Skip to main content
NCBI home page
Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1999 Feb;90(2):180–187. doi: 10.1111/j.1349-7006.1999.tb00731.x

Activation of Caspases in p53‐induced Transactivation‐independent Apoptosis

Chongfeng Gao 1, Nobuo Tsuchida 1,✉
PMCID: PMC5926047  PMID: 10189888

Abstract

Though p53‐induced apoptosis plays an important role in tumor suppression, the mechanism(s) by which p53 induces apoptosis is stillunclear. To elucidate the p53‐induced apoptotic pathway, we examined the role of p53 transactivation activity and caspase in J138V5C cells carrying a human temperature‐sensitive (ts) p53 mutant (138Ala→Val). The results showed that p53‐induced apoptosis was not blocked by cycloheximide, which effectively prevented the expression of p53 target genes, indicating that transactivation was not essential for p53‐induced apoptosis in this system. Western blotanalysis showed that PARP, CPP32 and ICH‐1 precursors were cleaved during apoptosis. The CPP32‐preferential tetrapeptide inhibitor Ac‐DEVD‐CHO blocked the cleavage of ICH‐1 and PARP precursors, suggesting that CPP32 or some other DEVD‐sensitive caspase(s) is the upstream activator of ICH‐1. We also examined the role of the Fas pathway by using Fas and Fas ligand‐neutralizing antibodies. Both antibodies failed to block p53‐induced apoptosis, suggesting that the Fas pathway was not essential for p53‐induced apoptosis in this system. Taken together, our results indicate that p53‐induced, transactivation‐independent apoptosis in Jurkat cells involves sequential activation of CPP32 or some other DEVD‐sensitive caspase(s) and ICH‐1, via a Fas‐independent pathway.

Keywords: p53, Apoptosis, Caspase, Fas, Jurkat

Full Text

The Full Text of this article is available as a PDF (428.2 KB).

REFERENCES

  • 1. ) Green , M. S. , Bennett , W. P. , Hollstein , M. and Harris , C. C.Mutation in the p53 tumor suppressor gene: clue to cancer etiology and molecular pathogenesis . Cancer Res. , 54 , 4855 – 4878 ( 1994. ). [PubMed] [Google Scholar]
  • 2. ) Levine , A. J.p53, the cellular gatekeeper for growth and division . Cell , 88 , 323 – 331 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 3. ) Ko , L. J. and Prives , C.p53: puzzle and paradigm . Genes Dev. , 10 , 1054 – 1072 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) El‐Deiry , W. S. , Tokino , T. , Velculescu , V. E. , Levy , R. , Parsons , R. , Trent , J. M. , Lin , D. , Mercer , E. , Kinzler , K. W. and Vogelstein , B.WAF1, a potential mediator of p53 tumor suppression . Cell , 75 , 817 – 825 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 5. ) Harper , J. W. , Adami , G. R. , Wei , N. , Keyomarsi , K. and Elledge , S. J.The p21 cdk‐interacting protein cip1 is a potent inhibitor of G1 cyclin‐dependent kinase . Cell , 75 , 805 – 816 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Xing , Y. , Hanmon , G. J. , Zhang , H. , Casso , D. , Kobayashi , R. and Beach , D.p21 is a universal inhibitor of cyclin kinase . Nature , 366 , 701 – 704 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 7. ) Sabbatini , P. , Lin , J. , Levine , A. J. and White , E.Essential role for p53‐mediated transcription in E1A‐induced apoptosis . Genes Dev. , 9 , 2184 – 2192 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Friedlander , P. , Haupt , Y. , Prives , C. and Oren , M.A mutant p53 that discriminates between p53‐responsive genes cannot induce apoptosis . Mol. Cell. Biol. , 16 , 4961 – 4971 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. ) Miyashita , T. and Reed , J. C.Tumor suppressor p53 is a direct transcriptional activator of the human bax gene . Cell , 80 , 293 – 299 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Buckbinder , L. , Talbott , R. , Velasco‐Miguel , S. , Takenaka , I. , Faha , B. , Seizinger , B. R. and Kley , N.Induction of the growth inhibitor IGF‐binding protein 3 by p53 . Nature , 377 , 646 – 649 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Caelles , C. , Helmberg , A. and Karin , M.p53 dependent apoptosis in the absence of transcriptional activation of p53‐target genes . Nature , 370 , 220 – 223 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 12. ) Haup , Y. , Rowan , S. , Shaulian , E. , Vousden , K. H. and Oren , M.Induction of apoptosis in HeLa cells by transactivation‐deficient p53 . Genes Dev. , 9 , 2170 – 2183 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 13. ) Walker , K. K. and Levine , A. J.Identification of a p53 functional domain that is necessary for efficient growth suppression . Proc. Natl. Acad. Sci. USA , 93 , 15335 – 15340 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. ) Chen , X. , Ko , L. J. and Prives , C.p53 level, functional domains and DNA damage determine the extent of apoptotic response of tumor cells . Genes Dev. , 10 , 2438 – 2451 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Wang , X. W. , Vermeulen , W. , Coursen , J. D. , Gibson , M. , Lupold , S. E. , Forrester , K. , Xu , G. , Elmore , L. , Yeh , H. , Hoeijmakers , J. J. and Harris , C. C.The XPB and XPD DNA helicases are components of the p53‐mediated apoptosis pathway . Genes Dev. , 10 , 1219 – 1232 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 16. ) Alnemri , E. , Livingston , D. J. , Nicholson , D. W. , Salvesen , G. , Thornberry , N. A. , Wong , W. W. and Yuan , J. Y.Human ICE/CED‐3 protease nomenclature . Cell , 87 , 171 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Tewari , M. , Quan , L. T. , Orquote Rourke , K. , Desnoyer , S. , Zeng , Z. , Beidler , D. R. , Poirier , G. G. , Salvesen , G. S. and Dixit , V. M.Yama/CPP32b, a mammalian homolog of CED‐3, is a CrmA‐inhibitable protease that cleaves the death substrate poly(ADP‐ribose) polymerase . Cell , 81 , 801 – 809 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Srivnivasula , S. M. , Ahmad , M. , Fernandes‐Alnemri , T. , Litwack , G. and Alnemri , E. S.Molecular ordering of the Fas‐apoptotic pathway: the Fas/apo‐1 protease Mch5 is a CrmA‐inhibitable protease that activates multiple Ced‐3/ICE‐like cysteine proteases . Proc. Natl. Acad. Sci. USA , 93 , 14486 – 14491 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. ) Martin , S. J. and Green , D. R.Protease activation during apoptosis: death by a thousand cuts ? Cell , 82 , 349 – 352 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Enari , M. , Talanian , R. V. , Wong , W. W. and Nagata , S.Sequential activation of ICE‐like and CPP32‐like protease during Fas‐mediated apoptosis . Nature , 380 , 723 – 726 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Geley , S. , Hartmann , B. L. , Hattmannstorfer , R. , Loffler , M. , Ausserlechner , M. J. , Bernhard , D. , Sgonc , R. , Strasser‐Wozak , E. M. C. , Ebner , M. , Auer , B. and Kofler , R.p53‐induced apoptosis in the human T‐ALL cell line CCRF‐CEM . Oncogene , 15 , 2429 – 2437 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Sabbatini , D. , Han , J. , Chiou , S. K. , Nicholson , D. W. and White , E.Interleukin 1βconverting enzyme‐like proteases are essential for p53‐mediated transcriptionally dependent apoptosis . Cell Growth Differ. , 8 , 643 – 653 ( 1997. ). [PubMed] [Google Scholar]
  • 23. ) Lotem , J. and Sachs , L.Cytokine suppression of protease activation in wild‐type p53‐dependent and p53–independent apoptosis . Proc. Natl. Acad. Sci. USA , 94 , 9349 – 9353 ( 1997. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. ) Yamato , K. , Yamamoto , M. , Hirano , Y. and Tsuchida , N.A human temperature‐sensitive p53 mutant p53 val‐138: modulation of the cell cycle, viability and expression of p53‐responsive genes . Oncogene , 11 , 1 – 6 ( 1995. ). [PubMed] [Google Scholar]
  • 25. ) Fuchs , E. J. , Mckenna , K. A. and Bedi , A.p53‐dependent DNA damage‐induced apoptosis requires Fas/apo‐1‐independent activation of CPP32b . Cancer Res. , 57 , 2550 – 2554 ( 1997. ). [PubMed] [Google Scholar]
  • 26. ) Soussi , T. , Fromentel , C. C. and May , P.Structural aspects of p53 protein in relation to gene evolution . Oncogene , 5 , 945 – 952 ( 1990. ). [PubMed] [Google Scholar]
  • 27. ) Gong , J. , Traganos , F. and Darzynkiewilz , Z.A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry . Anal. Biochem. , 218 , 314 – 319 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 28. ) Nagata , S.Apoptosis by death factor . Cell , 88 , 355 – 365 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Friesen , C. , Herr , I. , Krammer , P. H. and Debatin , K. M.Involvement of the CD95(Apo‐1/Fas)receptor/ligand system in drug‐induced apoptosis in leukemia cells . Nat. Med. , 2 , 574 – 577 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Fulda , S. , Sieverts , H. , Friesen , G. , Her , I. and Debatin , K. M.The CD95(Apo‐1/Fas) system mediates drug‐induced apoptosis in neuroblastoma cells . Cancer Res. , 57 , 3823 – 3829 ( 1997. ). [PubMed] [Google Scholar]
  • 31. ) Hueber , A. , Zornig , M. , Lyon , D. , Suda , T. , Nagata , S. and Evan , G.Requirement for the CD95 receptor‐ligand pathway in c‐myc‐induced apoptosis . Science , 278 , 1305 – 1308 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 32. ) Wang , J. F.Analysis of downstream effectors of p53 on cell growth arrest and apoptosis induced by a temperature‐sensitive Val138 mutant . J. Med. Dent. Sci. , 45 , 141 – 149 ( 1998. ). [PubMed] [Google Scholar]
  • 33. ) Hall , A. R. and Milner , J.Structural and kinetic analysis of p53‐induced DNA complexes and comparison of human and murine p53 . Oncogene , 10 , 561 – 567 ( 1995. ). [PubMed] [Google Scholar]
  • 34. ) Lane , D. P. , Stephen , C. W. , Midgley , C. A. , Sparks , A. , Hupp , T. R. , Daniels , D. A. , Greaves , R. , Reid , A. , Vojtesek , B. and Picksley , S. M.Epitope analysis of the murine p53 tumor suppressor protein . Oncogene , 12 , 2461 – 2466 ( 1996. ). [PubMed] [Google Scholar]
  • 35. ) Salvesen , G. and Dixit , V. M.Caspase: intracellular signaling by proteolysis . Cell , 9 , 443 – 446 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 36. ) Li , H. , Bergeron , L. , Cryns , N. , Pasternack , M. S. , Zhu , H. , Shi , L. , Greenberg , L. and Yuan , J.Activation of caspase‐2 in apoptosis . J. Biol. Chem. , 272 , 21010 – 21017 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 37. ) Yin , Y. , Terauchi , Y. , Solomon , G. G. , Aizawa , S. , Rangarajan , P. N. , Yazaki , Y. , Kadowaki , T. and Barrett , J. C.Involvement of p85 in p53‐dependent apoptotic response to oxidative stress . Nature , 391 , 707 – 710 ( 1998. ). [DOI] [PubMed] [Google Scholar]
  • 38. ) Villunger , A. , Egle , A. , Kos , M. , Hartmann , B. L. , Geley , S. , Kofler , R. and Greil , R.Drug‐induced apoptosis is associated with enhanced Fas (Apo‐1/CD95) ligand expression but independently of Fas (Apo‐1/CD95) signaling in human T‐acute lymphatic leukemia cells . Cancer Res. , 57 , 3331 – 3334 ( 1997. ). [PubMed] [Google Scholar]

Articles from Japanese Journal of Cancer Research : Gann are provided here courtesy of Wiley

RESOURCES