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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.

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ABSTRACT.Polyploidization of Meth-A and B16-F10 cells by demecolcine was examined using flow cytometry (FCM). In the presence of demecolcine, both cell lines were polyploidized to more than 16c DNAcontent. A marked difference was observed in the durations of S phase of polyploidy. The S-phase duration of Meth-A cells was doubly increased with ploidy, but that of B16F10 cells remained constant. When the rate of DNAsynthesis in the polyploidizing cells was examined through the BrdU-uptake experiments, it was confirmed that the level of DNA-synthesis rate was constant in Meth-A cells but increased in B16F10cells. The cellular content of c-Myc protein in polyploidized cells was also examined using anti-c-Myc monoclonal antibody. The c-Myc level of Meth-A cells was constant regardless of the ploidy but that of B16F10 cells increased with ploidy. Thus, the cMyccontent seems to be related to the duration of S phase in polyploidy.A relationship between DNAsynthesis rate and DNA content has been reported for Chinese hamster cells by Graves and McMillan (6). They concluded that the duration of S phase in mammaliancells is almost constant regardless of the DNAcontent. This was supported by the finding of the increasing BrdU uptake in polyploid CHO cells ( F10 mixture (7:3) supplemented with 10% fetal bovine serum, streptomycin (100 mg/ml) and penicillin (50 units/ml).B16F10 cells, a highly metastatic subline of mouse B16 melanomacells, were maintained under the same conditions as above except that they were cultured in monolayers. Both cell lines were cultured at low density. Synchronization of cell lines. Exponentially growing Meth-A and B16F10 cells were plated in Petri dishes (100 mm diameter, NUNC) at a density of about 2x105 and 1 x 105 cells/dish, respectively, with the medium being changed 24 h after seeding. Twelvehours thereafter, the cells were exposed to hydroxyurea (HU) at a final concentration of 0.6 mM.Five hours thereafter, the Meth-A cells were washed twice by centrifugation and the B16F10cells were rinsed twice with medium. Twohours after HUrelease, the cells were exposed to demecolcine as follows.Polyploidization of synchronized cell lines. The synchronized cells were exposed to demecolcine at a final concentration of0.1^g/ml. At various times, as shown in Fig. 2, Meth-A and B16F10 cells were fixed with 20% ethanol/PBS^") (divalent cation-free phosphate buffered saline) and stored at 4°C. B16F10cells were trypsinized (0.17% trypsin and 30mM

Keywords: apoptosis/high polyploid/tumor ABSTRACT. It is well known that DNA-ploidy is useful independent prognosticator of malignancy. However, the biological significance of polyploid cells and the relation between polyploidy and prognosis is not well understood. We analyzed DNAploidy by flow cytometry in Meth-A cells (a cultured sarcoma cell line) after treatment with K252a, a protein kinase inhibitor, and showed induction of polyploidization.Apoptotic cell death of the high polyploid cells was verified by flow cytometry, morphological observation and gel analysis of DNAintegrity. Expression of tumor-suppressor nuclear protein p53 investigated by immunohistochemistry was increased 10-fold or more in cells with 16C (C=haploid DNAcontent) relative to cells with 2C, suggesting that the overexpression of p53 was involved in the apoptosis. These results may be of clinical relevance since it has been known that both DNAploidy and p53 expression have prognostic significance.

ABSTRACT. Demecolcine (Colcemid), an inhibitor of spindle fiber formation in M phase, induced apoptosis in V79 cells. At a concentration of 0.01^g/ml demecolcine, V79 cells proliferated exponentially as well as controls, although temporal Mphase accumulation occurred 6 h after the addition of demecolcine. At 0.1 /*g/ml, the cells became hyperploid after remaining in the Mphase for some time. Apoptosis occurred in V79 cells exposed to demecolcine at a concentration of 0.03 /ug/m\. Apoptosis was defined as the appearance of a sub-Gl peak in DNAhistograms and a ladder pattern of fragmented DNAin gelelectrophoresis. Demecolcine (Colcemid) as well as colchicine prevents the formation of spindle fibers in M phase (8, 9 Apoptosis is a mode of cell death that involves the active participation of the cell in its own destruction and is distinct from necrosis. The general characteristics of apoptosis are well established. They consist of distinct morphological and biochemical changes that include cell shrinkage, cell surface blebbing, segmentation of the nucleus and extensive degradation of DNAinto oligonucleosomal-sized fragments (6, 10).Oneof the most commonmeansof detecting apoptosis is DNAgel electrophoresis, in which DNAfragments can be seen as a "ladder" of bands equivalent to multiples of mononucleosomes or oligonucleosomes. Flow cytometry (FCM)can identify apoptotic cells as a subdiploid peak, after staining with DNAspecific dyes (1,12). MATERIALS AND METHODCells. V79 cells (Chinese hamster lung cell line) were maintained in a humidified atmosphere of 5%CO2at 37°C as a monolayer culture in a Leibovitz's L15 : Ham's F10 mixture (7 : 3) supplemented with 10% newborn calf serum (M.A. Bioproducts), streptomycin (100 //g/ml) and penicillin (50 units /ml). The cells were cultured at low densities.Drug treatment. Exponentially growing V79 cells were plated in Petri dishes (60 mmdiameter, NUNC)at a density of about 1 x 105 cells/dish, then the medium was changed 24 h after seeding. Twelve hours thereafter, the cells were exposed to demecolcine (Sigma Chemical Co.) at various concentrations. At 6, 12, 24 and 48 hours thereafter, the cells were harvested and prepared for flow cytometry, cell growth measurements, nuclear morphology, and DNAgel-electrophoresis. CellpreparationforFCM. At various times after the drug addition, the V79 cells were washed twice with PBS(~> (divalent cation-free phosphate-buffered saline) and trypsinized (0.17% trypsin and 30 mMEDTA). The cells were fixed with 20% ethanol, then incubated with 0.25% RNase (Type II-A, Sigma Chemical Co.) for 3 h at 4°C. Immediately before measurements, the cells were stained with PI (propidium iodide, 7.5 x 10~5 M) and red fluorescence was examined by means of FCM.Under these staining conditions, the signal due to residual double stranded RNAis negligible and the relative intensity of red fluorescence corresponds to the DNAcontent (7). Flow cytometry. Fluorescence from individual cells was measured using a Cytofluorograf system 50H (Ortho Instruments) equipped with ...