Characterization of mutant human fibroblast cultures transformed with simian virus 40.
Fibroblast cell strains derived from a normal individual and from eight patients with various genetic mutations were transformed by a small-plaque variant of simian virus 40 (SV40, strain 776), cloned and studied after long-term in vitro maintenance. Seven of the cultures continued to express the mutant phenotype. Cultures derived from a patient with phosphoglycerate kinase I deficiency exhibited reappearance of normal enzyme activity after transformation. Compared to untransformed controls, all transformed cultures displayed decreased population doubling times, an increase in the relative number of cycling cells and increased saturation density on solid substrates, and did not show evidence of cellular senescence after long-term cultivation. Unlike previous studies on wild-type SV40-transformed human fibroblasts, the majority of cultures transformed by the small-plaque variant of SV40 did not exhibit signs of crisis. The cells also exhibited a decreased dependence on serum and were able to grow in semi-solid medium. The different transformed cultures expressed variable levels of SV40 large T-antigen, synthesized some infectious SV40 virus, and contained both unique arrangements and quantities of covalently integrated and episomal SV40 DNA. No correlation was observed between the rate of growth and synthesis of infectious virus in the different transformed clones. These studies indicate that this small-plaque variant of SV40 can be used effectively to generate long-lived human cultures, which generally retain their mutant phenotype. Transformation with this SV40 variant permits the generation of large quantities of clonal cell cultures for the biochemical and molecular analysis of their genetic defects.
Miranda
A F
AF
Columbia University, College of Physicians and Surgeons, New York, NY 10032.
Duigou
G J
GJ
Hernandez
E
E
Fisher
P B
PB
D000293:Adolescent; D000957:Antigens, Viral, Tumor; D002470:Cell Survival; D002472:Cell Transformation, Viral; D002675:Child, Preschool; D002875:Chromosomes; D004279:DNA, Viral / Q000096:biosynthesis; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007223:Infant; D008297:Male; D008938:Mitosis; D009154:Mutation; D010641:Phenotype; D013539:Simian virus 40 / Q000276:immunology; D012867:Skin; D013997:Time Factors; D014779:Virus Replication
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral