Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1996 Mar;70(3):1375–1383. doi: 10.1128/jvi.70.3.1375-1383.1996

Apoptosis is induced by N-myc expression in hepatocytes, a frequent event in hepadnavirus oncogenesis, and is blocked by insulin-like growth factor II.

K Ueda 1, D Ganem 1
PMCID: PMC189956  PMID: 8627653

Abstract

Induction of hepatocellular carcinoma in woodchucks by woodchuck hepatitis virus is associated with the activation of N-myc gene expression, usually by viral DNA integration in cis to the N-myc locus. We have examined the consequences of N-myc up-regulation in rodent hepatic cells in culture. Mouse alpha ML hepatocytes infected with a retroviral vector overexpressing the woodchuck N-myc2 gene display a higher proliferation rate than parental alpha ML cells but are morphologically unchanged and do not form colonies in soft agar. However, they display an increased propensity to undergo apoptosis, an effect that is markedly augmented by serum deprivation. Expression of the woodchuck hepatitis virus X gene in alpha ML cells does not alter the growth phenotype of the cells and has no effect upon N-myc-dependent apoptosis. However, apoptosis in N-myc2-expressing alpha ML cells is strongly inhibited by insulin-like growth factor II (IGF II). IGF II gene expression is also strongly up-regulated during hepatic carcinogenesis in vivo in virally infected animals and has been speculated to be part of an autocrine growth-stimulatory pathway. Our results suggest that IGF II may play another role in the development of virus-induced hepatoma: the prevention of programmed cell death triggered by deregulated N-myc expression.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Beasley R. P. Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer. 1988 May 15;61(10):1942–1956. doi: 10.1002/1097-0142(19880515)61:10<1942::aid-cncr2820611003>3.0.co;2-j. [DOI] [PubMed] [Google Scholar]
  2. Buendia M. A. Hepatitis B viruses and hepatocellular carcinoma. Adv Cancer Res. 1992;59:167–226. doi: 10.1016/s0065-230x(08)60306-1. [DOI] [PubMed] [Google Scholar]
  3. Cariani E., Lasserre C., Seurin D., Hamelin B., Kemeny F., Franco D., Czech M. P., Ullrich A., Brechot C. Differential expression of insulin-like growth factor II mRNA in human primary liver cancers, benign liver tumors, and liver cirrhosis. Cancer Res. 1988 Dec 1;48(23):6844–6849. [PubMed] [Google Scholar]
  4. Chen H. S., Kaneko S., Girones R., Anderson R. W., Hornbuckle W. E., Tennant B. C., Cote P. J., Gerin J. L., Purcell R. H., Miller R. H. The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J Virol. 1993 Mar;67(3):1218–1226. doi: 10.1128/jvi.67.3.1218-1226.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christofori G., Naik P., Hanahan D. A second signal supplied by insulin-like growth factor II in oncogene-induced tumorigenesis. Nature. 1994 Jun 2;369(6479):414–418. doi: 10.1038/369414a0. [DOI] [PubMed] [Google Scholar]
  6. Ellis R. E., Yuan J. Y., Horvitz H. R. Mechanisms and functions of cell death. Annu Rev Cell Biol. 1991;7:663–698. doi: 10.1146/annurev.cb.07.110191.003311. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Fourel G., Couturier J., Wei Y., Apiou F., Tiollais P., Buendia M. A. Evidence for long-range oncogene activation by hepadnavirus insertion. EMBO J. 1994 Jun 1;13(11):2526–2534. doi: 10.1002/j.1460-2075.1994.tb06542.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fourel G., Transy C., Tennant B. C., Buendia M. A. Expression of the woodchuck N-myc2 retroposon in brain and in liver tumors is driven by a cryptic N-myc promoter. Mol Cell Biol. 1992 Dec;12(12):5336–5344. doi: 10.1128/mcb.12.12.5336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fourel G., Trepo C., Bougueleret L., Henglein B., Ponzetto A., Tiollais P., Buendia M. A. Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours. Nature. 1990 Sep 20;347(6290):294–298. doi: 10.1038/347294a0. [DOI] [PubMed] [Google Scholar]
  11. Ganem D., Varmus H. E. The molecular biology of the hepatitis B viruses. Annu Rev Biochem. 1987;56:651–693. doi: 10.1146/annurev.bi.56.070187.003251. [DOI] [PubMed] [Google Scholar]
  12. Hansen L. J., Tennant B. C., Seeger C., Ganem D. Differential activation of myc gene family members in hepatic carcinogenesis by closely related hepatitis B viruses. Mol Cell Biol. 1993 Jan;13(1):659–667. doi: 10.1128/mcb.13.1.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harrington E. A., Bennett M. R., Fanidi A., Evan G. I. c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines. EMBO J. 1994 Jul 15;13(14):3286–3295. doi: 10.1002/j.1460-2075.1994.tb06630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jhappan C., Stahle C., Harkins R. N., Fausto N., Smith G. H., Merlino G. T. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell. 1990 Jun 15;61(6):1137–1146. doi: 10.1016/0092-8674(90)90076-q. [DOI] [PubMed] [Google Scholar]
  15. Kodama K., Ogasawara N., Yoshikawa H., Murakami S. Nucleotide sequence of a cloned woodchuck hepatitis virus genome: evolutional relationship between hepadnaviruses. J Virol. 1985 Dec;56(3):978–986. doi: 10.1128/jvi.56.3.978-986.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kulkarni G. V., McCulloch C. A. Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts. J Cell Sci. 1994 May;107(Pt 5):1169–1179. doi: 10.1242/jcs.107.5.1169. [DOI] [PubMed] [Google Scholar]
  17. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  19. Nagaya T., Nakamura T., Tokino T., Tsurimoto T., Imai M., Mayumi T., Kamino K., Yamamura K., Matsubara K. The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma. Genes Dev. 1987 Oct;1(8):773–782. doi: 10.1101/gad.1.8.773. [DOI] [PubMed] [Google Scholar]
  20. Ogston C. W., Jonak G. J., Rogler C. E., Astrin S. M., Summers J. Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks. Cell. 1982 Jun;29(2):385–394. doi: 10.1016/0092-8674(82)90155-6. [DOI] [PubMed] [Google Scholar]
  21. Popper H., Roth L., Purcell R. H., Tennant B. C., Gerin J. L. Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc Natl Acad Sci U S A. 1987 Feb;84(3):866–870. doi: 10.1073/pnas.84.3.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rao L., Debbas M., Sabbatini P., Hockenbery D., Korsmeyer S., White E. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7742–7746. doi: 10.1073/pnas.89.16.7742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reed J. C. Bcl-2 and the regulation of programmed cell death. J Cell Biol. 1994 Jan;124(1-2):1–6. doi: 10.1083/jcb.124.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robinson W. S. Molecular events in the pathogenesis of hepadnavirus-associated hepatocellular carcinoma. Annu Rev Med. 1994;45:297–323. doi: 10.1146/annurev.med.45.1.297. [DOI] [PubMed] [Google Scholar]
  25. Roe T., Reynolds T. C., Yu G., Brown P. O. Integration of murine leukemia virus DNA depends on mitosis. EMBO J. 1993 May;12(5):2099–2108. doi: 10.1002/j.1460-2075.1993.tb05858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schirmacher P., Held W. A., Yang D., Chisari F. V., Rustum Y., Rogler C. E. Reactivation of insulin-like growth factor II during hepatocarcinogenesis in transgenic mice suggests a role in malignant growth. Cancer Res. 1992 May 1;52(9):2549–2556. [PubMed] [Google Scholar]
  27. Seeger C., Baldwin B., Hornbuckle W. E., Yeager A. E., Tennant B. C., Cote P., Ferrell L., Ganem D., Varmus H. E. Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host. J Virol. 1991 Apr;65(4):1673–1679. doi: 10.1128/jvi.65.4.1673-1679.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shih C., Burke K., Chou M. J., Zeldis J. B., Yang C. S., Lee C. S., Isselbacher K. J., Wands J. R., Goodman H. M. Tight clustering of human hepatitis B virus integration sites in hepatomas near a triple-stranded region. J Virol. 1987 Nov;61(11):3491–3498. doi: 10.1128/jvi.61.11.3491-3498.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sladowski D., Steer S. J., Clothier R. H., Balls M. An improved MTT assay. J Immunol Methods. 1993 Jan 4;157(1-2):203–207. doi: 10.1016/0022-1759(93)90088-o. [DOI] [PubMed] [Google Scholar]
  30. Takagi H., Sharp R., Hammermeister C., Goodrow T., Bradley M. O., Fausto N., Merlino G. Molecular and genetic analysis of liver oncogenesis in transforming growth factor alpha transgenic mice. Cancer Res. 1992 Oct 1;52(19):5171–5177. [PubMed] [Google Scholar]
  31. Truant R., Antunovic J., Greenblatt J., Prives C., Cromlish J. A. Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation. J Virol. 1995 Mar;69(3):1851–1859. doi: 10.1128/jvi.69.3.1851-1859.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Varmus H. E., Padgett T., Heasley S., Simon G., Bishop J. M. Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA. Cell. 1977 Jun;11(2):307–319. doi: 10.1016/0092-8674(77)90047-2. [DOI] [PubMed] [Google Scholar]
  33. Wagner A. J., Kokontis J. M., Hay N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Genes Dev. 1994 Dec 1;8(23):2817–2830. doi: 10.1101/gad.8.23.2817. [DOI] [PubMed] [Google Scholar]
  34. Wagner A. J., Small M. B., Hay N. Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2. Mol Cell Biol. 1993 Apr;13(4):2432–2440. doi: 10.1128/mcb.13.4.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang X. W., Forrester K., Yeh H., Feitelson M. A., Gu J. R., Harris C. C. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2230–2234. doi: 10.1073/pnas.91.6.2230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wei Y., Fourel G., Ponzetto A., Silvestro M., Tiollais P., Buendia M. A. Hepadnavirus integration: mechanisms of activation of the N-myc2 retrotransposon in woodchuck liver tumors. J Virol. 1992 Sep;66(9):5265–5276. doi: 10.1128/jvi.66.9.5265-5276.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. White E., Cipriani R., Sabbatini P., Denton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J Virol. 1991 Jun;65(6):2968–2978. doi: 10.1128/jvi.65.6.2968-2978.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. White E. Death-defying acts: a meeting review on apoptosis. Genes Dev. 1993 Dec;7(12A):2277–2284. doi: 10.1101/gad.7.12a.2277. [DOI] [PubMed] [Google Scholar]
  39. Williams G. T. Programmed cell death: apoptosis and oncogenesis. Cell. 1991 Jun 28;65(7):1097–1098. doi: 10.1016/0092-8674(91)90002-g. [DOI] [PubMed] [Google Scholar]
  40. Williams G. T., Smith C. A. Molecular regulation of apoptosis: genetic controls on cell death. Cell. 1993 Sep 10;74(5):777–779. doi: 10.1016/0092-8674(93)90457-2. [DOI] [PubMed] [Google Scholar]
  41. Wu J. C., Merlino G., Fausto N. Establishment and characterization of differentiated, nontransformed hepatocyte cell lines derived from mice transgenic for transforming growth factor alpha. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):674–678. doi: 10.1073/pnas.91.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yang D. Y., Rogler C. E. Analysis of insulin-like growth factor II (IGF-II) expression in neoplastic nodules and hepatocellular carcinomas of woodchucks utilizing in situ hybridization and immunocytochemistry. Carcinogenesis. 1991 Oct;12(10):1893–1901. doi: 10.1093/carcin/12.10.1893. [DOI] [PubMed] [Google Scholar]
  43. Yang D., Alt E., Rogler C. E. Coordinate expression of N-myc 2 and insulin-like growth factor II in precancerous altered hepatic foci in woodchuck hepatitis virus carriers. Cancer Res. 1993 May 1;53(9):2020–2027. [PubMed] [Google Scholar]
  44. Zoulim F., Saputelli J., Seeger C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol. 1994 Mar;68(3):2026–2030. doi: 10.1128/jvi.68.3.2026-2030.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Lohuizen M., Berns A. Tumorigenesis by slow-transforming retroviruses--an update. Biochim Biophys Acta. 1990 Dec 11;1032(2-3):213–235. doi: 10.1016/0304-419x(90)90005-l. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES