| Project/Area Number |
06680699
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Cell biology
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| Research Institution | Science University of Tokyo |
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Principal Investigator |
NAKAJIMA Takuma Science University of Tokyo, Biological Science and Technology, Research Assistant, 基礎工学部, 助手 (90256678)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Adenovirus E1A / Adenovirus E1B / apoptosis / topoisomerase IIalpha / Ubiquitin / proteolyses / nuclei / アデノウイルスE1A / アデノウイルスE1B |
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| Research Abstract |
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.
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