Alterations of enzymatic activities during muscle differentiation in vitro

Shainberg, Asher; Yagil, Gad; Yaffe, David

doi:10.1016/0012-1606(71)90017-0

Cited by 405 publications

(134 citation statements)

References 39 publications

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“…after fusion has begun these L8 myotubes will spontaneously contract. This pattern of development rather faithfully mimics myotube formation in vivo as well as that which has been reported in primary explants of avian and rat myoblasts (5,6).…”

Section: And 2)supporting

confidence: 81%

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Kaufman¹,

Parks²

1977

Proc. Natl. Acad. Sci. U.S.A.

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A nonfusing variant, fu-1, of the L8 line of rat myoblasts was isolated and characterized with respect to its growth in vitro and developmental properties. Comparative analyses of density-dependent inhibition of growth, serum requirements, cell adhesiveness, colony formation-in soft agar, and hexose transport in L8 and fu-1 cells support the conclusion that the fu-1 cells are transformed. In addition, fu-1, but not Le, cells promote the development of tumors in athymic nude mice. fu-1 cells also do not make increased levels o creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) or myosin and they express an endogenous type-C virus. Both L" and fu-1 cells express myokinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activities in single cells. In contrast to fu-1 cells, the parent L8 line has increased creatine kinase and myosin after fusion and spontaneously contracts; expression of an endogenous virus could not be detected in these cells. These results suggest that loss of the ability to differentiate normally is associated with the loss of the normal control of cell division of myoblasts grown in vitro and in vivo.Myoblasts, the precursors of highly differentiated skeletal muscle, proliferate in cell culture as single cells. Upon reaching a critical stage in their development, the myoblasts withdraw from the cell cycle and fuse into multinucleate cells (myotubes) (1)(2)(3)(4) of rat myoblasts (fu-i) that is defective in fusion from the myogenic L8 line. Analysis of several parameters that are usually attributed to transformed cells in vitro suggests that the nonfusing variant fu-i cells are transformed when compared to the parent L8 cells. In addition, only fu-l cells will form tumors in athymic mice. We have also found that the fu-i cells express an endogenous C-type virus; the L8 cells do not express this virus.MATERIALS AND METHODS Cell Culture Conditions. The L8 cells, kindly provided by D. Yaffe, were cloned and maintained by serial passage. Cloned cells were retrieved from frozen stocks as needed. fu-i cells were selected from the cloned L8 line as described in the text. All cells were grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% horse serum (ISI), 0.5% chick embryo extract, penicillin at 100 units/ml, and streptomycin at 100 ,ug/ml, at 370 and in a 10% CO2 atmosphere. Stock cultures of 2 X 105 cells were seeded in 10 ml of medium and grown on 100-mm Falcon tissue culture dishes coated with 0.1% gelatin. To maintain the myogenic capacity of L8 cells, it is essential to subculture the cells before they become confluent. Cells were routinely harvested by detachment with 0.05% trypsin in Ca2+,Mg2+-free Earle's solution. In experimental cultures, 3 ml of medium containing 1 to 2 X 105 cells were seeded in 50-mm dishes; medium was changed every day or as indicated. The cells were found free of mycoplasma.Uptake of 2-Deoxyglucose. Cells (2 X 105) were plated in 3 ml of medium on 50-mm dishes coated with gelatin. To determine uptake of 2-deoxyglucose (dGlc), the cel...

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Section: And 2)supporting

confidence: 81%

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Kaufman¹,

Parks²

1977

Proc. Natl. Acad. Sci. U.S.A.

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“…Desmin and creatine kinase have been attributed functional roles in mature myocytes and are highly expressed in muscle (Shainberg et al, 1971;Li et al, 1997). Desmin expression in proliferating myoblasts is governed by a 5 0 proximal promoter region (myoblastspecific region), and its elevated expression during differentiation is governed by a 5 0 distal enhancer (myotube-specific region) that contains an E-box commonly found in muscle-specific genes (Wentworth et al, 1991;Li and Paulin, 1993).…”

Section: Discussionmentioning

confidence: 99%

“…We tested this concept further by investigating the effect of TGF-b2 on creatine kinase activity, a long-established and quantifiable marker of myoblast differentiation (Shainberg et al, 1971). Creatine kinase levels were quantified directly from samples taken from the same strain A cultures used to examine the down-regulation of desmin by flow cytometric analysis shown in Figure 4.…”

Section: Down-regulation Of Desmin Expression By Tgf-b2 Is Reversiblementioning

confidence: 99%

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Characterization of proliferating human skeletal muscle‐derived cells in vitro: Differential modulation of myoblast markers by TGF‐β2

Stewart¹,

Masi²,

Cumming³

et al. 2003

Journal Cellular Physiology

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Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor b2 (TGF-b2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGFb2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-b. These data collectively indicate that TGF-b2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-b2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.

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“…Inhibition increased with prolonged incubation with cycloheximide (Table V). Also, myotube formation is remarkably decreased if protein synthesis is inhibited by cycloheximide [35,36]. Myotube formation is also inhibited in the presence of puromycin [36].…”

Section: Fusion After Modification Of Plasma Membrane Vesiclesmentioning

confidence: 99%

Fusion of isolated myoblast plasma membranes. An approach to the mechanism

Dahl

Schudt

Gratzl

1978

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Summary 105Fusion of plasma membranes isolated from myoblasts grown in culture has been investigated.1. Membrane fusion was specifically dependent on Ca 2 + at physiological concentrations. However, at higher concentrations of cations, fusion could be triggered not only by Ca2+, but by Mg2+ and Sr 2 + as weH.2. The amount of fusion was directly proportional to temperature. 3. Fusion was found to depend on the state of maturation of the my oblast membranes.4. Experiments with chemically and enzymatically modified mem branes and with membranes derived from myoblasts grown in the presence of inhibitors of protein biosynthesis suggest the participation of proteinaceous membrane components in the fusion mechanism.

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Alterations of enzymatic activities during muscle differentiation in vitro

Cited by 405 publications

References 39 publications

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.

Characterization of proliferating human skeletal muscle‐derived cells in vitro: Differential modulation of myoblast markers by TGF‐β2

Fusion of isolated myoblast plasma membranes. An approach to the mechanism

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