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Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts 总被引:38,自引:0,他引:38
Stable myoblast cell lines were isolated after a brief exposure of mouse fibroblasts (10T1/2 cells) to 5-azacytidine. We show that transfection of 10T1/2 cells with DNA from these azacytidine-induced myoblasts (or from mouse C2C12 myoblasts) results in myogenic conversion of approximately 1 in 15,000 transfected colonies. In contrast, transfection of 10T1/2 cells with DNA from nonmyogenic cells (parental 10T1/2 cell DNA) does not give rise to myoblast colonies. These results indicate that an azacytidine-induced structural modification (presumably demethylation) in the DNA of a single locus is sufficient to convert 10T1/2 cells into determined myoblasts. 相似文献
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Expression of MRF4, a myogenic helix-loop-helix protein, produces multiple changes in the myogenic program of BC3H-1 cells.
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Expression of MRF4, a myogenic regulatory factor of the basic helix-loop-helix type, produced multiple changes in the myogenic program of the BC3H-1 cell line. BC3H-1 cells that stably expressed exogenous MRF4 were prepared and termed BR cell lines. Upon differentiation, the BR cells were found to have three muscle-specific properties (endogenous MyoD expression, myoblast fusion, and fast myosin light-chain 1 expression) that the parent BC3H-1 cells did not have. Of the four known myogenic regulatory factors (MyoD, myogenin, Myf-5, and MRF4), only MRF4 was capable of activating expression of the endogenous BC3H-1 myoD gene. In addition, the pattern of Myf-5 expression in BR cells was the opposite of that in BC3H-1 cells. Myf-5 expression was low in BR myoblasts and showed a small increase upon myotube formation, whereas Myf-5 expression was high in BC3H-1 myoblasts and decreased upon differentiation. Though the MRF4-transfected BR cells fused to form large myotubes and expressed fast myosin light-chain 1, the pattern of myosin heavy-chain isoform expression was the same in the BR and the nonfusing parent BC3H-1 cells, suggesting that factors in addition to the MyoD family members regulate myosin heavy-chain isoform expression patterns in BC3H-1 cells. In contrast to the changes produced by MRF4 expression, overexpression of Myf-5 did not alter BC3H-1 myogenesis. The results suggest that differential expression of the myogenic regulatory factors of the MyoD family may be one mechanism for generating cells with diverse myogenic phenotypes. 相似文献
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It has been shown previously that heterokaryons between myoblasts and non-myogenic cells disturb myogenic differentiation (Hirayama et al. (2001); Cell Struct. Funct. 26, 37-47), suggesting that some myogenesis inhibitory factors exist in non-myogenic cells. Skeletal myoblasts and adipose cells are derived from a common mesodermal stem cell, indicating that both cells have a closer relationship in the developmental lineage than the other somatic cells. To investigate the functional relationship between myoblasts and adipose cells, heterokaryons between quail myoblasts and 3T3-L1 cells, a mouse preadipocyte cell line, were prepared and examined for characteristics of myogenic differentiation. Myogenic differentiation was inhibited in the heterokaryons between quail myoblasts and well-differentiated (adipocytes) 3T3-L1 cells. On the contrary, normal myogenic differentiation proceeded in the heterokaryons between quail myoblasts and undifferentiated (preadipocytes) 3T3-L1 cells. Further investigation showed that the mouse myogenin gene from 3T3-L1 cells was transactivated in the heterokaryons between quail myoblasts and undifferentiated 3T3-L1 cells. The results demonstrated that undifferentiated 3T3-L1 cells have no myogenesis inhibitory factors but acquire these during terminal differentiation into adipocytes. 相似文献
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SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes 总被引:8,自引:0,他引:8
Sonia Lujvidin Ora Fuchs Uri Nudel David Yaffe 《Differentiation; research in biological diversity》1990,43(3):192-203
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. 相似文献
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The synthesis, turnover, and expression of all the major high mobility group (HMG) chromosomal proteins was studied in different rat skeletal myogenic cell lines. Whereas pulse-chase experiments revealed a similar half-life (greater than 2 cell generations) for all the HMG proteins in both L8 myoblasts and myotubes, [3H]lysine incorporation data indicated a 2- to 4-fold greater incorporation of the label in the HMG proteins in proliferating myoblasts relative to the nondividing myotubes. Analysis of the HMG-1, -14, and -17 mRNAs during myogenesis showed a significant down-regulation in L6 and L8 myotubes compared to the myoblasts. However, the timing of the shift and the extent of down-regulation was cell type-dependent, being more pronounced in L6 myotubes at fusion compared to 4 days postfusion in L8 myotubes. By contrast, L8-derived fusion-defective fu-1 cells over the same period of growth showed no change in HMG-14/17 mRNA levels. HMG-I(Y) protein isoforms, noted for the first time in rat myoblasts, like their counterparts, seemed to be stable and showed a precipitous reduction in their mRNAs during myogenesis. The results suggest a cell type-specific correlation between HMG expression and cell proliferation; they also argue for their role in maintenance of the cell's state of differentiation. 相似文献
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Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products. 总被引:59,自引:5,他引:54
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T Braun E Bober G Buschhausen-Denker S Kohtz K H Grzeschik H H Arnold S Kotz 《The EMBO journal》1989,8(12):3617-3625
The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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The small GTPase protein Rac1 is involved in a wide range of biological processes, yet its role in cell differentiation is mostly unknown. Here we show that Rac1 activity is high in proliferating myoblasts and decreases during the differentiation process. To analyze the involvement of Rac1 in muscle differentiation, different forms of the protein were expressed in muscle cells. A constitutively activated form of Rac1 (Rac1Q61L) inhibited the activity of MyoD in promoting muscle differentiation, whereas a dominant negative form of Rac1 (Rac1T17N) induced the activity of MyoD in promoting muscle differentiation. Expression of Rac1T17N imposed myogenic differentiation on myoblasts growing under mitogenic conditions. In inquiring whether Rac1 affected the withdrawal of myoblasts from the cell cycle, we analyzed the expression of cyclin D1 and p21(WAF1) and the phosphorylation state of the retinoblastoma protein. According to these markers and bromodeoxyuridine incorporation, C2 myoblasts expressing Rac1T17N exited the cell cycle earlier than control C2 cells. Myoblasts expressing Rac1Q61L did not permanently withdraw from the cell cycle. An indication of the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in Rac1-mediated myoblast proliferation was obtained by the use of MAPK kinase inhibitors U0126 and PD098059. These inhibitors arrested C2-Rac1Q61L cell cycling. Taken together, our results show that Rac1 activation interferes with myoblast exit from the cell cycle via or in concert with the MAPK pathway. 相似文献
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J R Florini K A Magri D Z Ewton P L James K Grindstaff P S Rotwein 《The Journal of biological chemistry》1991,266(24):15917-15923
Differentiation of muscle cells to form postmitotic myotubes is usually viewed as being negatively controlled by medium components, sometimes designated "mitogens." However, we have found that a family of mitogenic agents, the insulin-like growth factors (IGFs), are potent stimulators of differentiation in myoblasts which act by inducing expression of the myogenin gene. We show here that this action of the IGFs occurs even when these growth factors are not added to the cell medium; upon transfer to low-serum "differentiation medium," myoblasts begin active expression of the IGF-II gene, at both the mRNA and protein levels. Furthermore, autocrine secretion of IGF-II is essential for the process of terminal differentiation of the cells. These conclusions are based upon four lines of evidence. (1) The rate of spontaneous differentiation in several sublines of myogenic cells correlates with their level of expression of IGF-II. (2) C2 and Sol 8 cells, which secrete high levels of IGF-II, are relatively insensitive to exogenous IGFs, in contrast to L6 lines, which exhibit lower levels of IGF-II gene expression. (3) An antisense oligodeoxyribonucleotide complementary to the first five codons of IGF-II inhibits myogenic differentiation in the absence but not in the presence of exogenous IGF-II. (4) Spontaneous differentiation in response to autocrine IGF-II involves the same mechanism that occurs in cells stimulated by the IGFs, i.e. elevation of expression of the myogenin gene. 相似文献
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Effect of myogenic and adipogenic differentiation on expression of colony-stimulating factor genes.
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M A Harrington J H Falkenburg R Daub H E Broxmeyer 《Molecular and cellular biology》1990,10(9):4948-4952
The influence of cellular differentiation on colony-stimulating factor gene expression was examined in myogenically and adipogenically determined cell lines derived from 5-azacytidine-treated C3H10T1/2 C18 (10T1/2) mouse embryo fibroblasts. These studies demonstrate that colony-stimulating factor gene expression can be modulated by myogenic and adipogenic determination and terminal differentiation. 相似文献
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Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway 总被引:40,自引:0,他引:40
Stable myogenic cell lines have been derived at a high frequency by transfection of a cloned multipotential mouse embryo cell line, C3H 10T1/2, with cloned human DNA linked to a selectable neomycin resistance gene. The myogenic phenotype remains linked to neomycin resistance during secondary transfections. Although proliferative in growth conditions, these cell lines maintain the ability to differentiate and express muscle-specific proteins. We conclude that there is a simple genetic basis for myogenic determination and that a single gene, myd, converts 10T1/2 cells to a myoblast lineage. Southern blot analysis demonstrates nonidentity of myd and the MyoD1 gene. Northern blot analysis shows that myd-transfected myogenic lineages express MyoD1 mRNA while parental 10T1/2 cells do not. These results suggest that a dependent regulatory gene pathway mediates myogenic determination and differentiation. 相似文献
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Shibota Y Kaneko Y Kuroda M Nishikawa A 《Differentiation; research in biological diversity》2000,66(4-5):227-238
For the clarification of larval-to-adult muscle conversion, the authors established primary culture methods for adult- and larval-type myoblasts in the frog, Xenopus laevis, and examined the hormonal response in each case. The cell types were enzymatically dissociated from adult frog leg and tadpole tail muscles, respectively. The cells became attached to culture plates, proliferated, and fused with each other to form multinucleated myotubes within one week. Five significant differences between the two cell types were noted. (1) Adult cells showed greater proliferation activity than larval cells, the former increasing 5.5-fold over 6 days while the latter increase only 2.5-fold. (2) Differentiation (fusion) of larval type myoblasts started earlier. Cell fusion began on day 2 or 3 in larval cells and on day 4 in adult cells. (3) The metamorphic hormone, triiodo-L-thyronine (T3) decreased larval cell numbers to 56% of that of control-cultures on day 7 but had no effect on adult cell number. DNA synthetic activity (3H-thymidine incorporation) in larval cells decreased under T3 (10(-8) M) to 45% of the control level on day 7. (4) Differentiation of adult myoblasts into myotubes was promoted by T3, whereas that of larval cells diminished by half. (5) Myotube death was induced by T3 specifically in larval but not in adult cultures. In addition to the myotube death, double staining with TUNEL (in situ DNA nick end labeling) and anti-desmin antibody indicated that T3 induces myoblast (desmin+ cell) death specifically in larval but not in adult cells. It is thus evident that the conversion of a larval-type myogenic system during metamorphosis becomes possible through nearly totally specific control of cell division, cell differentiation, and programmed cell death at a precursor cell level by T3. 相似文献
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Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy 总被引:32,自引:4,他引:28
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《The Journal of cell biology》1994,125(6):1275-1287
The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases. 相似文献
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Traditionally, muscle cell lines are cultured on glass coverslips and differentiated to investigate myoblast fusion and differentiation. Efficient differentiation of myoblasts produces a dense network of myotubes with the correct organisation for contraction. Here we have tested the ability of artificially generated, precisely controlled peptide surfaces to enhance the efficiency of myoblast differentiation. We focused on specific short peptides from α-laminin-2 (IKVSV, VQLRNGFPYFSY and GLLFYMARINHA) as well as residues 15–155 from FGF1. We tested if these peptides in isolation, and/or in combination promoted muscle differentiation in culture, by promoting fusion and/or by improving sarcomere organisation. The majority of these peptides promoted fusion and differentiation in two different mouse myogenic cell lines and in primary human myoblasts. The additive effects of all four peptides gave the best results for both mouse cell lines tested, while primary human cell cultures differentiated equally well on most peptide surfaces tested. These data show that a mixture of short biomimetic peptides can reliably promote differentiation in mouse and human myoblasts. 相似文献
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Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin 总被引:7,自引:2,他引:5
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《The Journal of cell biology》1994,124(5):827-841
The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process. 相似文献