DEGRADATION OF TOPOISOMERASE II-ALPHA DURING ADENOVIRUS E1A-INDUCED APOPTOSIS IS MEDIATED BY THE ACTIVATION OF THE UBIQUITIN PROTEOLYSIS SYSTEM

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mamma ry tumor virus long terminal repeat, elicits apoptosis after induction of E1A(12S) in response to dexamethasone. The level of topoisomerase II alpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncoge ne 10, 651-662). Topoisomerase II alpha prepared by immunoprecipitatio n or extraction of the nuclear matrix was degraded much more efficient ly in the S10 extract prepared from MA1 cells treated with dexamethaso ne for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase II alpha was su ppressed specifically by inhibitors for the proteasome and was much re duced in the 42-h extract prepared from MA1-derivative cell lines expr essing E1B19k or Bcl-2. The proteolytic activity was lost after fracti onation of the 42-h S10 extract into the S70 and P70 fractions by cent rifugation at 70,000 x g for 6 h but partially recovered when these fr actions were combined. Polyubiquitinated forms of topoisomerase II alp ha could be detected by incubating it in the S70 or S100 extract, whic h lacks most of the proteasome activity. The ubiquitination activity i n S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component (s) in the ubiquitin proteolysis pathway, responsible for ubiquitinati on and degradation of topoisomerase II alpha, is activated or induced during the latent phase of E1A-induced apoptosis.