。N-端198个氨基酸片段可以检测到泛素化，而中心和C-端片段不能检测到泛素化。这些结果表明，在E1a诱导的细胞凋亡潜伏期，泛素蛋白分解途径中负责泛素化和拓扑异构酶IIα降解的一个成分(S)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.