Degradation of Topoisomerase IIα during Adenovirus E1A-induced Apoptosis Is Mediated by the Activation of the Ubiquitin Proteolysis System

Abstract

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A 12S in response to dexamethasone. The level of topoisomerase II␣ 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) Oncogene 10, 651-662). Topoisomerase II␣ prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone 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␣ was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 ؋ g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase II␣ 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 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 ubiquitination and degradation of topoisomerase II␣, is activated or induced during the latent phase of E1A-induced apoptosis.

The role of the ubiquitin-proteasome pathway in apoptosis

“…Topoisomerase IIa, for example, is subject to degradation by this pathway during the course of adenovirus-mediated apoptosis. 112,113 Since degregulated expression of topoisomerase IIa is now being reported to result in apoptosis, 114 its accumulation as a result of proteasome inhibition may contribute to this process. It is very likely that, as the understanding of apoptosis grows, additional pathways will be recognized as being impacted upon by proteasome inhibition and contributing to programmed cell death.…”

Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway

“…Cell extract and proteasome-depleted cell extract were prepared as described (Nakajima et al, 1996). The cell extract was incubated with 36 mg/ml recombinant p27 Kipl in a reaction buer (50 mM Tris-HCl [pH 8.3], 5 mM MgCl 2 , 2 mM DTT, 1 mM ATP, 10 mM phosphocreatine and 40 mg/ml creatine phosphokinase) at 378C for 3 h. The p27…”

c-Fos Degradation by the Proteasome

“…In mammalian cells, apoptosis can be repressed by antisense oligonucleotides that diminish the level of ubiquitinated nuclear proteins or by specific inhibitors of the proteasome (17)(18)(19). Recently, proteasome-mediated degradation of topoisomerase II␣ and IB␤ have been linked to adenovirus E1A-induced apoptosis and activation-induced T cell death, respectively (20,21).…”