Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40

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Abstract
Most SV40 transformed BALB/c 3T3 [mouse fibroblast] clones employed for biochemical studies were used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, one transformed clone FNE, which was isolated after SV40 infection of BALB/c 3T3 cells in factor free medium, was characterized. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity and synthesis of viral-specific RNA. Other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5-0.7 copies at passage 40. Initially, the cells were negative for all type C virus. Cells carried at low density for 13-20 passages (65-100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. Subtle but perhaps important differences in viral gene expression can occur as a function of passage. The importance of evaluating the interactions between SV40 and endogenous type C viruses was shown.