ABSTRACT. To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by demecolcine and K-252a, inhibitors of spindle-fiber formation and protein kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a week. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.Key words: cell cycle analysis/V79 cells/polyploidization/K-252a/demecolcine V79 Chinese hamster lung cells were polyploidized at the same rate by demecolcine and K-252a, inhibitors of protein kinases and spindle-fiber formation in M phase, respectively (Fujikawa-Yamamoto et al., 1993), but their morphology differed between multi-and mono-nuclei detected in the cells polyploidized by demecolcine and K-252a, respectively (Fujikawa-Yamamoto et al., 1999b). It is of interest to determine whether or not the cells polyploidized by different mechanisms behave in a different manner after the drug removal.A relationship between the rate of DNA synthesis and DNA content has been reported for Chinese hamster cells by Graves and McMillan (1984) wherein the duration of S phase is almost constant regardless of the DNA content. This was supported by the finding of increased BrdU uptake in polyploid CHO cells (Takanari et al., 1985). Although several studies have reported a constant duration of the S phase in the polyploidization of cultured cells (Brenneisen et al., 1994;Fujikawa-Yamamoto et al., 1997b;Graves et al., 1984;Jordan et al., 1996;Usui et al., 1991;Watters et al., 1994;Zhang et al., 1996), except for Meth-A cells (Fujikawa-Yamamoto et al., 1997b), the cell cycle parameters, that is, the duration of G1, S and G2/M phases, of polyploid cells in a steady state of growth have not been studied well because of difficulty in achieving such a state.In this study, the behavior of V79 cells polyploidized by demecolcine and K-252a was examined by flowcytometry (FCM) for about one month after the removal of the drugs and the cell cycle parameters determined for the pseudo diploid and tetraploid.
Materials and Methods
CellsV79 (Chinese hamster lung cell line) cells were maintained in a humidified atmosphere of 5% CO2 at 37°C as a monolayer culture in Leibovitz's L15 : Ham's F10 mixture (7:3) supplemented with 10% fetal calf serum (M.A. Bioproducts, Walkersville, Md, USA), streptomycin (100 µg/ml) and penicillin (50 units/ml). The cells were cultured at low density.
