Octaploid Meth-A cells are established from a highly polyploidized cell population

Abstract.  Tetraploid Meth-A cells were polyploidized by demecolcin, an inhibitor of spindle fibre formation in M phase, and then released from the drug 1, 2, 3 and 4 days after the addition. Octaploid cells were successfully established from cell populations including hexadecaploid cells produced by 2, 3 and 4 days of exposure to demecolcin. One-day-treated cells were polyploidized octaploid cells, but they returned to tetraploid cells. All of the octaploid Meth-A cells showed essentially the same features. The octaploid Meth-A cells had eight homologous chromosomes and double the DNA content of the parent tetraploid cells. The doubling time of octaploid Meth-A cells was 30.2 h, somewhat longer than the 28.3 and 24.0 h of tetraploid and diploid cells, respectively. The fractions of cells in the G₁, S and G₂/M phases were essentially the same in diploid, tetraploid and octaploid Meth-A cells. The cell volume of octaploid Meth-A cells was about two times that of the tetraploid cells. It was concluded that octaploid Meth-A cells were established from transient hexadecaploid cells produced by the polyploidization of tetraploid cells that had been established from diploid cells.     

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.     

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.     

Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K-252a

Abstract. Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G₁, S and G₂/M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.     

Polyploid giant cells provide a survival mechanism for p53 mutant cells after DNA damage

The relationships between delayed apoptosis, polyploid 'giant' cells and reproductive survivors were studied in p53-mutated lymphoma cells after DNA damage. Following severe genotoxic insult with irradiation or chemotherapy, cells arrest at the G(2)-M cell cycle check-point for up to 5 days before undergoing a few rounds of aberrant mitoses. The cells then enter endoreduplication cycles resulting in the formation of polyploid giant cells. Subsequently the majority of the giant cells die, providing the main source of delayed apoptosis; however, a small proportion survives. Kinetic analyses show a reciprocal relationship between the polyploid cells and the diploid stem line, with the stem line suppressed during polyploid cell formation and restituted after giant cell disintegration. The restituted cell-line behaves with identical kinetics to the parent line, once re-irradiated. When giant cells are isolated and followed in labelling experiments, the clonogenic survivors appear to arise from these cells. These findings imply that an exchange exists between the endocyclic (polyploid) and mitotic (diploid or tetraploid) populations during the restitution period and that giant cells are not always reproductively dead as previously supposed. We propose that the formation of giant cells and their subsequent complex breakdown and subnuclear reorganization may represent an important response of p53-mutated tumours to DNA damaging agents and provide tumours with a mechanism of repair and resistance to such treatments.     

Tetraploidization increases sensitivity to Aurora B kinase inhibition

Aurora kinases are overexpressed in many cancers and are targets for anticancer drugs. The yeast homolog of Aurora B kinase, IPL1, was found to be a ploidy-specific lethality gene. Given that polyploidization is a common feature of many cancers, we hypothesized polyploidization also sensitizes mammalian cells to inhibition of Aurora kinases. Using two models of apparent diploid vs. tetraploid cell lines (one based on the hepatocellular carcinoma cell line Hep3B and another on untransformed mouse fibroblasts), we found that tetraploid cells were more sensitive to Aurora B inhibition than their diploid counterparts. Apoptosis could be induced in tetraploid cells by two different Aurora B inhibitors. Furthermore, tetraploid cells were sensitive to Aurora B inhibition but were not affected by Aurora A inhibition. Interestingly, the underlying mechanism was due to mitotic slippage and the subsequent excessive genome reduplication. In support of this, abolition of cytokinesis with dihydrocytochalasin B resulted in similar effects on tetraploid cells as Aurora B inhibition. These results indicate that inhibition of Aurora B or cytokinesis can promote apoptosis effectively in polyploid cancer cells.     

Glucocorticoid receptors and cell cycle progression in human melanoma cell lines

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole-cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content per cell correlated with the reduction in final plating efficiency of glucocorticoid-treated cells, suggesting a receptor-mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M-5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 microM dexamethasone for 1 h. Evidence is presented of a real arrest of M-5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1-S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by flow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M-5A and NKI-26 cells suggested differentiation of the cells during glucocorticoid-induced arrest.     

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

Abstract. 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.     

Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine

Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized-tetraploid transition might occur infrequently. Cell cycle parameters of 4nH1K 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. The cell volume of 4nH1K cells was about twice of that of diploid cells, indicating that 4nH1K cells contained about twice as much total intracellular material as 2nH1 cells. The morphology of the 4nH1K cells was flagstone-like, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that morphological transformation occurred during the diploid-tetraploid transition. 4nH1K cells exhibited alkaline phosphatase activity and formed teratocarcinomas, implying that they were pluripotent. These characteristics of 4nH1K cells were similar to those of tetraploid 4nH1 cells that have been established through polyploidization by demecolcine, suggesting that 4nH1K and 4nH1 cells are similar irrespective of the different mechanisms of polyploidization.     

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Ploidity and cell cycle progression during treatment with gold chloride, auranofin and sodium aurothiomalate. Studies on a human epithelial cell line and its sub-strains made resistant to the antiproliferative effects of these gold compounds

The possible influence of gold(III) chloride and the two gold(I)-containing anti-arthritic drugs, auranofin and sodium aurothiomalate, on cellular ploidity and cell cycle progression was investigated on cultured human epithelial cells. Four different cell lines were used: the parent line (HE) and three sub-strains which previously had acquired resistance to the antiproliferative effects of either 350 mumol gold chloride/l culture medium (HEAu350), 2 mumol auranofin/l (HEAF) or 300 mumol sodium aurothiomalate/l (HEMyo). DNA-histograms were obtained by flow cytometry examinations during a 9-days' exposure to either of these gold-containing compounds and concentrations. The HE, HEAF and HEMyo cells had similar ploidities, close to tetraploid. The HEAu350 cells had altered ploidity to distinct tetraploid. The distribution of the resistant cells with the cell cycle phases was not different from that of untreated HE cells. The HE cells, when treated with auranofin or sodium aurothiomalate, accumulated in the G2-phase of the cell cycle. In addition, a new cedecoploid peak appeared. No such changes were observed on gold chloride exposure or in HE controls grown without drug supplement. The effects of auranofin and sodium aurothiomalate on cell cycle progression of the HE cells possibly indicate a tendency to polyploidity, and furthermore that inhibition of cellular mitosis is one mechanism of the antiproliferative effect common to the two drugs.     

Kinetics of endomitosis in primary murine megakaryocytes

Megakaryocytes (MKs) develop from diploid progenitor cells via successive rounds of DNA synthesis in the absence of cell division, a process termed endomitosis (EnM). While the mechanism underlying EnM is not known, studies in yeast and leukemic cell lines have suggested that it may be due to reduced levels of cyclin B1 or cdc2, leading to a decrease in mitotic kinase activity. Using flow cytometry to study EnM highly purified marrow-derived MK precursors, we found that: (1) on average, 36% of 8N-32N MKs expressed abundant cyclin B during G2/M. The percentage of cells in G2/M decreased in >64N MKs, suggesting the limit of EnM, (2) the level of cyclin B per G2/M MK increased linearly with ploidy, (3) cyclin B expression oscillated normally in polyploid MKs, (4) MPM-2, a phosphoepitope created by the action of mitotic kinases and specific to M-phase cells, was expressed in a significant fraction of polyploid MKs, and (5) there was an apparent increase of cyclin B in G1-phase in polyploid MKs. This study provides the first qualitative kinetic data regarding the cell cycle status of MKs within individual ploidy classes. It also demonstrates the feasibility of using anti-cyclin B antibody and flow cytometry to resolve G1 from G2/M populations in polyploid MKs. Finally, these findings establish that neither a relative nor absolute deficiency of mitotic kinase components is responsible for EnM, suggesting that the departure from normal cell division kinetics seen in polyploid MKs is likely due to alterations in other cell cycle regulators.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

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.     

Radiation-induced genetic instability: no association with changes in radiosensitivity or cell cycle checkpoints in C3H 10T1/2 mouse fibroblasts

We investigated various phenotypic characteristics of radiation-induced morphologically transformed C3H 10T1/2 mouse fibroblasts. The cells were treated with 8 Gy x-rays, and type II/III foci were isolated. Cell lines were developed from these foci, and subsequently clones were established from these focal lines. The clones were examined for DNA content, radiosensitivity and inducible cell cycle arrests. Besides the morphological changes associated with the transformed state, the major difference between the isolated focal lines or derived clones and the parental C3H 10T1/2 line was one of ploidy. The transformed cells often displayed aneuploid and multiple polyploid populations. No change in the radiosensitivity of the transformed cells was observed. Furthermore, the two major radiation- and staurosporine-induced G1 and G2 cell cycle arrests observed in the parental cell line were also observed in the morphological transformants, suggesting that checkpoint function was normal. Received: 15 May 1997 / Accepted in revised form: 21 October 1997     

Radiosensitization by Chir-124, a selective Chk1 inhibitor: Effects of p53 and cell cycle checkpoints

Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G2-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR- induced G2-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53-/- and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53-/- cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21-/- HCT116 cells displayed a similar pattern of response as p53-/- cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.     

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 G₁, S, and G₂/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.     

Expression of cell kinetics and death during monocyte–macrophage differentiation: effects of Actinomycin D and Vinblastine treatments

The different effects of two cytostatic drugs, Actinomycin D and Vinblastine, during macrophage-like differentiation induced in THP-1 monocytic cell line by phorbol ester phorbol 12-myristate 13-acetate (PMA) (6, 30, and 60 nM), were studied by morpho-cytochemical approaches. In PMA-unstimulated monocytic cells, the cytostatic effects of Actinomycin D (an antimetabolic drug) were characterized by a drastic reduction of the G2/M cells accompanied by dramatic death of the G1 cells; on the contrary, Vinblastine (a microtubule-depolymerizating drug) induced an accumulation of the G2/M cells with the appearance of aneugenic micronuclei and scarce cell death mainly from the G1 cells. After 60 nM PMA stimulation, the culture was mostly composed by macrophagic cells characterized by low proliferation and the appearance of mono-/binucleated polyploid cells; in this condition, the cytotoxicity of the two drugs, more effective for Vinblastine, induced cell death in the different ploidy classes (2c, 4c, 8c). Cell death appeared to be of apoptotic nature, but with some morpho-phenotypic differences due to the action mechanism of the drugs and dependent on cell culture growth and differentiation. As a consequence of the different block-action of the two drugs on the cell cycle phases and in relation to the different subcellular targets, the effects changed during the transition from not-adhering/proliferating monocytes to adhering/low-proliferating differentiated macrophages.     

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4?days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2?weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3?days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.