Pluripotency of a polyploid H1 (ES) cell system without leukaemia inhibitory factor.

OBJECTIVES: Tetraploid cells are strictly biologically inhibited from composition of embryos; by the same token, only diploid cells compose embryos. However, the distinction between diploid and tetraploid cells in development has not been well explained. To examine pluripotency of polyploid ES cells, a polyploid embryonic stem (ES)-cell system was prepared. MATERIALS AND METHODS: Diploid, tetraploid, pentaploid, hexaploid, octaploid and decaploid H1 (ES) cells (2H1, 4H1, 5H1, 6H1, 8H1 and 10H1 cells, respectively) were cultured for about 460 days in L15F10 medium without leukaemia inhibitory factor (LIF). The cells cultured under LIF-free conditions were denoted as 2H1(-), 4H1(-), 5H1(-), 6H1(-), 8H1(-) and 10H1(-) cells, respectively. Pluripotency and gene expression were examined. RESULTS: Ploidy alteration of H1(-) cells was similar to that of H1 cells. The polyploid H1(-) cells showed positive activity of alkaline phosphatase, suggesting that they maintained pluripotency in vitro without LIF. The polyploid H1(-) cells formed teratocarcinomas in mouse abdomen, suggesting they could differentiate in mouse abdomen in vivo. 2H1, 4H1 and polyploid H1(-) cells expressed nanog, oct3/4 and sox2 genes, suggesting that they fulfilled the criteria of ES cells. Nanog gene was significantly over-expressed in 4H1 and polyploid H1(-) cells, suggesting that overexpression of nanog gene was a characteristic of polyploid H1 cells. CONCLUSION: Polyploid H1 (ES) cells retained pluripotency in vitro, without LIF with nanog over-expression.

DNA-unstable decaploid mouse H1 (ES) cells established from DNA-stable pentaploid H1 (ES) cells polyploidized using demecolcine.

OBJECTIVES: DNA content of diploid H1 (ES) cells (2H1 cells) has been shown to be stable in long-term culture; however, tetraploid and octaploid H1 (ES) cells (4H1 and 8H1 cells, respectively) were DNA-unstable. Pentaploid H1 (ES) cells (5H1 cells) established recently have been found to be DNA-stable; how, then is cell DNA stability determined? To discuss ploidy stability, decaploid H1 (ES) cells (10H1 cells) were established from 5H1 cells and examined for DNA stability. MATERIALS AND METHODS: 5H1 cells were polyploidized using demecolcine (DC) and 10H1 cells were obtained by one-cell cloning. RESULTS: Number of chromosomes of 10H1 cells was 180 and durations of their G(1), S, and G(2)/M phases were 3, 7 and 6 h respectively. Volume of 10H1 cells was double that of 5H1 cells and morphology of 10H1 cells was flagstone-like in shape. 10H1 cells exhibited alkaline phosphatase activity and their DNA content decayed in 91 days of culture. 10H1 cells injected into mouse abdomen formed solid tumours that contained several kinds of differentiated cells with lower DNA content, suggesting that 10H1 cells were pluripotent and DNA-unstable. Loss of DNA stability was explained using a hypothesis concerning DNA structure of polyploid cells as DNA reconstructed through ploidy doubling was arranged in mirror symmetry in a new configuration. CONCLUSION: In the pentaploid-decaploid transition of H1 cells, cell cycle parameters and pluripotency were retained, but morphology and DNA stability were altered.

Establishment of a tetraploid cell line from mouse H-1 (ES) cells highly polyploidized with demecolcine.

OBJECTIVE: Establishment of tetraploid ES cells. MATERIALS AND METHODS: Mouse H-1 (ES) cells were polyploidized by demecolcine and released from the drug. RESULTS: A tetraploid cell line (4nH1 cells) was established from mouse H-1 (ES) cells (2nH1 cells) highly polyploidized by treatment with demecolcine. Cell cycle parameters of 4nH1 cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. Mode of chromosome number of 4nH1 cells was 76, about twice that of 2nH1 cells. Cell volume of 4nH1 cells was about twice of that of diploid cells, indicating that 4nH1 cells contained about twice as much total intracellular material as 2nH1 cells. Morphology of the 4nH1 cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that the transformation had occurred during the diploid-tetraploid transition. 4nH1 cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they would be pluripotent. CONCLUSION: A pluripotent tetraploid cell line (4nH1 cells) was established.

The reversion to diploid cells from established triploid V79 cells.

The triploid V79 cells are stable under usual culture conditions, and do not revert to being diploid. Here, the triploid-diploid transition of triploid V79 cells has been successfully induced in suspension culture in culture dishes with untreated surfaces. The diploid cells began to appear in a population of triploid V79 cells cultured under these conditions for 4 weeks. All of the triploid cells were transformed to diploid through subsequent monolayer culture for 5 weeks. It was confirmed that the revertant diploid cells had the same characteristics as original diploid V79 cells, with respect to DNA histograms, cell volume and chromosome number. Thus, it seems that suspension culturing is an important factor that induces the triploid-diploid transition.

Centrosome hyperamplification and chromosomal damage after exposure to radiation.

OBJECTIVE: In order to elucidate the effects of radiation on centrosome hyperamplification (CH), we examined the centrosome duplication cycle in KK47 bladder cancer cells following irradiation. METHODS: KK47 cells were irradiated with various doses of radiation and were examined for CH immunostaining for gamma-tubulin. RESULTS: Nearly all control cells contained one or two centrosomes, and mitotic cells displayed typical bipolar spindles. The centrosome replication cycle is well regulated in KK47. Twenty-four hours after 5-Gy irradiation, approximately 80% of irradiated cells were arrested in G2 phase, and at 48 h after irradiation, 56.9% of cells contained more than two centrosomes. Laser scanning cytometry performed 48 h after irradiation showed the following two pathways: (1) unequal distribution of chromosomes to daughter cells, or (2) failure to undergo cytokinesis, resulting in polyploidy. With mitotic collection, M-phase cells with CH could be divided into G1 cells with micronuclei and polyploidal cells. Fluorescence in situ hybridization analysis showed clear signs of chromosomal instability (CIN) at 48 h after irradiation. The present study had two major findings: (1) continual duplication of centrosomes occurred in the cell cycle-arrested cells upon irradiation, leading to centrosome amplification; (2) cytokinesis failure was due to aberrant mitotic spindle formation caused by the presence of amplified centrosomes. Abnormal mitosis with amplified centrosomes was detected in the accumulating G2/M population after irradiation, showing that this amplification of centrosomes was not caused by failure to undergo cytokinesis, but rather that abnormal mitosis resulting from amplification of centrosomes leads to cytokinesis block. CONCLUSION: These results suggest that CH is a critical event leading to CIN following exposure to radiation.

Centrosome hyperamplification and chromosomal instability in bladder cancer.

OBJECTIVE: Chromosomal instability (CIN) is a common feature of malignant tumors. Centrosome hyperamplification (CH) occurs frequently in human cancers, and may be a contributing factor in CIN. In this study, we investigated the relationship between CH and CIN in bladder cancer. METHODS: Clinical samples obtained by transurethral resection from 22 patients with bladder cancer were examined (histological grade G1, 5 cases; G2, 6 cases; G3, 11 cases). CH was evaluated by immunohistochemistry using anti-pericentrin antibody. CIN was evaluated by fluorescence in situ hybridization (FISH). FISH probes for pericentromeric regions of chromosomes 3, 7, and 17 were hybridized to touch preparations of nuclei from frozen tissues. We also analyzed the centrosome replication cycle of bladder cancer by laser scanning cytometry (LSC). RESULTS: Of the 22 cases examined, 18 (81.8%) had centrosome hyperamplification: CH 0, 4 cases (18.1%); CH I, 5 cases (22.7%); CH II, 5 cases (22.7%); CH III, 8 cases (36.4%). The grade of CH was directly proportional to the histological grade (p=0.03, chi(2) test). LSC analysis showed that the centrosome replication cycle was well regulated in pathologically low-grade bladder cancer, which did not have chromosomal instability. In contrast, we found marked variability of centrosomes in pathologically high-grade bladder cancer, which had chromosomal instability. CH and CIN were both detected in pathologically high-grade tumors. The grade of CH was directly proportional to the CIN grade (p=0.0079, chi(2) test). CONCLUSION: The results of the present study suggest that CH may be involved in CIN in bladder cancer.

Establishment of a tetraploid Meth-A cell line through polyploidization by demecolcine but not by staurosporine, K-252A and paclitaxel.

Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.

Temperature dependence in proliferation of tetraploid Meth-A cells in comparison with the parent diploid cells.

The temperature dependency for the growth of tetraploid Meth-A cells established from diploid cells was examined in comparison with the parent diploid cells. Proliferation of the tetraploid cells was markedly suppressed below 35 degrees C. At above 40 degrees C, both the diploid and tetraploid Meth-A cells ceased growing. Flow cytometry (FCM) analysis showed that the hyperploid cell fraction increased in the tetraploid Meth-A cell population at low temperatures. The fluidity of cell membranes at different temperatures was measured by means of electron spin resonance (ESR), and it was almost the same between the diploid and tetraploid Meth-A cells. It was suggested that the decreased proliferation below 35 degrees C of the tetraploid Meth-A cells might be due to the increased volume of the cells.

Different responses of polyploidized V79 cells after removal of two drugs, demecolcine and K-252a.

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.

Intranuclear localization of proliferating cell nuclear antigen during the cell cycle in renal cell carcinoma.

OBJECTIVE: To investigate, with laser scanning cytometry (LSC), proliferating cell nuclear antigen (PCNA) expression during the cell cycle in renal cell carcinoma. STUDY DESIGN: DNA ploidy and intracellular localization of PCNA in renal cell carcinoma were determined using LSC and immunohistochemistry. The subjects were nine patients who had received surgery for renal cell carcinoma. After DNA ploidy analysis, the glass slides were restained by immunohistochemistry of PCNA. LSC allowed direct observation of PCNA localization during the cell cycle because we could obtain immunohistochemical staining of PCNA as a function of cell cycle phase for individual cells. RESULTS: PCNA was not demonstrated in the nuclei of G0/G1 cells. PCNA expression increased from the S phase of the cell cycle. PCNA rapidly degraded at the end of the G2 phase. In the late G2 and M phase, PCNA was not detected in almost any nucleus. CONCLUSION: LSC allows morphologic observation of the intracellular distribution of PCNA during the cell cycle in renal cell carcinoma.

Involvement of protein kinase C in taxol-induced polyploidization in a cultured sarcoma cell line.

Taxol was found to inhibit the proliferation and to induce the polyploidization of cultured methylcholanthrene-induced sarcoma cells (Meth-A cells). To investigate whether protein kinase C is involved in taxol-induced polyploidization, phorbol 12-myristate 13-acetate (PMA), which regulates the activity of protein kinase C, was used along with taxol to treat the cells. We found that PMA did not interfere with the proliferation and did not induce polyploidization by itself. However, at low concentration, taxol, which by itself did not induce polyploidization, clearly induced polyploidization in the presence of PMA. To explore the mechanism by which PMA potentiates polyploidization, the levels of the G1 checkpoint-related proteins cyclin E and cdk2, and those of the G2 checkpoint-related proteins cyclin B and cdc2 were determined by flow cytometry. We found that both G1 and G2 checkpoint-related proteins increased during the induction of polyploidization. To verify the relationship between protein kinase C and tubulin polymerization, flow cytometry was used to determine the total content of tubulin protein, and morphological observation was used to examine spindle organization. PMA did not affect the taxol-induced increase in tubulin protein, but markedly potentiated taxol-induced spindle disorganization. These findings suggest that protein kinase C plays an important role in regulating the induction of polyploidization in Meth-A cells.

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry.

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.

Cell membrane changes of structure and function in protein kinase inhibitor-induced polyploid cells.

Exogenous cyclic AMP has been thought to be a chemical without marked pharmacological effect until now, as it is not capable of penetrating the cell membrane in most eucaryotic cells. The present study obtained results consistent with those of most previous studies, showing that exogenous cyclic AMP itself did not interfere with the cell cycle even at the high dose of 100 microM. However, it was found that K252a, a potent inhibitor of protein kinases including protein kinase C, induced DNA re-replication, i.e. DNA synthesis at a elevated DNA ploidy in cells that had not undergone cytokinesis (leading to polyploidization), and that exogenous cyclic AMP markedly potentiated the K252a-induced polyploidization at a very low dose similar to the effective dose of membrane-permeable cyclic AMP analogue dibutyryl cyclic AMP. These findings suggested that the cell membrane changed during the formation of polyploid cells. This supposition was confirmed by scanning electron microscopy to observe structural changes and by determination of cellular attachment to investigate functional changes.

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression.

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine.

The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.

Apoptotic cell death of high polyploid cells in a cultured sarcoma cell line.

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 DNA ploidy 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 DNA integrity. Expression of tumor-suppressor nuclear protein p53 investigated by immunohistochemistry was increased 10-fold or more in cells with 16C (C = haploid DNA content) 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 DNA ploidy and p53 expression have prognostic significance.

Saikosaponin b2 induces differentiation without growth inhibition in cultured B16 melanoma cells.

Treatment with 5 microM of saikosaponin (SS) b2 for 30 days was found to induce differentiation of B16 melanoma cells, with potentiation of expressions of melanogenesis and tyrosinase. To explore the mechanism of this effect, we observed the cell growth, cell cycle and morphology, and found that SSb2 did not affect any of these parameters. That is, SSb2 induced the differentiation of B16 melanoma cells without growth inhibition or cytotoxicity under conditions of low dose and long-term treatment. Phorbol ester, a protein kinase C (PKC) activator, markedly inhibited the expressions of melanogenesis and tyrosinase in both the control B16 melanoma cells and the long-term treated B16 melanoma cells. Down-regulation of the PKC activity may be involved in the effects of SSb2.

Spontaneous polyploidization results in apoptosis in a Meth-A tumor cell line.

Cultured Meth-A cells always include a small fraction of large cells, which had a DNA content above 4c (polyploid cells). The process from the formation to the disintegration of polyploid Meth-A cells was measured by means of time-lapse videography. Polyploid Meth-A cells arose spontaneously from normal cells (polyploidization), then died by apoptosis. The fraction of polyploid cells gradually increased in seven day-exponential cultures with a low concentration of demecolcine, which is a specific inhibitor of cell division. The results revealed that the polyploid Meth-A cells are generated from normal cells by failing cell division and that they die by apoptosis.

Different manner of DNA synthesis in polyploidizations of meth-A and B16F10 cell lines.

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 DNA content. 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 DNA synthesis 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 B16F10 cells. 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 c-Myc content seems to be related to the duration of S phase in polyploidy.

Saikosaponin b2-induced apoptosis of cultured B16 melanoma cell line through down-regulation of PKC activity.

Saikosaponin (SS) b2 was found to inhibit the proliferation of B16 melanoma cells. To explore this mechanism, we employed flow cytometry to determine the distribution of DNA content. The cell cycle of B16 melanoma cells was accumulated in the G1 phase followed by induction of apoptosis. This suggests that SSb2-induced proliferation inhibition is caused by G1 phase accumulation and that apoptosis induction is G1-phase-accumulation dependent. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, did not interfere with the proliferation and did not induce apoptosis of B16 melanoma cells by itself. However, PMA significantly abolished these effects of SSb2 in including proliferation inhibition and apoptosis induction. Down-regulation of the PKC activity may be involved in the effect of SSb2.