Differentiation of Quail Myoblasts Transformed with a Temperature Sensitive Mutant of Rous Sarcoma Virus. I. Relationship between Differentiation and Tyrosine Kinase of src Gene Product.

Abstract

ABSTRACT. Quail embryonic pectoral myoblasts fuse with each other at 35.5°C and 41°C to essentially equal extents. Whenthe myoblasts were transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV), their fusion and biochemical processes of differentiation became temperature-sensitive: their fusion occurred at 41°C, the non-permissive temperature, but not at 35.5°C, the permissive temperature, suggesting that the fusion was regulated by the viral transforming gene. Fusion of the transformed cells proceeded more rapidly and synchronously than that of the parent cells at 41°C, and was completely suppressed at the permissive temperature, unlike that of the parent cells. These transformed cells were used to examine the relationship between myogenicdifferentiation and the tyrosine kinase activity of the src gene product. In spite of the temperature sensitivity of transformation, results showed that expressions of the src gene at 35.5°C and 41°C were similar. However, the level of tyrosine-phosphorylated protein was decreased at 41°C. Moreover, myoblast fusion could occur at 35.5°C in the presence of herbimycin A, an inhibitor of the tyrosine kinase activity of the src gene product. These results indicate that the tyrosine kinase activity of the src gene product is closely associated with regulation of myogenicdifferentiation of the cells.Myogenesis involves various biochemical and morphological differential events. These events are distinct and can be seen even in vitro using established line cells derived from myoblasts. Manyworkers have thus used myogenic cells as models in studies on the regulation of differentiation (23,32,39,40). Biochemical differentiations of myoblasts have been studied especially extensively from an early stage of muscle research. Myogenic differentiation is of interest not only with respect to characteristic biochemical events, but also with respect to cell fusion. We have been studying artificial cell fusion induced by HVJ(Sendai virus) and have examined changes of the membrane induced by the virus (18-20). For comparison with this artificial cell fusion, we examined the process of myotube formation, one of the sequential processes in muscle differentiation, to obtain more information on the phenomenonof cell fusion at molecular and cellular levels.During myogenesis, myoblasts proliferate and become aligned and committed to differentiation, and then they fuse simultaneously with each other in a socalled 'fusion burst' (39). There have been many studies on myoblast fusion by various experimental approaches (6, 8, 9, ll, 21, 22, 25, 38), butitsmechanismisstillobscure. Onereason for this seems to be that no suitable experimental system in which the fusion reaction can be controlled is available. Hitherto, the mechanismof myoblast fusion has mainly been studied using established cell lines derived from myoblasts, such as the L6 rat myoblast line, and the fusion reaction has been controlled with calcium. Calcium, however, is associated not only with various physiological react...

Purification and Characterization of a Lectin-Like Substance from Silkworm Faeces.

“…Namely, differentiation of QM-RSVcells is controlled by temperature. These phenomenaare associated with the protein kinase activity of the src gene product of the virus used for cell transformation (8,9). Unique activity was recognized in QM-RSV cells treated with the extract of silkworm faeces.…”

“…Primary quail myoblast (QM) cells and QM-RSV cells were prepared as described previously (8,9). Briefly, pectoral muscles from quail embryos were minced and myoblasts were dissociated by treatment with trypsin.…”

“…In addition, this medium was supplemented with fetal calf serum (FCS) at 20% for use as growth mediumand at 5%for use as differentiation medium. The cells were grown at 35.5°C, and induced to differentiate at 41°C (8,9). For treatment of QM-RSV cells with the crude extract of silkworm faeces, purified fraction, or concanavalin A (Con A), the cells were seeded at a density of2 x 105 cells per 35-mm diameter plastic dish coated with collagen (Type I-P, Nitta Gelatin, Yao City, Osaka, Japan) in 2 ml of growth medium.…”

“…They were precultured at 35.5°C for 24 hr in growth medium and then cultured at 35.5°C or 41°C in differentiation medium containing final concentrations of 200^g/ml or 400 /ig/ml of the crude extract, 100^g/ml of purified fraction, or 10 ptg /ml of Con A. QMcells were seeded in plastic dishes coated with collagen and cultured at 37°C. QMcells fuse more slowly than QMRSVcells and myotubes are formed within 72 hr after change of differentiation medium (8,9). MDCK and HeLacells were cultured in minimum essential medium (MEM) containing penicillin (100 units/ml), streptomycin (0.1 mg/ml), and 10% calf serum (CS).…”

“…QM-RSV cells proliferate at 35.5°C, the permissive temperature for RSVat which the cells do not show myogenic differentiation, but differentiate at 41°C, the nonpermissive temperature for RSV: on the shift up to 41°C, the cells fuse with each other, and show biochemical differentiation with formation of multinucleated myotubes within 24 hr (8,9). This system is thus very convenient for examination of myogenic differentiation, and we have been using it to study the mechanismof myoblast fusion.…”