Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Myogenic Differentiation of p53- and Rb-deficient Immortalized and Transformed Bovine Fibroblasts in Response to MyoD

  • Jin, Xun (Laboratory of Cell Growth and Function Regulation, Korea University) ;
  • Lee, Joong-Seub (Laboratory of Cell Growth and Function Regulation, Korea University) ;
  • Kwak, Sungwook (Laboratory of Cell Growth and Function Regulation, Korea University) ;
  • Jung, Ji-Eun (Laboratory of Cell Growth and Function Regulation, Korea University) ;
  • Kim, Tae-Kyung (Laboratory of Cell Growth and Function Regulation, Korea University) ;
  • Xuo, Chenxiong (Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Hong, Zhongshan (Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Li, Zhehu (Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Kim, Sun-Myoung (Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Whang, Kwang Youn (Division of Bioscience and Technology, College of Life and Environmental Sciences, Korea University) ;
  • Hong, Ki-Chang (Division of Bioscience and Technology, College of Life and Environmental Sciences, Korea University) ;
  • You, Seungkwon (Division of Bioscience and Technology, College of Life and Environmental Sciences, Korea University) ;
  • Choi, Yun-Jaie (Laboratory of Animal Cell Biotechnology, School of Agricultural Biotechnology, Seoul National University) ;
  • Kim, Hyunggee (Laboratory of Cell Growth and Function Regulation, Korea University)
  • Received : 2005.10.12
  • Accepted : 2005.12.25
  • Published : 2006.04.30
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Abstract

We have established in culture a spontaneously immortalized bovine embryonic fibroblast (BEF) cell line that has lost p53 and $p16^{INK4a}$ functions. MyoD is a muscle-specific regulator capable of inducing myogenesis in a number of cell types. When the BEF cells were transduced with MyoD they differentiated efficiently to desmin-positive myofibers in the presence of 2% horse serum and 1.7 nM insulin. The myogenic differentiation of this cell line was more rapid and obvious than that of C2C12 cells, as judged by morphological changes and expression of various muscle regulatory factors. To confirm that lack of the p53 and $p16^{INK4a}$ pathway does not prevent MyoD-mediated myogenesis, we established a cell line transformed with SV40LT (BEFV) and introduced MyoD into it. In the presence of 2% horse serum and 1.7 nM insulin, the MyoD-transduced BEFV cells differentiated like the MyoD-transduced BEFS cells, and displayed a similar pattern of expression of muscle regulatory proteins. Taken together, our results indicate that MyoD overexpression overcomes the defect in muscle differentiation associated with immortalization and cell transformation caused by the loss of p53 and Rb functions.

Keywords

Acknowledgement

Supported by : ARPC, Ministry of Education and Human Resources Development in Korea

References

  1. Armant, D. R. (2005) Blastocysts don't go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells. Dev. Biol. 280, 260-280 https://doi.org/10.1016/j.ydbio.2005.02.009
  2. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., et al. (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349-352 https://doi.org/10.1126/science.279.5349.349
  3. Braun, T., Bober, E., Buschhausen-Denker, G., Kohtz, S., Grzeschik, K. H., et al. (1989) Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products. EMBO J. 8, 3617-3625
  4. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51, 987-1000 https://doi.org/10.1016/0092-8674(87)90585-X
  5. De, S. R., Zahra, D. G., Duncan, E. L., and Reddel, R. R. (1995) Immortalization of human fibroblasts by liposome-mediated transfer of SV40 early region genes. Methods Cell Sci. 17, 75-81 https://doi.org/10.1007/BF00986654
  6. Dias, P., Parham, D. M., Shapiro, D. N., Tapscott, S. J., and Houghton, P. J. (1992) Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility. Cancer Res. 52, 6431-6439
  7. Felix, C. A., Kappel, C. C., Mitsudomi, T., Nau, M. M., Tsokos, M., et al. (1992) Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma. Cancer Res. 52, 2243-2247
  8. Frolov, M. V. and Dyson, N. J. (2004) Molecular mechanisms of E2F-dependent activation and pRB-mediated repression. J. Cell Sci. 117, 2173-2181 https://doi.org/10.1242/jcs.01227
  9. Hahn, W. C. (2002) Immortalization and Transformation of Human Cells. Mol. Cells 13, 351-361
  10. Hammond, E. M. and Giaccia, A. J. (2005) The role of p53 in hypoxia-induced apoptosis, Biochem. Biophys. Res. Commun. 331, 718-725 https://doi.org/10.1016/j.bbrc.2005.03.154
  11. Hayflick, L. and Moorhead, P. S. (1961) The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585-621 https://doi.org/10.1016/0014-4827(61)90192-6
  12. Jha, K. K., Banga, S., Palejwala, V., and Ozer, H. L. (1998) SV40-mediated immortalization. Exp. Cell Res. 245, 1-7 https://doi.org/10.1006/excr.1998.4272
  13. Keller, C., Arenkiel, B. R., Coffin, C. M., El-Bardeesy, N., DePinho, R. A., et al. (2004) Alveolar rhabdomyosarcomas in conditional Pax3:Fkhr mice: cooperativity of Ink4a/ARF and Trp53 loss of function. Genes Dev. 18, 2614-2626 https://doi.org/10.1101/gad.1244004
  14. Kim, H., Farris, J., Christman, S. A., Kong, B. W., Foster, L. K., et al. (2002a) Events in the immortalizing process of primary human mammary epithelial cells by the catalytic subunit of human telomerase. Biochem. J. 365, 765-772 https://doi.org/10.1042/bj20011848
  15. Kim, H., You, S., Farris, J., Kong, B. W., Christman, S. A., et al. (2002b) Expression profiles of p53, p16INK4a and telomere regulating genes in the replicative senescent human, mouse and chicken fibroblast cells. Exp. Cell Res. 272, 199-208 https://doi.org/10.1006/excr.2001.5420
  16. Ko, L. J. and Prives, C. (1996) p53: puzzle and paradigm. Genes Dev. 10, 1054-1072 https://doi.org/10.1101/gad.10.9.1054
  17. Lee, S. M., Youn, B. H., Kim, C. S., Kim C. S., Kang, C. H., et al. (2005) Gamma-Irradiation and doxorubicin treatment of normal human cells cause cell cycle arrest via different pathways. Mol. Cells 20, 331-338
  18. Levine, A. J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323-331 https://doi.org/10.1016/S0092-8674(00)81871-1
  19. Lukas, J., Parry, D., Aagaard, L., Mann, D. J., Bartkova, J., et al. (1995) Retinoblastomaprotein-dependent cell-cycle inhibition by the tumor suppressor p16. Nature 375, 503-506 https://doi.org/10.1038/375503a0
  20. Noble, J. R., Zhong, Z. H., Neumann, A. A., Melki, J. R., Clark, S. J., et al. (2004) Alterations in the p16(INK4a) and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts. Oncogene 23, 3116-3121 https://doi.org/10.1038/sj.onc.1207440
  21. Nowak, J. A., Malowitz, J., Girgenrath, M., Kostek, C. A., Kravetz, A. J., et al. (2004) Immortalization of mouse myogenic cells can occur without loss of p16INK4a, $p19^{ARF}$, or p53 and is accelerated by inactivation of Bax. BMC Cell Biol. 5, 1-14 https://doi.org/10.1186/1471-2121-5-1
  22. Porrello, A., Cerone, M. A., Coen, S., Gurtner, A., Fontemaggi, G., et al. (2000) p53 regulates myogenesis by triggering the differentiation activity of pRb. J. Cell Biol. 151, 1295-1304 https://doi.org/10.1083/jcb.151.6.1295
  23. Puri, P. L., Bhakta, K., Wood, L. D., Costanzo, A., Zhu, J., et al. (2002) A myogenic differentiation checkpoint activated by genotoxic stress. Nat. Genet. 32, 585-593 https://doi.org/10.1038/ng1023
  24. Richler, C. and Yaffe, D. (1970) The in vitro cultivation and differentiation capacities of myogenic cell lines. Dev. Biol. 23, 1-22 https://doi.org/10.1016/S0012-1606(70)80004-5
  25. Rothenberg, E. V. and Taghon, T. (2005) Molecular genetics of T cell development. Annu. Rev. Immunol. 23, 601-649 https://doi.org/10.1146/annurev.immunol.23.021704.115737
  26. Sherr, C. J. and DePinho, R. A. (2000) Cellular senescence: mitotic clock or culture shock? Cell 102, 407-410 https://doi.org/10.1016/S0092-8674(00)00046-5
  27. Shieh, S.-Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334 https://doi.org/10.1016/S0092-8674(00)80416-X
  28. Takahashi, Y., Oda, Y., Kawaguchi, K., Tamiya, S., Yamamoto, H., et al. (2004) Altered expression and molecular abnormalities of cell-cycle-regulatory proteins in rhabdomyosarcoma. Mod. Pathol. 17, 660-669 https://doi.org/10.1038/modpathol.3800101
  29. Tapscott, S. J., Thayer, M., and Weintraub, H. (1993) Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis. Science 259, 1450-1453 https://doi.org/10.1126/science.8383879
  30. Taylor, L. M., James, A., Schuller, C. E., Brce, J., Lock, R. B., et al. (2004) Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization. J. Biol. Chem. 279, 43634-43645 https://doi.org/10.1074/jbc.M402388200
  31. Weintraub, H., Tapscott, S. J., Davis, R. L., Thayer, M. J., Adam, M. A., et al. (1989) Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA 86, 5434-5438
  32. White, J. D., Rachel, C., Vermeulen, R., Davies, M., and Grounds, M. D. (2002) The role of p53 in vivo during skeletal muscle post-natal development and regeneration: studies in p53 knockout mice. Int. J. Dev. Biol. 46, 577-582
  33. Wright, W. E., Sassoon, D. A., and Lin, V. K. (1989) Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56, 607-617 https://doi.org/10.1016/0092-8674(89)90583-7
  34. Yaffe, D. (1968) Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. USA 61, 477-483
  35. Yeager, T. R. and Reddel, R. R. (1999) Constructing immortalized human cell lines. Curr. Opin. Biotechnol. 10, 465-469 https://doi.org/10.1016/S0958-1669(99)00011-7
  36. You, S., Moon, J. H., Kim, T. K., Kim, S. C., Kim, J. W., et al. (2004) Cellular characteristics of primary and immortal canine embryonic fibroblast cells. Exp. Mol. Med. 36, 325-335 https://doi.org/10.1038/emm.2004.43