Alteration of myoblast phenotype by dimethyl sulfoxide.

Abstract

Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 2.7.3.2) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.Murine erythroleukemia and neuroblastoma cells in culture differentiate in the presence of dimethyl sulfoxide (Me2SO) (1, 2) or polar compounds of the polymethylene bisacetamide series (3, 4). We examined the effects of Me2SO on differentiation of L8 myoblasts, an established cell line of rat skeletal muscle (5). At confluency, these cells fuse to form multinucleate myotubes with concomitant accumulation of muscle-specific proteins, including creatine phosphokinase (EC 2.7.3.2) (CPK) (5, 6). Exposure of logarithmically growing L8 cells to 2% (vol/vol) Me2SO for 48 hr completely prevented both fusion of myoblasts and accumulation of CPK. However, fusion and increase in enzyme activity were not prevented when Me2SO was applied to high-density (prefusion stage) cultures. Even though fusion and rise in CPK activity did not occur, our data showed that Me2SO did not prevent cell differentiation, but, rather, induced a phenotypic conversion from myoblast to fibroblast-like cell. Hexamethylene bisacetamide, a potent inducer of erythroleukemia cell differentiation, had similar effects (unpublished data). MATERIALS AND METHODSCell Cultivation. A subline of L8 myoblast (5) planted in 100-mm dishes. Clonal cultures were left undisturbed for 2 or 3 weeks; all other cultures were refed every third day. In all studies, Me2SO-containing medium was applied to duplicate cultures either 24 hr after initial planting or at the time when the first multinucleate myotubes became visible.Cytology. For determination of mitotic indices, cultures grown on coverslips were fixed in 100% methanol and stained for 10 min with 2% aceto-orcein. The percent of mitotic cells was determined in at least 2000 cells in each preparation. Fusion indices were determined by counting the percent of nuclei incorporated into myotubes in 2000-4000 cells in each preparation, which was fixed in 100% methanol and stained with 0.1% crystal violet in 20% ethanol for 15 sec. Thymidine-labeling indices were measured in autoradiograms of coverslipgrown cultures that were pulse-labeled for 15 min with 10 MCi of tritiated thymidine per ml (specific activity 29 Ci/mmol, New England Corp., Bo...

Heterogeneous response to differentiation induction with different polar compounds in a clonal rat rhabdomyosarcoma cell line (BA-HAN-1C)

“…In contrast, the effects of DMSO, HMBA and NaBut were exclusively confined to an increase in creatine kinase activity used as a biochemical marker of differentiation, whereas no increase in morphological differentiation was observed and the formation of terminally differentiated postmitotic mytotube-like giant cells even significantly decreased. A corresponding inhibition of myotube formation had already been reported for non-tumorigenic myoblast cell lines after exposure to DMSO, HMBA and NaBut (Miranda et al, 1978;Blau & Epstein, 1979;Fiszman et al, 1980;Endo & Nadal-Ginard, 1987 8x 10-6± 5 x 10-6 15x 10-6+ 4x 10-6 DMSO 3.4 1.5 23 20…”

“…Polar compounds such as dimethylsulphoxide (DMSO) (Friend et al, 1971;Collins et al, 1978;Tarella et al, 1982;Tsao et al, 1982), hexamethylene bisacetamide (HMBA) (Reuben et al, 1976;Ramsay et al, 1986;Mansfield et al, 1988;Snyder et al, 1988), sodium butyrate (NaBut) (Prasad & Sinha, 1976;Dexter et al, 1981;Augeron & Laboisse, 1984;Nordenberg et al, 1987;Lee et al, 1988), and N-monomethylformamide (NMF) (Dexter et al, 1982;Dibner et al, 1985;Cordeiro & Savarese, 1986;Iwakawa et al, 1987) have only been sporadically described (Dexter 1977;Garvin et al, 1986). Furthermore, DMSO, HMBA and NaBut have been reported to inhibit terminal differentiation and myotube-formation in non-tumorigenic myoblast cell lines (Miranda et al, 1978;Blau & Epstein, 1979;Fiszman et al, 1980;Endo & Nadal-Ginard, 1987). Therefore, we investigated the susceptibility of the rhabdomyosarcoma cell line BA-HAN-IC to differentiation induction with polar compounds.…”

Induction by Butyrate of Differentiated Properties in Cloned Murine Rhabdomyosarcoma Cells

“…Leder and Leder [31] have also reported that DMSO and butyrate may induce by different mechanisms. Furthermore, the report that DMSO inhibits the fusion process in chick embryonic myoblasts [32] indicates that DMF, a polar solvent with chemical properties very similar to DMSO, might also be antagonistic to the myogenic process. Furthermore, the report that DMSO inhibits the fusion process in chick embryonic myoblasts [32] indicates that DMF, a polar solvent with chemical properties very similar to DMSO, might also be antagonistic to the myogenic process.…”

Medium composition for effective slow freezing of embryonic cell lines derived from marine medaka (Oryzias dancena)

“…These results suggest that the addition of FBS in freezing medium is undoubtedly required for stable freezing of embryonic cell lines from O. dancena species. It was reported that DMSO has cytotoxic effects by inducing the alteration of membrane fluidity and cytoskeleton (Brayton 1986;Hunt 2011;Katsuda et al 1987;Miranda et al 1978).…”