Advertisement

Journal of Biomedical Science

, Volume 5, Issue 3, pp 185–191 | Cite as

Bcl-2 blocks apoptotic signal of transforming growth factor-β in human hepatoma cells

  • Yu-Lun Huang
  • Chen-Kung Chou
Original Paper
  • 33 Downloads

Abstract

Transforming growth factor-β (TGF-β) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF-β induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF-β in three well differentiated human hepatoma cell lines. Two TGF-β sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF-β resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-XL, Bcl-XS and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF-β-induced apoptosis. TGF-β treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF-β-F-induced apoptosis by interfering TGF-β generated signals leading to induce reactive oxygen species production.

Key Words

TGF-β Bcl-2 Apoptosis Antioxidative enzyme Reactive oxygen species 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Akao Y, Otshuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular localization ofbcl-2: Detection in nuclear outer membrane, endoplasmic reticulum, and mitochondrial membranes. Cancer Res 54:2468–2471;1994.PubMedGoogle Scholar
  2. 2.
    Allsopp TE, Wyatt S, Paterson HF, Davies AM. The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 73:295–307;1993.CrossRefPubMedGoogle Scholar
  3. 3.
    Assoian RK, Komoryia A, Meyers CA, Sporn MB. Transforming growth factor β in human platelets: Identification of a major storage site, purification and characterization. J Biol Chem 258:7155–7160;1983.PubMedGoogle Scholar
  4. 4.
    Boise LH, M Gonzalez-Garcia CE, Postema LD, Lindsten LA, Turka XM, Nuñez G, Thompson C. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic death. Cell 74:597–608;1993.CrossRefPubMedGoogle Scholar
  5. 5.
    Burow S, Valet G. Flow-cytometric characterization of stimulation, free radical formation, peroxidase activity and phagocytosis of human granulocytes with 2,7-dichlorofluorescein (DCF). Eur J Cell Biol 43:128–133;1987.PubMedGoogle Scholar
  6. 6.
    Charlotte F, L'Hermine A, Martin N, Geleyn Y, Nollet M, Faulard P, Zafrani ES. Immuno-histochemical detection of bcl-2 protein in normal and pathological human liver. Am J Pathol 144:460–465;1994.PubMedGoogle Scholar
  7. 7.
    Chen RH, Ebner R, Derynck R. Inactivation of the type-II receptor reveals two receptor pathways for the diverse TGF-β activities. Science 260:1335–1338;1993.PubMedGoogle Scholar
  8. 8.
    Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299;1979.CrossRefPubMedGoogle Scholar
  9. 9.
    Chuang LY, Hung WC, Chang CC, Tsai JH. Characterization of apoptosis induced by transforming growth factor β1 in human hepatoma cells. Anticancer Res 14:147–152;1994.PubMedGoogle Scholar
  10. 10.
    deJong D, Prins FA, Mason DY, Reed JC, van Ommen GB, Kluin PM. Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res 54:256–260;1994.PubMedGoogle Scholar
  11. 11.
    Frolik CA, Dart LL, Meyers CA, Smith DM, Sporn MB. Purification and initial characterization of a type β transforming growth factor from human placenta. Proc Natl Acad Sci USA 80:3676–3680;1983.PubMedGoogle Scholar
  12. 12.
    Fukuda K, Kojiro M, Chiu JF. Induction of apoptosis by transforming growth factor β1 in the rat hepatoma cell line MCA-RH7777: A possible association with tissue transglutaminase expression. Hepatology 18:945–953;1993.PubMedGoogle Scholar
  13. 13.
    Gajewski TF, Thompson CB. Apoptosis meets signal transduction: Elimination of a Bad influence. Cell 87:589–592;1996.CrossRefPubMedGoogle Scholar
  14. 14.
    Garcia I, Martinou I, Tsujimoto Y, Martinou JP. Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 258:302–304;1992.PubMedGoogle Scholar
  15. 15.
    Chen RH, Ebner R, Derynck R. Inactivation of the type-II receptor reveals two receptor pathways for the diverse TGF-β activities. Science 260:1335–1338;1993.PubMedGoogle Scholar
  16. 16.
    Graham F, Van der Eb A. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456–457;1973.CrossRefPubMedGoogle Scholar
  17. 17.
    Hale AJ, Smith CA, Sutherland LC, Stoneman VE, Longthorne VL, Culhane AC, Williams GT. Apoptosis: Molecular regulation of cell death. Eur J Biochem 236:1–26;1996.CrossRefPubMedGoogle Scholar
  18. 18.
    Hockenbery D, Nunez G, Milliman C, Schreiber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334–336;1990.CrossRefPubMedGoogle Scholar
  19. 19.
    Hockenbergy DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75:241–251;1993.CrossRefPubMedGoogle Scholar
  20. 20.
    Ito N, Kawata S, Tamura S, Takaishi K, Shirai Y, Kiso S, Yabuuchi I, Matsuda Y, Nishooka M, Tarui S. Elevated levels of transforming growth factor β messenger RNA and its polypeptide in human hepatocellular carcinoma. Cancer Res 51:4080–4083;1991.PubMedGoogle Scholar
  21. 21.
    Kane DJ, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, Ord T, Bredesen DE. Bcl-2 inhibition of neural death: Decreased generation of reactive oxygen species. Science 262:1274–1277;1993.PubMedGoogle Scholar
  22. 22.
    Kayanoki Y, Fujii J, Suzuki K, Kawata S, Matsuzawa Y, Taniguchi N. Suppression of antioxidative enzyme expression by transforming growth factor-β1 in rat hepatocytes. J Biol Chem 269:15488–15492;1994.PubMedGoogle Scholar
  23. 23.
    Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 275:1132–1136;1997.PubMedGoogle Scholar
  24. 24.
    Krajewski S, Tanaka S, Takayama S, Schibler MJ, Fenton W, Reed JC. Investigation of the subcellular distribution of the bcl-2 oncoprotein: Residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Cancer Res 53:4701–4714;1993.PubMedGoogle Scholar
  25. 25.
    Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3:614–620;1997.CrossRefPubMedGoogle Scholar
  26. 26.
    Lechleider RJ, deCaestecker MP, Dehejia A, Polymeropoulos MH, Roberts AB. Characterization of functional domains within Smad4/DPC4. J Biol Chem 272:13690–13696;1997.CrossRefPubMedGoogle Scholar
  27. 27.
    Lin JK, Chou CK. In vitro apoptosis in the human hepatoma cell line induced by transforming growth factor β1. Cancer Res 52:385–388;1992.PubMedGoogle Scholar
  28. 28.
    Lithgow T, Van Driel R, Beryram JF, Strasser A. The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the mitochondrial membrane. Cell Growth Differ 5:411–417;1994.PubMedGoogle Scholar
  29. 29.
    Liu F, Hata A, Baker JC, Doody J, Cárcamo J, Harland RM, Massagué JA. Human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381:620–623;1996.CrossRefPubMedGoogle Scholar
  30. 30.
    Massagué J. Type beta transforming growth factor from feline sarcoma virus-transformed rat cells, isolation and biological properties. J Biol Chem 259:9756–9761;1984.PubMedGoogle Scholar
  31. 31.
    Massagué J. Transforming growth factor β signaling: Receptors, transducers, and Mad proteins. Cell 85:947–950;1996.CrossRefPubMedGoogle Scholar
  32. 32.
    Masui T, Wakefield LM, Lechner JL, La Veck MA, Sporn MB, Harris CC. Type β transforming growth factor is the primary differentiation-inducing serum factor from normal human bronchial epithelial cells. Proc Natl Acad Sci USA 83:2438–2442;1986.PubMedGoogle Scholar
  33. 33.
    Miyashita T, Reed JC. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 81:151–157;1993.PubMedGoogle Scholar
  34. 34.
    Monaghan P, Robertson D, Amos TA, Dyer MJ, Mason DY, Greaves MF. Ultrastructural localization of bcl-2 protein. J Histochem Cytochem 40:1819–1825;1992.PubMedGoogle Scholar
  35. 35.
    Moses HL, Tucker RG, Leof EB, Coffey RJ, Halper J, Shipley GD. Type β transforming growth factor is a growth stimulator and a growth inhibitor. Cancer Cells (Cold Spring Harbor) 3:65–71;1985.Google Scholar
  36. 36.
    Nunez G, London L, Hockenbery D, Alexander M, McKearn JP, Korsmeyer SJ. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol 144:3602–3610;1990.PubMedGoogle Scholar
  37. 37.
    Oberhammer F, Bursch W, Parzefall W, Breit P, Erber E, Stadler M, Schulte-Hermann R. Effect of transforming growth factor β on cell death of cultured rat hepatocytes. Cancer Res 51:2478–2485;1991.PubMedGoogle Scholar
  38. 38.
    Oberhammer F, Pavelka M, Sharma S, Tiefenbacher R, Purchio AF, Bursch W, Schulte-Hermann R. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor β1. Proc Natl Acad Sci USA 89:5408–5412;1992.PubMedGoogle Scholar
  39. 39.
    Oberhammer F, Bursch W, Tiefenbacher R, Fröschl G, Pavelka M, Purchio T, Schulte-Hermann R. Apoptosis is induced by transforming growth factor β1 with 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 18:1238–1246;1993.CrossRefPubMedGoogle Scholar
  40. 40.
    Ohba M, Shibanuma M, Kuroki T, Nose K. Production of hydrogen peroxide by transforming growth factor β1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol 126:1079–1088;1994.CrossRefPubMedGoogle Scholar
  41. 41.
    Oltvai ZN, Millman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619;1993.PubMedGoogle Scholar
  42. 42.
    Park K, Kim SJ, Bang YJ, Park JG, Kim NK, Roberts AB, Sporn MB. Genetic changes in the transforming growth factor β (TGF-β) type II receptor gene in human gastric cancer cells. Correlation with sensitivity to growth inhibition by TGF-β. Proc Natl Acad Sci USA 91:8772–8776;1994.PubMedGoogle Scholar
  43. 43.
    Roberts AB, Anzano MA, Meyers CA, Wideman J, Blacher R, Pan T-CE, Stein S, Lehrman SR, Smith JM, Lamb LC, Sporn MB. Purification and properties type β transforming growth factor from bovine kidney. Biochemistry 22:5692–5698;1983.CrossRefPubMedGoogle Scholar
  44. 44.
    Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB. Type β transforming growth factor: A bifunctional regulator of cellular growth. Proc Natl Acad Sci USA 82:119–123;1985.PubMedGoogle Scholar
  45. 45.
    Sánchez A, Álvarez AM, Benito M, Fabregat I. Apoptosis induced by transforming growth factor-β in fetal hepatocyte primary cultures. J Biol Chem 271:7416–7422;1996.CrossRefPubMedGoogle Scholar
  46. 46.
    Sanderson N, Factor V, Nagy P, Kopp J, Kondaiah P, Wakefield L, Roberts AB. Hepatic expression of mature transforming growth factor β1 in transgenic mice results in multiple tissue lesions. Proc Natl Acad Sci USA 92:2572–2576;1995.PubMedGoogle Scholar
  47. 47.
    Selvakumaran M, Liebermann DA, Hoffman-Liebermann B. Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: Role in leukemogenesis. Mol Cell Biol 12:2493–2500;1992.PubMedGoogle Scholar
  48. 48.
    Selvakumaran M, Liebermann DA, Hoffman-Liebermann B. Myeloblastic leukemia cells conditionally blocked by myc-estrogen receptor chimeric transgenes for terminal differentiation coupled to growth arrest and apoptosis. Blood 81:2257–2262;1993.PubMedGoogle Scholar
  49. 49.
    Sentman CL, Shutter JR, Hockenbery D, Kanagawa O, Korsmeyer SJ. bcl-2 inhibits multiple forms of apoptosis but negative selection in thymocytes. Cell 67:879–888;1991.CrossRefPubMedGoogle Scholar
  50. 50.
    Shibuya H, Yamaguchi K, Shirakabe K, Tonegawa A, Gotoh Y, Ueno N, Irie K, Nishida E, Matsumoto K. TAB-1: An activation of the TAK1 MAPKKK in transforming growth factor β signal transduction. Science 272:1179–1182;1996.Google Scholar
  51. 51.
    Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JT. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181–184;1989.CrossRefPubMedGoogle Scholar
  52. 52.
    Sporn MB, Roberts AB, Wakefield LM, deCrombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Ciol 105:1039–1045;1987.Google Scholar
  53. 53.
    Steinman HM. The bcl-2 oncoprotein functions as a pro-oxidant. J Biol Chem 270:3487–3490;1995.PubMedGoogle Scholar
  54. 54.
    Terrell TG, Working PK, Chow CP, Green JD, Richter GW, Solez K, Ryffel B. Pathology of recombinant human transforming growth factor β1 in rats and rabbits. Int Rev Exp Pathol 34B:43–67;1994.Google Scholar
  55. 55.
    Thannickal VJ, Hassoun PM, White AC, Fanburg BL. Enhanced rate of H2O2 release from bovine pulmonary artery endothelial cells induced by transforming growth factor β1. Am J Physiol 265:L622-L626;1993.PubMedGoogle Scholar
  56. 56.
    Tsujimoto Y. Stress-resistance conferred by high level of bcl-2α protein in human B lymphoblastoid cell. Oncogene 4:1331–1336;1989.PubMedGoogle Scholar
  57. 57.
    Tsujimoto Y. Overexpression of the human Bcl-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci USA 86:1958–1962;1989.PubMedGoogle Scholar
  58. 58.
    Tu C-PD, Qian B. Human liver glutathione-S-transferase: complete primary sequence of an Ha subunit cDNA. Biochem Biophys Res Commun 141:229–237;1986.CrossRefPubMedGoogle Scholar
  59. 59.
    Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335:440–442;1988.CrossRefPubMedGoogle Scholar
  60. 60.
    Vaux DL, Haecker G, Strasser A. An evolutionary perspective on apoptosis. Cell 76:777–779;1993.CrossRefGoogle Scholar
  61. 61.
    Ventura F, Liu F, Doody J, Massagué J. Interaction of transforming growth factor-β receptor I with farnesyl-protein transferase-α in yeast and mammalian cells. J Biol Chem 271:13931–13934;1996.CrossRefPubMedGoogle Scholar
  62. 62.
    Walton MI, Whysong D, O'Connor PM, Hockenbery D, Korsmeyer SJ, Kohn KW. Constitutive expression of human Bcl-2 modulates nitrogen mustard and camptothecin induced apoptosis. Cancer Res 53:1853–1861;1993.PubMedGoogle Scholar
  63. 63.
    Wang HG, Rapp UR, Reed JC. Bcl-2 targets the protein kinase Raf-1 to mitochondria. Cell 87:629–638;1996.CrossRefPubMedGoogle Scholar
  64. 64.
    White E. Life, death, and the pursuit of apoptosis. Genes Dev 10:1–15;1996.PubMedGoogle Scholar
  65. 65.
    Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E, Matsumoto K. Identification of a member of the MAPKKK family as a potential mediator of transforming growth factor β signal transduction. Science 270:2008–2011;1995.PubMedGoogle Scholar
  66. 66.
    Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones PD, Wang X. Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science 275:1129–1132;1197.CrossRefGoogle Scholar

Copyright information

© National Science Council 1998

Authors and Affiliations

  • Yu-Lun Huang
    • 1
  • Chen-Kung Chou
    • 1
    • 2
    • 3
  1. 1.Institute of BiochemistryNational Yang-Ming UniversityTaiwan
  2. 2.Department of Medical ResearchVeterans General HospitalTaipeiTaiwan
  3. 3.Division of Molecular and Genomic MedicineNational Health Research InstitutesTaipeiTaiwan

Personalised recommendations