SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes.
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.
Iujvidin
S
S
Department of Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
Fuchs
O
O
Nudel
U
U
Yaffe
D
D
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D008938:Mitosis / Q000502:physiology; D009132:Muscles / Q000166:cytology* / Q000378:metabolism / Q000382:microbiology / Q000502:physiology; D009857:Oncogenes / Q000502:physiology; D010957:Plasmids; D051381:Rats; D012192:Retroviridae Infections / Q000235:genetics / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004247:DNA