George J. Todaro

The production of hyaluronate by spontaneously established cell lines and viral transformed lines of fibroblastic origin

Human diploid fibroblast strains and established mouse fibroblast lines continue to synthesize hyaluronate over many cell generations in culture. However, this property may be lost in the course of many years of serial culture of established lines as it is no longer detectable in three well-known mouse lines of fibroblastic origin, including L-cells. Transformation of the established mouse fibroblast line 3T3 by SV40 or polyoma virus and of human diploid fibroblast strains by SV40 leads in each case to a marked diminution in the rate of hyaluronate synthesis.

Antigenic and cultural properties of cells doubly transformed by polyoma virus and SV40

Abstract Cells of the established mouse line 3T3 were transformed in vitro by polyoma virus. Two of the resulting polyoma transformed lines were then transformed by SV40. Several doubly transformed clones were isolated and their properties were studied. Although most of them did not release either virus, all produced both virus-specific complement-fixing (CF) cellular antigens. The titers of each of these CF antigens in the double transformants were as high as those of the single antigens in cells transformed by only one of the viruses. Polyoma transformants and SV40 transformants of 3T3 may be distinguished by their colonial morphology. Colonies of the doubly transformed cells had, in general, characteristics of each of the single transformants. However, certain doubly transformed lines showed a preponderance of either the polyoma or SV40 character. The phenotypes of the doubly transformed lines demonstrate that each of the viruses is capable of acting independently of the other and each may have its distinctive effect on the same host cell.

Serum Albumin Supplemented Medium for Long Term Cultivation of Mammalian Fibroblast Strains.

Summary Serially transferred hamster or human fibroblasts maintained on synthetic medium containing 10% calf serum could be carried through about 10 and about 50-70 cell generations respectively, before growth ceased. Addition of crystallized serum albumin to the medium to a concentration of 20 mg/ ml affected the growth potential so that in both cases over 100 cell generations could be obtained. The human fibroblasts after 95 cell generations showed no chromosomal abnormalities while in the hamster fibroblasts some deviation from the diploid number did occur. In neither case was there evidence of the evolution of improved growth properties that characterize established cell lines.

ENHANCEMENT BY THYMIDINE ANALOGS OF SUSCEPTIBILITY OF CELLS TO TRANSFORMATION BY SV40.

Abstract The established mouse cell line, 3T3, is readily transformed by SV40. Infection of exponentially growing cultures results in a greater transformation frequency (3.6–6.8%) than does infection of stationary phase, non-DNA-synthesizing cells (1.8–2.6%). The thymidine analogs IUDR and BUDR are without effect on cell viability or transformation frequency when added to nongrowing cultures. Dividing cultures lose viability in the presence of these drugs to a degree dependent on the concentration and duration of exposure. Under conditions that lead to loss of viability of a portion of the population, surviving the cells are transformed by SV40 with a considerably higher frequency. Since this effect occurs whether the cells are exposed to the analogs before or after infection, they do not appear to act through modification of viral growth. At analog concentrations where cell killing is not too expensive, the absolute number of transformed colonies is increased, indicating a direct effect of the analogs on the susceptibility of the cells to transformation.

SUCCESSIVE TRANSFORMATIONS OF AN ESTABLISHED CELL LINE BY POLYOMA VIRUS AND SV40.

Two different oncogenic viruses, polyoma and SV40, are capable of transforming mouse cell line 3T3. The properties of the transformed cells produced by the two viruses are in some ways similar, but in other ways they are specific for the infecting virus. This fact permits testing whether a cell line transformed by the one oncogenic virus is still susceptible to the transforming action of the second virus. Two different clonally isolated polyoma-transformed lines when infected with SV40 give rise to cells with properties characteristic of SV40-transformed cells. The frequency of transformation, however, is considerably reduced compared to that of the parent cell line, 3T3.