Protein synthesis and secretion in a myogenic cell line

Schubert, David; Tarikas, Helgi; Humphreys, Susie; Heinemann, Stephen F.; Patrick, James W.

doi:10.1016/0012-1606(73)90161-9

Cited by 64 publications

(21 citation statements)

References 28 publications

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“…Rat L6 myogenic cells were shown to secrete several unidentified collagenase-sensitive, high-Mr proteins with high levels of proline and low levels of methionine (Schubert et al, 1973;Garrels, 1979. A non-fusing continuous human embryonal rhabdomyosarcoma cell line apparently synthesized primarily type III collagen as well as minor proportions of types I and V (Kreig et al, 1979).…”

Section: Discussionmentioning

confidence: 99%

“…previous reports have documented secretion of collagenous proteins by the clonal rat myoblast line L-6 (Schubert et al, 1973;Garrels, 1979;, by a human rhabdomyosarcoma (Krieg et al, 1979), and also by primary muscle cell cultures Kuhl et al, 1982;Duance et al, 1979a;Kundu et al, 1981a,b;von der Mark et al, 1981). Types I, III, IV and V collagens have also been detected in muscle tissue (Duance et al, 1977;Bailey et al, 1979a,b;Ketley et al, 1976).…”

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confidence: 91%

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Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts

Beach¹,

Rao²,

Festoff³

1985

Biochemical Journal

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We have previously shown that G8-1, a murine clonal skeletal-muscle cell line, produces a substrate-attached extracellular matrix [Beach, Burton, Hendricks & Festoff (1982) J. Biol. Chem. 257, 11437-11442]. To examine further the expression of extracellular-matrix proteins by muscle cells, we have analysed the collagenous proteins secreted by G8-1 myoblasts. We have found that collagens and/or procollagens, corresponding to genetic types I, III and IV (and possibly V), are produced and secreted by G8-J myoblasts. The major secreted collagenous polypeptides were identified as al type I and its precursors by using pulse-chase studies, pepsin and collagenase digestions and CNBr fragmentation. The presence of lesser amounts of the other collagens was determined by immunoprecipitation. These results demonstrate that clonal skeletal-muscle cells, in the absence of fibroblasts and an exogenous -collagen substrate, are able to synthesize and secrete several extracellular-matrix collagenous proteins in proportions similar to those which are commonly found in muscle tissue and mixed cultures of muscle cells and fibroblasts.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 91%

Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts

Beach¹,

Rao²,

Festoff³

1985

Biochemical Journal

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“…In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo-phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.The L6 rat myoblast cell line isolated by Yaffe (45) forms multinucleate myotubes and synthesizes elevated levels of several muscle-characteristic proteins when grown to confluence in physiological levels of calcium (10,19,30,31,(35)(36)(37)43 …”

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confidence: 99%

“…The L6 rat myoblast cell line isolated by Yaffe (45) forms multinucleate myotubes and synthesizes elevated levels of several muscle-characteristic proteins when grown to confluence in physiological levels of calcium (10,19,30,31,(35)(36)(37)43 …”

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confidence: 99%

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

Crerar¹,

Leather²,

David³

et al. 1983

Mol. Cell. Biol.

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To assess the functional role of RNA polymerase II in the regulation of transcription during muscle differentiation, we isolated and characterized a large number of independent cx-amanitin-resistant (AmaR) mutants of L6 rat myoblasts that express both wild-type and altered RNA polymerase II activities. We also examined their myogenic (Myo) phenotype by determining their ability to develop into mature myotubes, to express elevated levels of muscle creatine kinase, and to synthesize muscle-characteristic proteins as detected by two-dimensional polyacrylamide gel electrophoresis. We found a two-to threefold increase in the frequency of clones with a myogenic-defective phenotype in the AmaR (RNA polymerase II) mutants as compared to control ethyl methane sulfonate-induced, 6-thioguanine-resistant (hypoxanthine, guanine phosphoribosyl transferase) mutants or to unselected survivors also exposed to ethyl methane sulfonate. Subsequent analysis showed that about half of these myogenic-defective AmaR mutants had a conditional Myo(ama) phenotype; when cultured in the presence of amanitin, they exhibited a Myo-phenotype; in its absence they exhibited a Myo+ phenotype. This conditional Myo(ama) phenotype is presumably caused by the inactivation by amanitin of the wild-type amanitin-sensitive RNA polymerase II activity and the subsequent rise in the level of mutant amanitin-resistant RNA polymerase II activity. In these Myo(ama) mutants, the wild-type RNA polymerase II is normally dominant with respect to the Myo+ phenotype, whereas the mutant RNA polymerase II is recessive and results in a Myo-phenotype only when the wild-type enzyme is inactivated. These findings suggest that certain mutations in the amaR structural gene for the amanitin-binding subunit of RNA polymerase II can selectively impair the transcription of genes specific for myogenic differentiation but not those specific for myoblast proliferation.The L6 rat myoblast cell line isolated by Yaffe (45) forms multinucleate myotubes and synthesizes elevated levels of several muscle-characteristic proteins when grown to confluence in physiological levels of calcium (10,19,30,31,(35)(36)(37)43

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“…The myoblast fusion is one of the earliest recognizable phenotypic signs of muscle differentiation, for a burst of synthesis in muscle-specific proteins, such as myosin, actin and creatine kinase occurs after myotube formation (12,16,17).…”

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confidence: 99%

Cyclic Nucleotide Changes in Fusion-Arrested Chick Embryo Myoblasts during Differentiation <I>In Vitro</I>

Moriyama

Murayama

1977

Cell Struct. Funct.

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ABSTRACT. Changes in intracellular concentrations of adenosine 3', 5'-monophosphate (cAMP) and guanosine 3', 5'-monophosphate (cGMP) were investigated in primary cultures of chick embryonic muscle cells grown in the presence of 5'-bromodeoxyuridine (BUdR). Cell fusion was completely inhibited in BUdR-treated cultures; however, the effect was reversed when the BUdR-containing medium was replaced with normal medium. As in control cultures, a significant increase in the ratio of cAMP-to-cGMP was observed during the first few days in both the BUdR-treated and the BUdR-released cultures. The data indicate that cell fusion is not a necessary step for concentration changes in cyclic nucleotides, which means that these changes might be a cause but not an effect of myoblast fusion.Myoblasts of chick embryonic skeletal muscle in appropriate culture conditions form muscle fibers by successive fusion of mononucleated myoblasts into multinucleated myotubes. The myoblast fusion is one of the earliest recognizable phenotypic signs of muscle differentiation, for a burst of synthesis in muscle-specific proteins, such as myosin, actin and creatine kinase occurs after myotube formation (12,16,17).The intracellular concentration of cyclic nucleotides has been proposed as possibly regulating cell fusion. The correlation between myoblast fusion with endogenous (8, 26, 27) and exogenous (22, 24) adenosine 3', 5'-monophosphate (cAMP) level has been shown in cultured, as well as in developing embryonic muscle cells. Furthermore, our previous data (8) have suggested that in addition to an increase in cAMP, a decrease in guanosine 3', 5'-monophosphate (cGMP) might be involved in regulating cell fusion. From our results, as well as those of previous workers, questions have arisen on how closely the changes in cyclic nucleotides are linked to cell fusion.In the present study cyclic nucleotide levels were examined in fusion blocked cultures using 5'-bromodeoxyuridine (BUdR) to determine whether cell fusion steps are coupled with cyclic nucleotide changes. MATERIALS AND METHODSCultures. Cells were prepared from thigh muscles of 12-day old chick embryos by trypsinization, as described previously (8). After minimizing non-myogenic cells by the selective preplating method (14), myogenic cells were inoculated at a cell density of 1 x 106 per dish in a 60 mm diameter Petri dish (Falcon plastics) coated with 1 % gelatin. Each dish contained 2.5 ml of culture medium, consisted of 80 % Eagle's minimum essential medium, 15 % horse 339

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Protein synthesis and secretion in a myogenic cell line

Cited by 64 publications

References 28 publications

Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts

Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts

Myogenic differentiation of L6 rat myoblasts: evidence for pleiotropic effects on myogenesis by RNA polymerase II mutations to alpha-amanitin resistance.

Cyclic Nucleotide Changes in Fusion-Arrested Chick Embryo Myoblasts during Differentiation <I>In Vitro</I>

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