Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jul 1;17(13):3576–3586. doi: 10.1093/emboj/17.13.3576

Bax-mediated cell death by the Gax homeoprotein requires mitogen activation but is independent of cell cycle activity.

H Perlman 1, M Sata 1, A Le Roux 1, T W Sedlak 1, D Branellec 1, K Walsh 1
PMCID: PMC1170694  PMID: 9649428

Abstract

Tissues with the highest rates of proliferation typically exhibit the highest frequencies of apoptosis, but the mechanisms that coordinate these processes are largely unknown. The homeodomain protein Gax is down-regulated when quiescent cells are stimulated to proliferate, and constitutive Gax expression inhibits cell proliferation in a p21(WAF/CIP)-dependent manner. To understand how mitogen-induced proliferation influences the apoptotic process, we investigated the effects of deregulated Gax expression on cell viability. Forced Gax expression induced apoptosis in mitogen-activated cultures, but quiescent cultures were resistant to cell death. Though mitogen activation was required for apoptosis, neither the cdk inhibitor p21(WAF/CIP) nor the tumor suppressor p53 was required for Gax-induced cell death. Arrest in G1 or S phases of the cell cycle with chemical inhibitors also did not affect apoptosis, further suggesting that Gax-mediated cell death is independent of cell cycle activity. Forced Gax expression led to Bcl-2 down-regulation and Bax up-regulation in mitogen-activated, but not quiescent cultures. Mouse embryonic fibroblasts homozygous null for the Bax gene were refractive to Gax-induced apoptosis, demonstrating the functional significance of this regulation. These data suggest that the homeostatic balance between cell growth and death can be controlled by mitogen-dependent pathways that circumvent the cell cycle to alter Bcl-2 family protein expression.

Full Text

The Full Text of this article is available as a PDF (637.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Batistatou A., Greene L. A. Internucleosomal DNA cleavage and neuronal cell survival/death. J Cell Biol. 1993 Aug;122(3):523–532. doi: 10.1083/jcb.122.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bochaton-Piallat M. L., Gabbiani F., Redard M., Desmoulière A., Gabbiani G. Apoptosis participates in cellularity regulation during rat aortic intimal thickening. Am J Pathol. 1995 May;146(5):1059–1064. [PMC free article] [PubMed] [Google Scholar]
  3. Boyd J. M., Gallo G. J., Elangovan B., Houghton A. B., Malstrom S., Avery B. J., Ebb R. G., Subramanian T., Chittenden T., Lutz R. J. Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene. 1995 Nov 2;11(9):1921–1928. [PubMed] [Google Scholar]
  4. Chittenden T., Flemington C., Houghton A. B., Ebb R. G., Gallo G. J., Elangovan B., Chinnadurai G., Lutz R. J. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J. 1995 Nov 15;14(22):5589–5596. doi: 10.1002/j.1460-2075.1995.tb00246.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  6. Clayman G. L., Liu T. J., Overholt S. M., Mobley S. R., Wang M., Janot F., Goepfert H. Gene therapy for head and neck cancer. Comparing the tumor suppressor gene p53 and a cell cycle regulator WAF1/CIP1 (p21). Arch Otolaryngol Head Neck Surg. 1996 May;122(5):489–493. doi: 10.1001/archotol.1996.01890170025006. [DOI] [PubMed] [Google Scholar]
  7. Clowes A. W., Reidy M. A., Clowes M. M. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983 Sep;49(3):327–333. [PubMed] [Google Scholar]
  8. Colombel M., Olsson C. A., Ng P. Y., Buttyan R. Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle. Cancer Res. 1992 Aug 15;52(16):4313–4319. [PubMed] [Google Scholar]
  9. Dear T. N., Colledge W. H., Carlton M. B., Lavenir I., Larson T., Smith A. J., Warren A. J., Evans M. J., Sofroniew M. V., Rabbitts T. H. The Hox11 gene is essential for cell survival during spleen development. Development. 1995 Sep;121(9):2909–2915. doi: 10.1242/dev.121.9.2909. [DOI] [PubMed] [Google Scholar]
  10. Deckwerth T. L., Elliott J. L., Knudson C. M., Johnson E. M., Jr, Snider W. D., Korsmeyer S. J. BAX is required for neuronal death after trophic factor deprivation and during development. Neuron. 1996 Sep;17(3):401–411. doi: 10.1016/s0896-6273(00)80173-7. [DOI] [PubMed] [Google Scholar]
  11. Del Sal G., Ruaro M. E., Philipson L., Schneider C. The growth arrest-specific gene, gas1, is involved in growth suppression. Cell. 1992 Aug 21;70(4):595–607. doi: 10.1016/0092-8674(92)90429-g. [DOI] [PubMed] [Google Scholar]
  12. Deng C., Zhang P., Harper J. W., Elledge S. J., Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell. 1995 Aug 25;82(4):675–684. doi: 10.1016/0092-8674(95)90039-x. [DOI] [PubMed] [Google Scholar]
  13. Evan G. I., Wyllie A. H., Gilbert C. S., Littlewood T. D., Land H., Brooks M., Waters C. M., Penn L. Z., Hancock D. C. Induction of apoptosis in fibroblasts by c-myc protein. Cell. 1992 Apr 3;69(1):119–128. doi: 10.1016/0092-8674(92)90123-t. [DOI] [PubMed] [Google Scholar]
  14. Fabbretti E., Edomi P., Brancolini C., Schneider C. Apoptotic phenotype induced by overexpression of wild-type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A. Genes Dev. 1995 Aug 1;9(15):1846–1856. doi: 10.1101/gad.9.15.1846. [DOI] [PubMed] [Google Scholar]
  15. Farinelli S. E., Greene L. A. Cell cycle blockers mimosine, ciclopirox, and deferoxamine prevent the death of PC12 cells and postmitotic sympathetic neurons after removal of trophic support. J Neurosci. 1996 Feb 1;16(3):1150–1162. doi: 10.1523/JNEUROSCI.16-03-01150.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorski D. H., LePage D. F., Patel C. V., Copeland N. G., Jenkins N. A., Walsh K. Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells. Mol Cell Biol. 1993 Jun;13(6):3722–3733. doi: 10.1128/mcb.13.6.3722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gorski D. H., LePage D. F., Walsh K. Cloning and sequence analysis of homeobox transcription factor cDNAs with an inosine-containing probe. Biotechniques. 1994 May;16(5):856-8, 860-2, 865. [PubMed] [Google Scholar]
  18. Han D. K., Haudenschild C. C., Hong M. K., Tinkle B. T., Leon M. B., Liau G. Evidence for apoptosis in human atherogenesis and in a rat vascular injury model. Am J Pathol. 1995 Aug;147(2):267–277. [PMC free article] [PubMed] [Google Scholar]
  19. Harvey D. M., Levine A. J. p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts. Genes Dev. 1991 Dec;5(12B):2375–2385. doi: 10.1101/gad.5.12b.2375. [DOI] [PubMed] [Google Scholar]
  20. Hoang A. T., Cohen K. J., Barrett J. F., Bergstrom D. A., Dang C. V. Participation of cyclin A in Myc-induced apoptosis. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6875–6879. doi: 10.1073/pnas.91.15.6875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hockenbery D. M., Oltvai Z. N., Yin X. M., Milliman C. L., Korsmeyer S. J. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell. 1993 Oct 22;75(2):241–251. doi: 10.1016/0092-8674(93)80066-n. [DOI] [PubMed] [Google Scholar]
  22. Hockenbery D. Defining apoptosis. Am J Pathol. 1995 Jan;146(1):16–19. [PMC free article] [PubMed] [Google Scholar]
  23. Hockenbery D., Nuñez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature. 1990 Nov 22;348(6299):334–336. doi: 10.1038/348334a0. [DOI] [PubMed] [Google Scholar]
  24. Hoffman B., Liebermann D. A. Molecular controls of apoptosis: differentiation/growth arrest primary response genes, proto-oncogenes, and tumor suppressor genes as positive & negative modulators. Oncogene. 1994 Jul;9(7):1807–1812. [PubMed] [Google Scholar]
  25. Isner J. M., Kearney M., Bortman S., Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995 Jun 1;91(11):2703–2711. doi: 10.1161/01.cir.91.11.2703. [DOI] [PubMed] [Google Scholar]
  26. Kearney M., Pieczek A., Haley L., Losordo D. W., Andres V., Schainfeld R., Rosenfield K., Isner J. M. Histopathology of in-stent restenosis in patients with peripheral artery disease. Circulation. 1997 Apr 15;95(8):1998–2002. doi: 10.1161/01.cir.95.8.1998. [DOI] [PubMed] [Google Scholar]
  27. Knudson C. M., Tung K. S., Tourtellotte W. G., Brown G. A., Korsmeyer S. J. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science. 1995 Oct 6;270(5233):96–99. doi: 10.1126/science.270.5233.96. [DOI] [PubMed] [Google Scholar]
  28. Ko L. J., Prives C. p53: puzzle and paradigm. Genes Dev. 1996 May 1;10(9):1054–1072. doi: 10.1101/gad.10.9.1054. [DOI] [PubMed] [Google Scholar]
  29. Kowalik T. F., DeGregori J., Schwarz J. K., Nevins J. R. E2F1 overexpression in quiescent fibroblasts leads to induction of cellular DNA synthesis and apoptosis. J Virol. 1995 Apr;69(4):2491–2500. doi: 10.1128/jvi.69.4.2491-2500.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu Y., Kitsis R. N. Induction of DNA synthesis and apoptosis in cardiac myocytes by E1A oncoprotein. J Cell Biol. 1996 Apr;133(2):325–334. doi: 10.1083/jcb.133.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mader S. L. Influence of animal age on the beta-adrenergic system in cultured rat aortic and mesenteric artery smooth muscle cells. J Gerontol. 1992 Mar;47(2):B32–B36. doi: 10.1093/geronj/47.2.b32. [DOI] [PubMed] [Google Scholar]
  32. Maillard L., Van Belle E., Smith R. C., Le Roux A., Denéfle P., Steg G., Barry J. J., Branellec D., Isner J. M., Walsh K. Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty. Cardiovasc Res. 1997 Sep;35(3):536–546. doi: 10.1016/s0008-6363(97)00147-8. [DOI] [PubMed] [Google Scholar]
  33. Marazzi G., Wang Y., Sassoon D. Msx2 is a transcriptional regulator in the BMP4-mediated programmed cell death pathway. Dev Biol. 1997 Jun 15;186(2):127–138. doi: 10.1006/dbio.1997.8576. [DOI] [PubMed] [Google Scholar]
  34. Marcellus R. C., Teodoro J. G., Wu T., Brough D. E., Ketner G., Shore G. C., Branton P. E. Adenovirus type 5 early region 4 is responsible for E1A-induced p53-independent apoptosis. J Virol. 1996 Sep;70(9):6207–6215. doi: 10.1128/jvi.70.9.6207-6215.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marx S. O., Jayaraman T., Go L. O., Marks A. R. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res. 1995 Mar;76(3):412–417. doi: 10.1161/01.res.76.3.412. [DOI] [PubMed] [Google Scholar]
  36. McCurrach M. E., Connor T. M., Knudson C. M., Korsmeyer S. J., Lowe S. W. bax-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2345–2349. doi: 10.1073/pnas.94.6.2345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miura M., Zhu H., Rotello R., Hartwieg E. A., Yuan J. Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3. Cell. 1993 Nov 19;75(4):653–660. doi: 10.1016/0092-8674(93)90486-a. [DOI] [PubMed] [Google Scholar]
  38. Miyashita T., Harigai M., Hanada M., Reed J. C. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res. 1994 Jun 15;54(12):3131–3135. [PubMed] [Google Scholar]
  39. Miyashita T., Krajewski S., Krajewska M., Wang H. G., Lin H. K., Liebermann D. A., Hoffman B., Reed J. C. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994 Jun;9(6):1799–1805. [PubMed] [Google Scholar]
  40. Miyashita T., Reed J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995 Jan 27;80(2):293–299. doi: 10.1016/0092-8674(95)90412-3. [DOI] [PubMed] [Google Scholar]
  41. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27;74(4):609–619. doi: 10.1016/0092-8674(93)90509-o. [DOI] [PubMed] [Google Scholar]
  42. Perlman H., Maillard L., Krasinski K., Walsh K. Evidence for the rapid onset of apoptosis in medial smooth muscle cells after balloon injury. Circulation. 1997 Feb 18;95(4):981–987. doi: 10.1161/01.cir.95.4.981. [DOI] [PubMed] [Google Scholar]
  43. Qin X. Q., Livingston D. M., Kaelin W. G., Jr, Adams P. D. Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10918–10922. doi: 10.1073/pnas.91.23.10918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Quaggin S. E., Yeger H., Igarashi P. Antisense oligonucleotides to Cux-1, a Cut-related homeobox gene, cause increased apoptosis in mouse embryonic kidney cultures. J Clin Invest. 1997 Feb 15;99(4):718–724. doi: 10.1172/JCI119216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Raffo A. J., Perlman H., Chen M. W., Day M. L., Streitman J. S., Buttyan R. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen depletion in vivo. Cancer Res. 1995 Oct 1;55(19):4438–4445. [PubMed] [Google Scholar]
  46. Sata M., Perlman H., Muruve D. A., Silver M., Ikebe M., Libermann T. A., Oettgen P., Walsh K. Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response. Proc Natl Acad Sci U S A. 1998 Feb 3;95(3):1213–1217. doi: 10.1073/pnas.95.3.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sharkey M., Graba Y., Scott M. P. Hox genes in evolution: protein surfaces and paralog groups. Trends Genet. 1997 Apr;13(4):145–151. doi: 10.1016/s0168-9525(97)01096-2. [DOI] [PubMed] [Google Scholar]
  48. Shi L., Nishioka W. K., Th'ng J., Bradbury E. M., Litchfield D. W., Greenberg A. H. Premature p34cdc2 activation required for apoptosis. Science. 1994 Feb 25;263(5150):1143–1145. doi: 10.1126/science.8108732. [DOI] [PubMed] [Google Scholar]
  49. Stratford-Perricaudet L. D., Makeh I., Perricaudet M., Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest. 1992 Aug;90(2):626–630. doi: 10.1172/JCI115902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Walsh K., Perlman H. Molecular strategies to inhibit restenosis: modulation of the vascular myocyte phenotype. Semin Interv Cardiol. 1996 Sep;1(3):173–179. [PubMed] [Google Scholar]
  51. Wang J., Walsh K. Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science. 1996 Jul 19;273(5273):359–361. doi: 10.1126/science.273.5273.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wei G. L., Krasinski K., Kearney M., Isner J. M., Walsh K., Andrés V. Temporally and spatially coordinated expression of cell cycle regulatory factors after angioplasty. Circ Res. 1997 Mar;80(3):418–426. [PubMed] [Google Scholar]
  53. Weir L., Chen D., Pastore C., Isner J. M., Walsh K. Expression of gax, a growth arrest homeobox gene, is rapidly down-regulated in the rat carotid artery during the proliferative response to balloon injury. J Biol Chem. 1995 Mar 10;270(10):5457–5461. doi: 10.1074/jbc.270.10.5457. [DOI] [PubMed] [Google Scholar]
  54. Xiong W. C., Montell C. Defective glia induce neuronal apoptosis in the repo visual system of Drosophila. Neuron. 1995 Mar;14(3):581–590. doi: 10.1016/0896-6273(95)90314-3. [DOI] [PubMed] [Google Scholar]
  55. Yamashita J., Itoh H., Ogawa Y., Tamura N., Takaya K., Igaki T., Doi K., Chun T. H., Inoue M., Masatsugu K. Opposite regulation of Gax homeobox expression by angiotensin II and C-type natriuretic peptide. Hypertension. 1997 Jan;29(1 Pt 2):381–387. doi: 10.1161/01.hyp.29.1.381. [DOI] [PubMed] [Google Scholar]
  56. Yang E., Korsmeyer S. J. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood. 1996 Jul 15;88(2):386–401. [PubMed] [Google Scholar]
  57. Zhan Q., Lord K. A., Alamo I., Jr, Hollander M. C., Carrier F., Ron D., Kohn K. W., Hoffman B., Liebermann D. A., Fornace A. J., Jr The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth. Mol Cell Biol. 1994 Apr;14(4):2361–2371. doi: 10.1128/mcb.14.4.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zoidl G., Blass-Kampmann S., D'Urso D., Schmalenbach C., Müller H. W. Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth. EMBO J. 1995 Mar 15;14(6):1122–1128. doi: 10.1002/j.1460-2075.1995.tb07095.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES