Abnormal mitosis triggers p53-dependent cell cycle arrest in human tetraploid cells

Erroneously arising tetraploid mammalian cells are chromosomally instable and may facilitate cell transformation. An increasing body of evidence shows that the propagation of mammalian tetraploid cells is limited by a p53-dependent arrest. The trigger of this arrest has not been identified so far. Here we show by live cell imaging of tetraploid cells generated by an induced cytokinesis failure that most tetraploids arrest and die in a p53-dependent manner after the first tetraploid mitosis. Furthermore, we found that the main trigger is a mitotic defect, in particular, chromosome missegregation during bipolar mitosis or spindle multipolarity. Both a transient multipolar spindle followed by efficient clustering in anaphase as well as a multipolar spindle followed by multipolar mitosis inhibited subsequent proliferation to a similar degree. We found that the tetraploid cells did not accumulate double-strand breaks that could cause the cell cycle arrest after tetraploid mitosis. In contrast, tetraploid cells showed increased levels of oxidative DNA damage coinciding with the p53 activation. To further elucidate the pathways involved in the proliferation control of tetraploid cells, we knocked down specific kinases that had been previously linked to the cell cycle arrest and p53 phosphorylation. Our results suggest that the checkpoint kinase ATM phosphorylates p53 in tetraploid cells after abnormal mitosis and thus contributes to proliferation control of human aberrantly arising tetraploids.     

Preferential generation of human T cell hybrids with a tetraploid fusion partner

The factors determining successful derivation of human T lymphocyte hybrids are largely unknown. This report describes diploid and tetraploid clones of the T cell line CEM which were fused with either a human T cell line (Jurkat) or with peripheral blood lymphocytes (PBL). Fusions of all CEMR clones with the Jurkat cell line yielded hybrids at a very high frequency (1 X 10(-4)). Fusion of diploid clones of CEM with PBL yielded no hybrids, whereas fusion of tetraploid clones of CEM with PBL resulted in growth frequencies of 1 to 3 X 10(-6). Enumeration of hybrids immediately after fusion indicated that in all cases, fused cells represented 5 to 10% of the population. That the ability to yield viable hybrids after fusion was a characteristic of tetraploid cells was indicated by the finding that tetraploid variants of a diploid clone could also yield viable hybrids after fusion. Possible mechanisms for the difference in results generated with diploid and tetraploid cells, and characteristics of the hybrid cells generated, are also discussed.     

Flow cytofluorometric analysis of a human aneuploid cell line growing in vitro and in intraperitoneal diffusion-chambers

An aneuploid established cell line originating from human skin (NCTC 3075) was cultivated in vitro culture and in intraperitoneal diffusion chambers in hamsters. In in vitro cell culture a near tetraploid cell line dominated. Shortly after implantation into the diffusion chambers in the peritoneal cavity of hamsters a selective lysis of cells with near tetraploid DNA content occurred, with a relative increase of a diploid subline. After 5 days in the hamster a tetraploid cell line again dominated as in in vitro culture. The use of flow cytofluorometry for ploidy analysis and changes in cell cycle traverse is demonstrated. The possible use of this model system in studies of response to therapy is discussed.     

Failure of cell cleavage induces senescence in tetraploid primary cells

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.     

Depletion of Endonuclease G Selectively Kills Polyploid Cells

Endonuclease G is a mitochondrio-nuclear located nuclease with dual- vital and lethal- functions. Besides its role in apoptosis execution, we have recently shown that depletion of endonuclease G leads to necrotic cell death in yeast. Here, we present further mechanistic elucidation of endonuclease G's vital functions. The deletion of the yeast Endonuclease G gene causes the complete elimination of tetraploid cells during exponential growth. Consistently, conditional knockdown of mammalian endonuclease G selectively kills tetraploid but not diploid clones of the human HCT116 colon carcinoma cell line. We conclude that endonuclease G is important for the viability of polyploid mammalian and yeast cells.     

Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.     

p53 dependent centrosome clustering prevents multipolar mitosis in tetraploid cells

Backgroundp53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy.Conclusionsp53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway.     

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.     

红豆杉离体细胞四倍体的诱导

细胞大规模培养被认为是目前生产紫杉醇最有希望的替代途径之一,获得更高产的细胞系,可降低细胞大规模培养生产紫杉醇的成本,加速其产业化进程。药用植物多倍体与二倍体相比具有根,茎,叶和花果的巨型性,抗逆性强,次生代谢产物含量提高等特性。本研究通过同步化培养和秋水仙素诱导处理,成功建立了红豆杉四倍体细胞系并且经过7个周期的继代证明了该细胞系的稳定性。结果显示,用浓度为750mg/L的秋水仙素处理3d可以得到稳定传代的四倍体细胞系,其四倍体细胞比例稳定在62.5%。进一步对其次生代谢产物紫杉醇的检测显示该四倍体细胞比对照二倍体细胞具有更高的合成紫杉醇的能力。     

When 2 + 2 = 5: The origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

When 2+2=5: the origins and fates of aneuploid and tetraploid cells

Aneuploid cells are frequently observed in human tumors, suggesting that aneuploidy may play an important role in the development of cancer. In this review, I discuss the processes that may give rise to aneuploid cells in normal tissue and in tumors. Aneuploid cells may arise directly from diploid cells through errors in chromosome segregation, as a consequence of incorrect microtubule-kinetochore attachments, or through failure of the spindle checkpoint. A second route to formation of aneuploid cells is through a tetraploid intermediate, where division of tetraploid cells can yield very high rates of chromosome missegregation as a consequence of multipolar spindle formation. Diploid cells may become tetraploid through a variety of mechanisms, including endoreduplication, cell fusion, and cytokinesis failure. Although aneuploid cells may arise from either diploid or tetraploid cells, the fate of the resulting aneuploid cells may be distinct. It is therefore important to understand the different pathways that can give rise to aneuploid cells, and how the varied origins of these cells affect their subsequent ability to survive or proliferate.     

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.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

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.     

Polyploidization in rat liver: the role of binucleate cells.

Liver cells were isolated from rats undergoing active formation of tetraploid cells and prelabelled in their DNA with 14C thymidine. Autoradiography of the isolated cells showed that binucleate diploid cells, a major component of the parenchymal cell population at this time, are also active in DNA synthesis. These cells probably pass through mitosis and generate mononucleate tetraploid cells, the dominant cell type of mature rat liver, since the frequency of binucleate tetraploid cells is very low at this stage in rat liver development. The biological significance of liver polyploidy is discussed and it is suggested that this lies in enhanced resistance to mutagenesis.     

Chromosome instability in Spodoptera frugiperda Sf-9 cell line

Homogeneous cell lines are essential in industry and research if reliable and reproducible data are to be obtained. The Spodoptera frugiperda (Sf-9) cell line routinely used for the production of recombinant proteins was found to be heterogeneous, containing a mixture of diploid and tetraploid cells. Using dilution-cloning techniques, diploid and tetraploid subpopulations were isolated from a Sf-9 parental cell line, and their cytogenetic state was monitored using Vinblastine to arrest cells in mitosis. Flow cytometry was used to obtain a snapshot of the predominant subpopulations present to verify the karyological results. The rate at which clonal populations digress into the heterogeneous state was found to be more rapid for the diploid subpopulation, with the emergence of tetraploid cells after only 11 passages, than for the tetraploid subpopulation, where diploid clones appeared after 18 passages. The chromosomes in both diploid and tetraploid subpopulations as well as the parental cell line were found to spontaneously fragment during growth and expansion processes, giving rise to variable chromosome numbers. DNA analysis of cell lines obtained from laboratories worldwide have shown that the Sf-9 cell line used for the production of many recombinant proteins is cytologically unstable, leading to varying degrees of polyploidal state depending on its culture history and supplier.     

Survival and size are differentially regulated by placental and fetal PKBalpha/AKT1 in mice

The importance of placental circulation is exemplified by the correlation of placental size and blood flow with fetal weight and survival during normal and compromised human pregnancies in such conditions as preeclampsia and intrauterine growth restriction (IUGR). Using noninvasive magnetic resonance imaging, we evaluated the role of PKBalpha/AKT1, a major mediator of angiogenesis, on placental vascular function. PKBalpha/AKT1 deficiency reduced maternal blood volume fraction without affecting the integrity of the fetomaternal blood barrier. In addition to angiogenesis, PKBalpha/AKT1 regulates additional processes related to survival and growth. In accordance with reports in adult mice, we demonstrated a role for PKBalpha/AKT1 in regulating chondrocyte organization in fetal long bones. Using tetraploid complementation experiments with PKBalpha/AKT1-expressing placentas, we found that although placental PKBalpha/AKT1 restored fetal survival, fetal PKBalpha/AKT1 regulated fetal size, because tetraploid complementation did not prevent intrauterine growth retardation. Histological examination of rescued fetuses showed reduced liver blood vessel and renal glomeruli capillary density in PKBalpha/Akt1 null fetuses, both of which were restored by tetraploid complementation. However, bone development was still impaired in tetraploid-rescued PKBalpha/Akt1 null fetuses. Although PKBalpha/AKT1-expressing placentas restored chondrocyte cell number in the hypertrophic layer of humeri, fetal PKBalpha/AKT1 was found to be necessary for chondrocyte columnar organization. Remarkably, a dose-dependent phenotype was exhibited for PKBalpha/AKT1 when examining PKBalpha/Akt1 heterozygous fetuses as well as those complemented by tetraploid placentas. The differential role of PKBalpha/AKT1 on mouse fetal survival and growth may shed light on its roles in human IUGR.     

The effects of cell size and ploidy on cell allocation in mouse chimaeric blastocysts

In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.