Transformation of human skeletal muscle cells by simian virus 40.
Transformation of human skeletal muscle cells by simian virus 40.
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猿猴病毒40对人类骨骼肌细胞的转化。
DOI：
10.1073/pnas.80.21.6581
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发表时间：
1983
影响因子：
11.1
通讯作者：
Fisher,PB
中科院分区：
文献类型：
--
作者：
Miranda,AF;Babiss,LE;Fisher,PB
人类肌病中涉及的生化改变的分子研究受到限制，因为来自原代组织的培养物的有限寿命和缓慢的生长速率。由于肿瘤病毒猿猴病毒40（SV 40）可以改变人类细胞的生长特性和寿命，我们用SV 40的小斑块变体感染了来自四个活检组织的骨骼肌培养物，并分析了克隆成肌细胞衍生物的生物学和生化特性。感染后早期，成肌细胞正常融合成多核肌管，未融合和融合的细胞均含有SV 40肿瘤抗原（T抗原）。在感染后6至8次传代培养后，成肌细胞融合的能力减弱，并产生具有增加的生长速率和饱和密度的克隆细胞系。转化培养物也失去了接触抑制生长，成为锚定独立。与未转化的成肌细胞不同，SV 40转化的克隆没有经历肌酸激酶活性的增加或肌酸激酶同工酶从BB形式到肌肉特异性MM形式的转变。通过Southern印迹杂交分析两个克隆的SV 40转化的成肌细胞系（KJ-SV 40和PK-SV 40）中SV 40 DNA整合模式的分析表明，KJ-SV 40含有至少一个SV 40 DNA整合到染色体DNA中的位点，PK-SV 40含有至少三个SV 40 DNA共价连接到细胞DNA的位点。来自PK-SV 40转化体的细胞裂解物和生长培养基含有感染性小噬斑变体SV 40，而KJ-SV 40不含有或不产生可检测的病毒。这些研究表明，人成肌细胞可以通过SV 40永生化。这一过程可能被证明是有用的，为生物化学和分子分析产生大量的遗传缺陷的人类细胞。
Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.