FR3T3 Cells Research Articles

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line.

The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3 , under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2'-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division.

The transformation of a rat cell line induced by a tumor promoter occurs without affecting sister chromatid exchange.

Malignant transformation of cells of an established rat cell line, FR3T3, can be achieved by the exclusive treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) over a stationary growth period. In the course of the transformation process, sister chromatid exchange (SCE) rates of treated cells were examined at various key-points and compared to control cells. It was found that neither a single TPA treatment nor repeated TPA application during 16-22 consecutive cell-doubling times (which induces substantial alterations of the phenotypes) increased SCE rates in the rat cells. Furthermore, transformed cell clones, which were selected from low-serum cultures and which were found to be tumorigenic in vivo, had SCE rates in the same range as the parental nontransformed FR3T3 cells. It is, therefore, concluded that malignant transformation of the rat cells - induced by TPA - occurs without affecting such large-scale DNA rearrangements as are detectable by means of the SCE method.

Simian virus 40 T antigen is detected only in cells in the G2 phase of the cell cycle in one group of rat transformants

The relationship between the cell cycle and the presence of the nuclear T antigen in simian virus 40-transformed rat cells independently derived from FR 3T3 cells has been investigated using a fluorescence-activated cell sorter and a combination of stainings for cellular DNA (4',6-diamidine-2-phenylindole dihydrochloride, DAPI) and for the viral T antigen (fluorescein-labeled IgG). Such an analysis revealed that in three out of six lines, T antigen could be detected only in the G2 phase of the cell cycle. In the other three lines, the accumulation of T antigen appeared to be independent of the position of the cell in the cycle. The emergence of either phenotype was strictly correlated with the physiological state of the cell (growing versus resting, respectively) at the time the transformants were established.

Relationship of simian virus 40 tumor antigens to virus-induced mutagenesis.

We analyzed the mutation frequency to 8-azaguanine (8AZ) resistance in rat FR3T3 cells acutely infected with simian virus 40 wild type and tsA and early deletion mutants and in a series of temperature-sensitive (N) and temperature-insensitive (A) transformants derived from Chinese hamster lung (CHL) cells. Upon acute infection, the frequency of mutation to 8AZ resistance was raised at most by two- to eightfold over the spontaneous frequency, and it was independent of the presence of a functional 90,000-molecular-weight T antigen or 20,000-molecular-weight t antigen or both. Similarly, in the stable transformants of CHL cells, no correlation was found between functional T antigens and mutation to 8AZ resistance. It therefore seems unlikely that simian virus 40-induced transformation results from any mutagenic activity of this virus.

Relationship of simian virus 40 tumor antigens to virus-induced mutagenesis.

We analyzed the mutation frequency to 8-azaguanine (8AZ) resistance in rat FR3T3 cells acutely infected with simian virus 40 wild type and tsA and early deletion mutants and in a series of temperature-sensitive (N) and temperature-insensitive (A) transformants derived from Chinese hamster lung (CHL) cells. Upon acute infection, the frequency of mutation to 8AZ resistance was raised at most by two- to eightfold over the spontaneous frequency, and it was independent of the presence of a functional 90,000-molecular-weight T antigen or 20,000-molecular-weight t antigen or both. Similarly, in the stable transformants of CHL cells, no correlation was found between functional T antigens and mutation to 8AZ resistance. It therefore seems unlikely that simian virus 40-induced transformation results from any mutagenic activity of this virus.

Cell multiplication in metazoans: evidence for negative control of initiation in rat fibroblasts.

Experiments were performed to clarify the circumstances under which a rat fibroblast-like cell line proliferated in culture or when injected into syngeneic hosts. Rat FR3T3 and SV40FR3T3 cells in culture multiply exponentially at comparable rates regardless of the horse serum concentration in the culture medium. The concentration of horse serum in the medium determines the length of the exponential phase of cell multiplication. Heat inactivation of the serum for periods as long as 24 hr does not impair the ability of the serum to sustain maximal cell multiplication rates. At low serum concentrations the exponential phase may be prolonged by refeeding the cultures daily with fresh medium. In medium supplemented with 50% fresh rat serum, FRT3T3 and SV403T3 cells are prevented from multiplying by yet undefined component(s) in this serum. This correlates well with the lack of tumor formation by these cells when inoculated into syngeneic hosts. The evidence obtained in culture strongly suggests that the multiplication ability of established cells in culture is a dominant constitutive character of these cells that is expressed whenever they are provided with optimal supply of nutrients and when no specific inhibitory conditions are present. Based on the data presented, we postulate that cell multiplication is a repressible function. These data also are compatible with a negative control mechanism for cell multiplication.