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. 1997 Feb 3;16(3):495–503. doi: 10.1093/emboj/16.3.495

Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

L Amicone 1, F M Spagnoli 1, G Späth 1, S Giordano 1, C Tommasini 1, S Bernardini 1, V De Luca 1, C Della Rocca 1, M C Weiss 1, P M Comoglio 1, M Tripodi 1
PMCID: PMC1169653  PMID: 9034332

Abstract

Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET protooncogene. The cytoplasmic portion of Met (referred to as cyto-Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human alpha-1-antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte-enriched transcription factors as well as hepatic products.

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Selected References

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

  1. Amicone L., Galimi M. A., Spagnoli F. M., Tommasini C., De Luca V., Tripodi M. Temporal and tissue-specific expression of the MET ORF driven by the complete transcriptional unit of human A1AT gene in transgenic mice. Gene. 1995 Sep 11;162(2):323–328. doi: 10.1016/0378-1119(95)00277-d. [DOI] [PubMed] [Google Scholar]
  2. Antoine B., Levrat F., Vallet V., Berbar T., Cartier N., Dubois N., Briand P., Kahn A. Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp Cell Res. 1992 May;200(1):175–185. doi: 10.1016/s0014-4827(05)80086-2. [DOI] [PubMed] [Google Scholar]
  3. Bussolino F., Di Renzo M. F., Ziche M., Bocchietto E., Olivero M., Naldini L., Gaudino G., Tamagnone L., Coffer A., Comoglio P. M. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol. 1992 Nov;119(3):629–641. doi: 10.1083/jcb.119.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cao Z., Umek R. M., McKnight S. L. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991 Sep;5(9):1538–1552. doi: 10.1101/gad.5.9.1538. [DOI] [PubMed] [Google Scholar]
  5. Cassio D., Weiss M. C. Expression of fetal and neonatal hepatic functions by mouse hepatoma-rat hepatoma hybrids. Somatic Cell Genet. 1979 Nov;5(6):719–738. doi: 10.1007/BF01542637. [DOI] [PubMed] [Google Scholar]
  6. Cooper C. S., Park M., Blair D. G., Tainsky M. A., Huebner K., Croce C. M., Vande Woude G. F. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature. 1984 Sep 6;311(5981):29–33. doi: 10.1038/311029a0. [DOI] [PubMed] [Google Scholar]
  7. De Simone V., De Magistris L., Lazzaro D., Gerstner J., Monaci P., Nicosia A., Cortese R. LFB3, a heterodimer-forming homeoprotein of the LFB1 family, is expressed in specialized epithelia. EMBO J. 1991 Jun;10(6):1435–1443. doi: 10.1002/j.1460-2075.1991.tb07664.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Defrances M. C., Wolf H. K., Michalopoulos G. K., Zarnegar R. The presence of hepatocyte growth factor in the developing rat. Development. 1992 Oct;116(2):387–395. doi: 10.1242/dev.116.2.387. [DOI] [PubMed] [Google Scholar]
  9. Frain M., Swart G., Monaci P., Nicosia A., Stämpfli S., Frank R., Cortese R. The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell. 1989 Oct 6;59(1):145–157. doi: 10.1016/0092-8674(89)90877-5. [DOI] [PubMed] [Google Scholar]
  10. Frisch S. M., Francis H. Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol. 1994 Feb;124(4):619–626. doi: 10.1083/jcb.124.4.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Giordano S., Ponzetto C., Di Renzo M. F., Cooper C. S., Comoglio P. M. Tyrosine kinase receptor indistinguishable from the c-met protein. Nature. 1989 May 11;339(6220):155–156. doi: 10.1038/339155a0. [DOI] [PubMed] [Google Scholar]
  12. Giordano S., Zhen Z., Medico E., Gaudino G., Galimi F., Comoglio P. M. Transfer of motogenic and invasive response to scatter factor/hepatocyte growth factor by transfection of human MET protooncogene. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):649–653. doi: 10.1073/pnas.90.2.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gottardi C. J., Caplan M. J. An ion-transporting ATPase encodes multiple apical localization signals. J Cell Biol. 1993 Apr;121(2):283–293. doi: 10.1083/jcb.121.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Landschulz W. H., Johnson P. F., Adashi E. Y., Graves B. J., McKnight S. L. Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev. 1988 Jul;2(7):786–800. doi: 10.1101/gad.2.7.786. [DOI] [PubMed] [Google Scholar]
  16. Michalopoulos G. K. Liver regeneration: molecular mechanisms of growth control. FASEB J. 1990 Feb 1;4(2):176–187. [PubMed] [Google Scholar]
  17. Nakamura T., Teramoto H., Ichihara A. Purification and characterization of a growth factor from rat platelets for mature parenchymal hepatocytes in primary cultures. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6489–6493. doi: 10.1073/pnas.83.17.6489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ni R., Tomita Y., Matsuda K., Ichihara A., Ishimura K., Ogasawara J., Nagata S. Fas-mediated apoptosis in primary cultured mouse hepatocytes. Exp Cell Res. 1994 Dec;215(2):332–337. doi: 10.1006/excr.1994.1349. [DOI] [PubMed] [Google Scholar]
  19. Nuñez G., London L., Hockenbery D., Alexander M., McKearn J. P., Korsmeyer S. J. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed] [Google Scholar]
  20. Ogasawara J., Watanabe-Fukunaga R., Adachi M., Matsuzawa A., Kasugai T., Kitamura Y., Itoh N., Suda T., Nagata S. Lethal effect of the anti-Fas antibody in mice. Nature. 1993 Aug 26;364(6440):806–809. doi: 10.1038/364806a0. [DOI] [PubMed] [Google Scholar]
  21. Park M., Dean M., Cooper C. S., Schmidt M., O'Brien S. J., Blair D. G., Vande Woude G. F. Mechanism of met oncogene activation. Cell. 1986 Jun 20;45(6):895–904. doi: 10.1016/0092-8674(86)90564-7. [DOI] [PubMed] [Google Scholar]
  22. Paul D. L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. doi: 10.1083/jcb.103.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ponzetto C., Bardelli A., Maina F., Longati P., Panayotou G., Dhand R., Waterfield M. D., Comoglio P. M. A novel recognition motif for phosphatidylinositol 3-kinase binding mediates its association with the hepatocyte growth factor/scatter factor receptor. Mol Cell Biol. 1993 Aug;13(8):4600–4608. doi: 10.1128/mcb.13.8.4600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ponzetto C., Bardelli A., Zhen Z., Maina F., dalla Zonca P., Giordano S., Graziani A., Panayotou G., Comoglio P. M. A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/scatter factor receptor family. Cell. 1994 Apr 22;77(2):261–271. doi: 10.1016/0092-8674(94)90318-2. [DOI] [PubMed] [Google Scholar]
  25. Raff M. C., Barres B. A., Burne J. F., Coles H. S., Ishizaki Y., Jacobson M. D. Programmed cell death and the control of cell survival: lessons from the nervous system. Science. 1993 Oct 29;262(5134):695–700. doi: 10.1126/science.8235590. [DOI] [PubMed] [Google Scholar]
  26. Rubin J. S., Chan A. M., Bottaro D. P., Burgess W. H., Taylor W. G., Cech A. C., Hirschfield D. W., Wong J., Miki T., Finch P. W. A broad-spectrum human lung fibroblast-derived mitogen is a variant of hepatocyte growth factor. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):415–419. doi: 10.1073/pnas.88.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rüther U., Tripodi M., Cortese R., Wagner E. F. The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice. Nucleic Acids Res. 1987 Sep 25;15(18):7519–7529. doi: 10.1093/nar/15.18.7519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sargent T. D., Wu J. R., Sala-Trepat J. M., Wallace R. B., Reyes A. A., Bonner J. The rat serum albumin gene: analysis of cloned sequences. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3256–3260. doi: 10.1073/pnas.76.7.3256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sawyers C. L., Denny C. T. Chronic myelomonocytic leukemia: Tel-a-kinase what Ets all about. Cell. 1994 Apr 22;77(2):171–173. doi: 10.1016/0092-8674(94)90307-7. [DOI] [PubMed] [Google Scholar]
  30. Schmidt C., Bladt F., Goedecke S., Brinkmann V., Zschiesche W., Sharpe M., Gherardi E., Birchmeier C. Scatter factor/hepatocyte growth factor is essential for liver development. Nature. 1995 Feb 23;373(6516):699–702. doi: 10.1038/373699a0. [DOI] [PubMed] [Google Scholar]
  31. Shay J. W., Pereira-Smith O. M., Wright W. E. A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res. 1991 Sep;196(1):33–39. doi: 10.1016/0014-4827(91)90453-2. [DOI] [PubMed] [Google Scholar]
  32. Sladek F. M., Zhong W. M., Lai E., Darnell J. E., Jr Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily. Genes Dev. 1990 Dec;4(12B):2353–2365. doi: 10.1101/gad.4.12b.2353. [DOI] [PubMed] [Google Scholar]
  33. Snider W. D. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell. 1994 Jun 3;77(5):627–638. doi: 10.1016/0092-8674(94)90048-5. [DOI] [PubMed] [Google Scholar]
  34. Stern C. D., Ireland G. W., Herrick S. E., Gherardi E., Gray J., Perryman M., Stoker M. Epithelial scatter factor and development of the chick embryonic axis. Development. 1990 Dec;110(4):1271–1284. doi: 10.1242/dev.110.4.1271. [DOI] [PubMed] [Google Scholar]
  35. Stevenson B. R., Siliciano J. D., Mooseker M. S., Goodenough D. A. Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol. 1986 Sep;103(3):755–766. doi: 10.1083/jcb.103.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takayama S., Sato T., Krajewski S., Kochel K., Irie S., Millan J. A., Reed J. C. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 1995 Jan 27;80(2):279–284. doi: 10.1016/0092-8674(95)90410-7. [DOI] [PubMed] [Google Scholar]
  37. Tsukada T., Tomooka Y., Takai S., Ueda Y., Nishikawa S., Yagi T., Tokunaga T., Takeda N., Suda Y., Abe S. Enhanced proliferative potential in culture of cells from p53-deficient mice. Oncogene. 1993 Dec;8(12):3313–3322. [PubMed] [Google Scholar]
  38. Vaux D. L., Cory S., Adams J. M. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature. 1988 Sep 29;335(6189):440–442. doi: 10.1038/335440a0. [DOI] [PubMed] [Google Scholar]
  39. Weidner K. M., Behrens J., Vandekerckhove J., Birchmeier W. Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J Cell Biol. 1990 Nov;111(5 Pt 1):2097–2108. doi: 10.1083/jcb.111.5.2097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wu J. C., Merlino G., Cveklova K., Mosinger B., Jr, Fausto N. Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1. Cancer Res. 1994 Nov 15;54(22):5964–5973. [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. Yao R., Cooper G. M. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science. 1995 Mar 31;267(5206):2003–2006. doi: 10.1126/science.7701324. [DOI] [PubMed] [Google Scholar]
  43. Zhen Z., Giordano S., Longati P., Medico E., Campiglio M., Comoglio P. M. Structural and functional domains critical for constitutive activation of the HGF-receptor (Met). Oncogene. 1994 Jun;9(6):1691–1697. [PubMed] [Google Scholar]
  44. van der Geer P., Hunter T., Lindberg R. A. Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol. 1994;10:251–337. doi: 10.1146/annurev.cb.10.110194.001343. [DOI] [PubMed] [Google Scholar]

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