Regulation of MyoD function in the dividing myoblast

Proliferating myoblasts express MyoD, yet no phenotypic markers are activated as long as mitogen levels are sufficient to keep the cells dividing. Depending upon mitogen levels, a decision is made in G1 that commits the myoblast to either continue to divide or to exit from the cell cycle and activate terminal differentiation. Ectopic expression of MyoD under the control of the RSV or CMV promoters causes 10T1/2 cells to rapidly exit the cell cycle and differentiate as single myocytes, even in growth medium, whereas expression of MyoD under the weaker SV40 promoter is compatible with proliferation. Co-expression of MyoD and cyclin D1, but not cyclins A, B, E or D3, blocks transactivation of a MyoD responsive reporter. Similarly, transfection of myoblasts with the cyclin-dependent kinase (cdk) inhibitors p16 and p21 supports some muscle-specific gene expression even in growth medium. Taken altogether, these results suggest cell cycle progression negatively regulates myocyte differentiation, possibly through a mechanism involving the D1 responsive cdks. We review evidence coupling growth status, the cell cycle and myogenesis. We describe a novel mitogen-sensitive mechanism that involves the cyclin D1-dependent direct interaction between the G1 cdks and MyoD in the dividing myoblast, which regulates MyoD function in a mitogen-sensitive manner.     

SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes

Primary skeletal muscle myoblasts have a limited proliferative capacity in cell culture and cease to proliferate after several passages. We examined the effects of several oncogenes on the immortalization and differentiation of primary cultures of rat skeletal muscle myoblasts. Retroviruses containing a SV40 large T antigen (LT) gene very efficiently immortalize myogenic cells. The immortalized cell lines retain a very high differentiation capacity and form, in the appropriate culture conditions, a very dense network of muscle fibers. As in primary culture, cell fusion is associated with the synthesis of large amounts of muscle-specific proteins. However, unlike normal myoblasts (and previously established myogenic cell lines), nuclei in the multinucleated fibers of SV40-immortalized cells synthesize DNA and enter mitosis. Thus, withdrawal from DNA synthesis is not obligatory for cell fusion and biochemical differentiation. Using a retrovirus coding for a temperature-sensitive SV40 LT, myogenic cell lines were produced in which the SV40 LT could be inactivated by a shift from 33 degrees C to 39 degrees C. The inactivation of LT induced massive cell fusion and synthesis of muscle proteins. The nuclei in those fibers did not synthesize DNA, nor did they undergo mitosis. This approach enabled the reproducible establishment of myogenic cell lines from very small populations of myoblasts or single primary myogenic clones. Activated p53 also readily immortalized cells in primary muscle cultures, however the cells of eight out of the nine cell lines isolated had a fibroblastic morphology and could not be induced to form multinucleated fibers.     

SV40 Large T Antigen Interferes with Adult Myosin Heavy Chain Expression, but Not with Differentiation of Human Satellite Cells

The growth of muscle fibers during late development as well as in regeneration following muscle injury is the result of the proliferation and differentiation of satellite cells. However, all human cells, including satellite cells, show a limit in their proliferation. In order to define a cellular system with enhanced proliferative capacity, human satellite cells were transfected with a construct containing large T antigen from SV40 under the control of the human vimentin promoter. Vimentin is normally expressed during proliferation, and its expression is down-regulated as differentiation proceeds. In transfected cells, the construct is regulated like the endogenous vimentin gene. The effect of exogenous T antigen expression on both the proliferation and differentiation of human satellite cells was investigated. T antigen expression reduced the doubling time of human satellite cells from 36 to 20 h and increased the final proliferative capacity from 46 to 69 mean population doublings. When differentiation was triggered, although T antigen did not prevent the formation of myotubes, fusion was delayed. A similar delay was observed in the appearance of myogenin protein, one of the HLH regulatory factors, but not in the corresponding mRNA. Finally, T antigen has an effect on adult myosin isoform expression, since both adult slow and fast isoforms were only detected in myotubes negative for T antigen. These results led us to propose a model of the possible interactions between T antigen and muscle-specific factors.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Differentiation of simian virus 40 transformed human mammary epithelial stem cell lines to myoepithelial-like cells is associated with increased expression of viral large T antigen

Cloned simian virus 40 (SV40)-transformed human breast epithelial cell lines can differentiate to myoepithelial-like cells, and these can be isolated as clonal cell lines. Immunofluorescent and immunocytochemical analysis of such cell lines growing on plastic surfaces, collagen gels, and as tumor-nodules in nude mice indicate that all the cell lines produce SV40 large T antigen, but that the production of this antigen is qualitatively increased in the myoepithelial-like cells and cell lines. The myoepithelial-like cell lines produce 4-6 times more immunoprecipitable large T antigen than the parental epithelial cells. The amount of mRNA for large T antigen is also increased by 3.5-5-fold in the myoepithelial-like cell lines when analysed by dot-blot or by Northern hybridisations. Thus, differentiation along the myoepithelial-like cell pathway is associated in these SV40-transformed cells with increased expression of the viral large T antigen. It is suggested that immortalization of primary breast epithelial cell cultures may be, in part, due to the expression of large T antigen preventing processes of terminal keratinization.     

Role of simian virus 40 gene A function in maintenance of transformation.

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the (ts) A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3-to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpremissive temperature. All of the wild-type virus-transformed cells exhbited a transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. Thers results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristics of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells.     

Hypoxia inhibits myogenic differentiation through accelerated MyoD degradation

Cells undergo a variety of biological responses when placed in hypoxic conditions, including alterations in metabolic state and growth rate. Here we investigated the effect of hypoxia on the ability of myogenic cells to differentiate in culture. Exposure of myoblasts to hypoxia strongly inhibited multinucleated myotube formation and the expression of differentiation markers. We showed that hypoxia reversibly inhibited MyoD, Myf5, and myogenin expression. One key step in skeletal muscle differentiation involves the up-regulation of the cell cycle-dependent kinase inhibitors p21 and p27 as well as the product of the retinoblastoma gene (pRb). Myoblasts cultured under hypoxic conditions in differentiation medium failed to up-regulate both p21 and pRb despite the G1 cell cycle arrest, as evidenced by p27 accumulation and pRb hypophosphorylation. Hypoxia-dependent inhibition of differentiation was associated with MyoD degradation by the ubiquitin-proteasome pathway. MyoD overexpression in C2C12 myoblasts overrode the differentiation block imposed by hypoxic conditions. Thus, hypoxia by inducing MyoD degradation blocked accumulation of early myogenic differentiation markers such as myogenin and p21 and pRb, preventing both permanent cell cycle withdraw and terminal differentiation. Our study revealed a novel anti-differentiation effect exerted by hypoxia in myogenic cells and identified MyoD degradation as a relevant target of hypoxia.     

Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential

Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.     

Conditional immortalization of normal and dysgenic mouse muscle cells by the SV40 large T antigen under the vimentin promoter control.

We have created new mouse muscle cell lines of an immortalized type, expressing normal differentiation at the myotube stage: sarcomeric organization, functional excitation-contraction coupling, and triadic differentiation. The DNA immortalizing recombinant utilizes a deletion mutant of the regulatory region of the human vimentin promoter controlling the expression of a SV40 thermosensitive large T antigen, in which the small t sequence has been deleted. Skeletal mouse replicative myoblasts synthesized predominantly vimentin. After myoblast fusion the vimentin gene is strongly repressed in multinucleated syncytia. Furthermore, the normal activity of the vimentin promoter in myoblasts is increased in the large T antigen-expressing cells. We observed that continuous and rapid division of myoblasts occurs at permissive temperature, suggesting that immortalization is achieved even though the small t antigen is absent. When fusion is induced by changing media conditions, large T antigen expression is totally repressed by the vimentin promoter. When the temperature is elevated to 39 degrees C, the preexisting large T antigen is inactivated. The resulting myotubes from normal mouse differentiate totally normally as indicated by their morphology, ultrastructure, and electrophysiological properties. Mutant (muscular dysgenesis) immortalized cells express the same properties as mutant primary counterparts with no contraction, no slow Ca2+ current, and no triadic differentiation. These immortalized cell lines are potentially very useful for further pharmacology, transplantation, and cell biology studies. The vimentin promoter control of immortalizing recombinant DNA can be used for any mammalian normal and mutant muscle cell lines.     

Inhibition of simian virus 40 T-antigen expression by cellular differentiation.

Murine 3T3T stem cells transfected with pSV3neo DNA were employed to study the effects of somatic cell differentiation on simian virus 40 (SV40) T-antigen expression. This experimental approach was used because the 3T3T cell line is a well-characterized in vitro adipocyte differentiation system and the pSV3neo plasmid contains the early region of the SV40 genome and a selective marker, G418 resistance. Cell clones containing stably integrated pSV3neo which expressed T antigen were isolated in G418-containing medium. Most of these cell clones differentiated poorly. However, several clones retained the ability to efficiently differentiate into adipocytes, and with these cell clones, it was established that adipocyte differentiation markedly repressed T-antigen expression. The differentiation-specific repression of T-antigen expression did not result from a loss of proliferative potential associated with terminal differentiation, because it was observed in adipocytes that could be restimulated to proliferate. In such cells, restimulation of cell growth induced reactivation of T-antigen expression. Repression of T-antigen expression was also demonstrated during differentiation of SV40 T-antigen-immortalized human keratinocytes. These results establish that the process of cellular differentiation can repress T-antigen expression in at least two distinct biological systems.