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. 1999 Sep 24;79(1):95–100. doi: 10.1038/sj.bjc.6690017

Bcl-2 overexpression blocks caspase activation and downstream apoptotic events instigated by photodynamic therapy

D J Granville 1,2, H Jiang 1, M T An 1, J G Levy 1,3, B M McManus 2, D W C Hunt 1,2
PMCID: PMC2362171  PMID: 10408699

Abstract

Treatment with the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) followed by irradiation with visible light induces apoptosis in human acute myelogenous leukaemia HL-60 cells. Photoactivation of BPD-MA induces procaspase 3 (CPP32/Yama/apopain) and procaspase 6 (Mch2) cleavage into their proteolytically active subunits in these cells. The Bcl-2 proto-oncogene product has been shown to protect cells from a number of proapoptotic stimuli. In the present study, the influence of Bcl-2 overexpression on cellular resistance to photoactivation of BPD-MA was studied. Overexpression of Bcl-2 in HL-60 cells prevented apoptosis-related events including caspase 3 and 6 activation, poly(ADP-ribose) polymerase cleavage and the formation of hypodiploid DNA produced by BPD-MA (0–200 ng ml−1) and light. However, Bcl-2 overexpression was less effective at preventing cell death that occurred after photoactivation at high levels (50–100 ng ml−1) compared with lower doses (10–25 ng ml−1) of BPD-MA. These results indicate that caspase 3 and 6 activation and their regulation by Bcl-2 may play important roles in photodynamic therapy (PDT)-induced cell killing. © 1999 Cancer Research Campaign

Keywords: apoptosis, photodynamic therapy, Bcl-2, caspase, resistance, leukaemic cells

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Selected References

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  1. Carthy C. M., Granville D. J., Watson K. A., Anderson D. R., Wilson J. E., Yang D., Hunt D. W., McManus B. M. Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. J Virol. 1998 Sep;72(9):7669–7675. doi: 10.1128/jvi.72.9.7669-7675.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Casciola-Rosen L., Nicholson D. W., Chong T., Rowan K. R., Thornberry N. A., Miller D. K., Rosen A. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med. 1996 May 1;183(5):1957–1964. doi: 10.1084/jem.183.5.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M. A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13(8):795–808. doi: 10.1002/cyto.990130802. [DOI] [PubMed] [Google Scholar]
  4. Enari M., Sakahira H., Yokoyama H., Okawa K., Iwamatsu A., Nagata S. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 1998 Jan 1;391(6662):43–50. doi: 10.1038/34112. [DOI] [PubMed] [Google Scholar]
  5. Estoppey S., Rodriguez I., Sadoul R., Martinou J. C. Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis. Cell Death Differ. 1997 Jan;4(1):34–38. doi: 10.1038/sj.cdd.4400205. [DOI] [PubMed] [Google Scholar]
  6. Fernandes-Alnemri T., Litwack G., Alnemri E. S. Mch2, a new member of the apoptotic Ced-3/Ice cysteine protease gene family. Cancer Res. 1995 Jul 1;55(13):2737–2742. [PubMed] [Google Scholar]
  7. Gajewski T. F., Thompson C. B. Apoptosis meets signal transduction: elimination of a BAD influence. Cell. 1996 Nov 15;87(4):589–592. doi: 10.1016/s0092-8674(00)81377-x. [DOI] [PubMed] [Google Scholar]
  8. Glück S., Chadderton A., Ho A. D. The selective uptake of benzoporphyrin derivative mono-acid ring A results in differential cell kill of multiple myeloma cells in vitro. Photochem Photobiol. 1996 Jun;63(6):846–853. doi: 10.1111/j.1751-1097.1996.tb09641.x. [DOI] [PubMed] [Google Scholar]
  9. Gomer C. J., Ferrario A., Hayashi N., Rucker N., Szirth B. C., Murphree A. L. Molecular, cellular, and tissue responses following photodynamic therapy. Lasers Surg Med. 1988;8(5):450–463. doi: 10.1002/lsm.1900080503. [DOI] [PubMed] [Google Scholar]
  10. Granville D. J., Jiang H., An M. T., Levy J. G., McManus B. M., Hunt D. W. Overexpression of Bcl-X(L) prevents caspase-3-mediated activation of DNA fragmentation factor (DFF) produced by treatment with the photochemotherapeutic agent BPD-MA. FEBS Lett. 1998 Jan 30;422(2):151–154. doi: 10.1016/s0014-5793(97)01616-5. [DOI] [PubMed] [Google Scholar]
  11. Granville D. J., Levy J. G., Hunt D. W. Photodynamic therapy induces caspase-3 activation in HL-60 cells. Cell Death Differ. 1997 Oct;4(7):623–628. doi: 10.1038/sj.cdd.4400286. [DOI] [PubMed] [Google Scholar]
  12. Granville D. J., Levy J. G., Hunt D. W. Photodynamic treatment with benzoporphyrin derivative monoacid ring A produces protein tyrosine phosphorylation events and DNA fragmentation in murine P815 cells. Photochem Photobiol. 1998 Mar;67(3):358–362. [PubMed] [Google Scholar]
  13. Halenbeck R., MacDonald H., Roulston A., Chen T. T., Conroy L., Williams L. T. CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. Curr Biol. 1998 Apr 23;8(9):537–540. doi: 10.1016/s0960-9822(98)79298-x. [DOI] [PubMed] [Google Scholar]
  14. He J., Agarwal M. L., Larkin H. E., Friedman L. R., Xue L. Y., Oleinick N. L. The induction of partial resistance to photodynamic therapy by the protooncogene BCL-2. Photochem Photobiol. 1996 Nov;64(5):845–852. doi: 10.1111/j.1751-1097.1996.tb01845.x. [DOI] [PubMed] [Google Scholar]
  15. Ibrado A. M., Huang Y., Fang G., Liu L., Bhalla K. Overexpression of Bcl-2 or Bcl-xL inhibits Ara-C-induced CPP32/Yama protease activity and apoptosis of human acute myelogenous leukemia HL-60 cells. Cancer Res. 1996 Oct 15;56(20):4743–4748. [PubMed] [Google Scholar]
  16. Ibrado A. M., Liu L., Bhalla K. Bcl-xL overexpression inhibits progression of molecular events leading to paclitaxel-induced apoptosis of human acute myeloid leukemia HL-60 cells. Cancer Res. 1997 Mar 15;57(6):1109–1115. [PubMed] [Google Scholar]
  17. Jamieson C. H., McDonald W. N., Levy J. G. Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells. Leuk Res. 1990;14(3):209–219. doi: 10.1016/0145-2126(90)90128-v. [DOI] [PubMed] [Google Scholar]
  18. Jamieson C., Richter A., Levy J. G. Efficacy of benzoporphyrin derivative, a photosensitizer, in selective destruction of leukemia cells using a murine tumor model. Exp Hematol. 1993 May;21(5):629–634. [PubMed] [Google Scholar]
  19. Kick G., Messer G., Plewig G., Kind P., Goetz A. E. Strong and prolonged induction of c-jun and c-fos proto-oncogenes by photodynamic therapy. Br J Cancer. 1996 Jul;74(1):30–36. doi: 10.1038/bjc.1996.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kluck R. M., Bossy-Wetzel E., Green D. R., Newmeyer D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997 Feb 21;275(5303):1132–1136. doi: 10.1126/science.275.5303.1132. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lazebnik Y. A., Takahashi A., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9042–9046. doi: 10.1073/pnas.92.20.9042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. doi: 10.1016/s0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  24. Liu X., Zou H., Slaughter C., Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell. 1997 Apr 18;89(2):175–184. doi: 10.1016/s0092-8674(00)80197-x. [DOI] [PubMed] [Google Scholar]
  25. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. doi: 10.1016/0022-1759(83)90303-4. [DOI] [PubMed] [Google Scholar]
  26. Nagata S. Apoptosis by death factor. Cell. 1997 Feb 7;88(3):355–365. doi: 10.1016/s0092-8674(00)81874-7. [DOI] [PubMed] [Google Scholar]
  27. Neamati N., Fernandez A., Wright S., Kiefer J., McConkey D. J. Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J Immunol. 1995 Apr 15;154(8):3788–3795. [PubMed] [Google Scholar]
  28. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  29. Noodt B. B., Berg K., Stokke T., Peng Q., Nesland J. M. Apoptosis and necrosis induced with light and 5-aminolaevulinic acid-derived protoporphyrin IX. Br J Cancer. 1996 Jul;74(1):22–29. doi: 10.1038/bjc.1996.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Orth K., Chinnaiyan A. M., Garg M., Froelich C. J., Dixit V. M. The CED-3/ICE-like protease Mch2 is activated during apoptosis and cleaves the death substrate lamin A. J Biol Chem. 1996 Jul 12;271(28):16443–16446. [PubMed] [Google Scholar]
  31. Pegoraro L., Palumbo A., Erikson J., Falda M., Giovanazzo B., Emanuel B. S., Rovera G., Nowell P. C., Croce C. M. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7166–7170. doi: 10.1073/pnas.81.22.7166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perry D. K., Smyth M. J., Wang H. G., Reed J. C., Duriez P., Poirier G. G., Obeid L. M., Hannun Y. A. Bcl-2 acts upstream of the PARP protease and prevents its activation. Cell Death Differ. 1997 Jan;4(1):29–33. doi: 10.1038/sj.cdd.4400200. [DOI] [PubMed] [Google Scholar]
  33. Reed J. C. Double identity for proteins of the Bcl-2 family. Nature. 1997 Jun 19;387(6635):773–776. doi: 10.1038/42867. [DOI] [PubMed] [Google Scholar]
  34. Reed J. C. Mechanisms of Bcl-2 family protein function and dysfunction in health and disease. Behring Inst Mitt. 1996 Oct;(97):72–100. [PubMed] [Google Scholar]
  35. Reed J. C., Miyashita T., Takayama S., Wang H. G., Sato T., Krajewski S., Aimé-Sempé C., Bodrug S., Kitada S., Hanada M. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem. 1996 Jan;60(1):23–32. doi: 10.1002/(SICI)1097-4644(19960101)60:1%3C23::AID-JCB5%3E3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
  36. Richter A. M., Kelly B., Chow J., Liu D. J., Towers G. H., Dolphin D., Levy J. G. Preliminary studies on a more effective phototoxic agent than hematoporphyrin. J Natl Cancer Inst. 1987 Dec;79(6):1327–1332. [PubMed] [Google Scholar]
  37. Rowan S., Fisher D. E. Mechanisms of apoptotic cell death. Leukemia. 1997 Apr;11(4):457–465. doi: 10.1038/sj.leu.2400626. [DOI] [PubMed] [Google Scholar]
  38. Sakahira H., Enari M., Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998 Jan 1;391(6662):96–99. doi: 10.1038/34214. [DOI] [PubMed] [Google Scholar]
  39. Schlegel J., Peters I., Orrenius S., Miller D. K., Thornberry N. A., Yamin T. T., Nicholson D. W. CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem. 1996 Jan 26;271(4):1841–1844. doi: 10.1074/jbc.271.4.1841. [DOI] [PubMed] [Google Scholar]
  40. Shimizu S., Eguchi Y., Kosaka H., Kamiike W., Matsuda H., Tsujimoto Y. Prevention of hypoxia-induced cell death by Bcl-2 and Bcl-xL. Nature. 1995 Apr 27;374(6525):811–813. doi: 10.1038/374811a0. [DOI] [PubMed] [Google Scholar]
  41. Srinivasula S. M., Fernandes-Alnemri T., Zangrilli J., Robertson N., Armstrong R. C., Wang L., Trapani J. A., Tomaselli K. J., Litwack G., Alnemri E. S. The Ced-3/interleukin 1beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2alpha are substrates for the apoptotic mediator CPP32. J Biol Chem. 1996 Oct 25;271(43):27099–27106. doi: 10.1074/jbc.271.43.27099. [DOI] [PubMed] [Google Scholar]
  42. Takahashi A., Alnemri E. S., Lazebnik Y. A., Fernandes-Alnemri T., Litwack G., Moir R. D., Goldman R. D., Poirier G. G., Kaufmann S. H., Earnshaw W. C. Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8395–8400. doi: 10.1073/pnas.93.16.8395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Telford W. G., King L. E., Fraker P. J. Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J Immunol Methods. 1994 Jun 3;172(1):1–16. doi: 10.1016/0022-1759(94)90373-5. [DOI] [PubMed] [Google Scholar]
  44. Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997 Jul 18;272(29):17907–17911. doi: 10.1074/jbc.272.29.17907. [DOI] [PubMed] [Google Scholar]
  45. Tsujimoto Y., Cossman J., Jaffe E., Croce C. M. Involvement of the bcl-2 gene in human follicular lymphoma. Science. 1985 Jun 21;228(4706):1440–1443. doi: 10.1126/science.3874430. [DOI] [PubMed] [Google Scholar]
  46. Wang X., Zelenski N. G., Yang J., Sakai J., Brown M. S., Goldstein J. L. Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during apoptosis. EMBO J. 1996 Mar 1;15(5):1012–1020. [PMC free article] [PubMed] [Google Scholar]
  47. Yang E., Zha J., Jockel J., Boise L. H., Thompson C. B., Korsmeyer S. J. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell. 1995 Jan 27;80(2):285–291. doi: 10.1016/0092-8674(95)90411-5. [DOI] [PubMed] [Google Scholar]
  48. Yang J., Liu X., Bhalla K., Kim C. N., Ibrado A. M., Cai J., Peng T. I., Jones D. P., Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. doi: 10.1126/science.275.5303.1129. [DOI] [PubMed] [Google Scholar]
  49. Zaidi S. I., Oleinick N. L., Zaim M. T., Mukhtar H. Apoptosis during photodynamic therapy-induced ablation of RIF-1 tumors in C3H mice: electron microscopic, histopathologic and biochemical evidence. Photochem Photobiol. 1993 Dec;58(6):771–776. doi: 10.1111/j.1751-1097.1993.tb04969.x. [DOI] [PubMed] [Google Scholar]

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