Tetraploid Cell Lines Research Articles

Persistence of Chronic Lymphocytic Leukemia Stem-like Populations under Simultaneous In Vitro Treatment with Curcumin, Fludarabine, and Ibrutinib: Implications for Therapy Resistance.

Leukemic stem cells (LSCs) possess similar characteristics to normal hematopoietic stem cells, including self-renewal capacity, quiescence, ability to initiate leukemia, and drug resistance. These cells play a significant role in leukemia relapse, persisting even after apparent remission. LSCs were first described in 1994 by Lapidot et al. Although they have been extensively studied in acute leukemia, more LSC research is still needed in chronic lymphocytic leukemia (CLL) to understand if reduced apoptosis in mature cells should still be considered as the major cause of this disease. Here, we provide new evidence suggesting the existence of stem-like cell populations in CLL, which may help to understand the disease as well as to develop effective treatments. In this study, we identified a potential leukemic stem cell subpopulation using the tetraploid CLL cell line I83. This subpopulation is characterized by diploid cells that were capable of generating the I83 tetraploid population. Furthermore, we adapted a novel flow cytometry analysis protocol to detect CLL subpopulations with stem cell properties in peripheral blood samples and primary cultures from CLL patients. These cells were identified by their co-expression of CD19 and CD5, characteristic markers of CLL cells. As previously described, increased alkaline phosphatase (ALP) activity is indicative of stemness and pluripotency. Moreover, we used this method to investigate the potential synergistic effect of curcumin in combination with fludarabine and ibrutinib to deplete this subpopulation. Our results confirmed the effectiveness of this ALP-based analysis protocol in detecting and monitoring leukemic stem-like cells in CLL. This analysis also identified limitations in eradicating these populations using in vitro testing. Furthermore, our findings demonstrated that curcumin significantly enhanced the effects of fludarabine and ibrutinib on the leukemic fraction, exhibiting synergistic effects (combination drug index, CDI 0.97 and 0.37, respectively). Our results lend support to the existence of potential stem-like populations in CLL cell lines, and to the idea that curcumin could serve as an effective adjuvant in therapies aimed at eliminating these populations and improving treatment efficacy.

Polyploidy as a strategy to increase taxane production in yew cell cultures: Obtaining and characterizing a Taxus baccata tetraploid cell line

Novel approaches to optimize the production of plant specialized metabolites are crucial to reach maximum productivity of plant biofactories. Plant polyploidization frequently enhances protein synthesis and thereby increases the biosynthesis of specialized metabolites. Paclitaxel is a valuable anticancer agent scarcely produced in nature. Therefore, plant biofactories represent a sustainable alternative source of this compound and related taxanes. With the aim of improving the productivity of Taxus spp. cell cultures, we induced polyploidy in vitro by treating immature embryos of Taxus baccata with colchicine. To obtain the polyploid cell lines, calli were induced from T. baccata plantlets previously treated with colchicine and ploidy levels were accurately identified using flow cytometry. In terms of cell morphology, tetraploid cells were about 3-fold bigger than the diploid cells. The expression of taxane pathway genes was higher in the tetraploid cell line compared to the diploid cells. Moreover, taxane production was 6.2-fold higher and the production peak was achieved 8 days earlier than in the diploid cell line, indicating a higher productivity. The obtained tetraploid cell line proved to be highly productive, constituting a step forward towards the development of a bio-sustainable production system for this chemotherapeutic drug.

Abstract 3171: Detecting ploidy specific vulnerability to resource restriction: An integrated in vitro and in silico approach

Abstract Changes in the number of copies of the genome, i.e. changes in ploidy are widespread among cancer cells. Increased ploidy is thought to benefit cancer cells by conferring robustness against deleterious consequences of genomic instability. However, whether there are any potential costs associated with increased ploidy has not been sufficiently investigated. One hypothesis holds that high ploidy cells may exhibit increased sensitivity to limitations in resources, due to the extra energy required for the replication of a larger genome. To address this hypothesis, we developed an integrated in vitro and in silico system to investigate the response to glucose restriction in diploid and tetraploid cells. Near diploid (2N) SUM159 breast cancer cells were fused to generate an isogenic tetraploid (4N) cell line. 2N or 4N cells were plated with varying initial concentrations of glucose. Cells grew for five days and media was not refreshed, so cells depleted the glucose in the media until they eventually died. Throughout the experiments, glucose concentration was measured at 24 hour intervals, and images were taken at 8 hour intervals allowing quantification of the number of alive or dead cells over time. These in vitro data were then used as inputs to the in silico portion of our system, which consists of an Ordinary Differential Equation (ODE) model of cancer cell growth. ODE model parameters were estimated using the measured outputs from the in vitro experiments. Parameters estimated from the ODE model gave insight into differences between the 2N and 4N cell lines. We found that 4N cells outcompeted 2N cells at high glucose concentrations, due to their ability to divide more rapidly when glucose was abundant. However, 2N cells were able to outcompete 4N cells at lower glucose concentrations. The advantage of 2N cells at low glucose was not due to an increased proliferative capacity. Instead, 2N cells were more resistant to cell death when glucose was limited. Our next steps will be to expand our system to assess ploidy specific sensitivity to other resources, such as phosphate or oxygen. The ODE model we have developed allows us to identify resource conditions that will select for tumor cells based on ploidy. In future this knowledge can be used to suggest therapeutics that can modulate resource availability within tumors, in order to steer tumor evolution towards less aggressive phenotypes. Citation Format: Richard J. Beck, Didem Ilter, Ana P. Gomes, Noemi Andor. Detecting ploidy specific vulnerability to resource restriction: An integrated in vitro and in silico approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3171.

Establishing Tetraploid Embryogenic Cell Lines of Magnolia officinalis to Facilitate Tetraploid Plantlet Production and Phenotyping.

The production of synthetic polyploids for plant breeding is compromised by high levels of mixoploids and low numbers of solid polyploid regenerants during in vitro induction. Somatic embryogenesis could potentially contribute to the maximization of solid polyploid production due to the single cell origin of regenerants. In the present study, a novel procedure for establishing homogeneous tetraploid embryogenic cell lines in Magnolia officinalis has been established. Embryogenic cell aggregate (ECA) about 100–200 μm across, and consisting of dozens of cells, regenerated into a single colony of new ECAs and somatic embryos following colchicine treatment. Histological analysis indicated that the few cells that survived some colchicine regimes still regenerated to form a colony. In some colonies, 100% tetraploid somatic embryos were obtained without mixoploid formation. New granular ECA from single colonies with 100% tetraploid somatic embryos were isolated and cultured individually to proliferate into cell lines. These cell lines were confirmed to be homogeneous tetraploid by flow cytometry. Many tetraploid somatic embryos and plantlets were differentiated from these cell lines and the stability of ploidy level through the somatic embryogenesis process was confirmed by flow cytometry and chromosome counting. The establishment of homogeneous polyploid cell lines, which were presumed to represent individual polyploidization events, might expand the phenotypic variations of the same duplicated genome and create novel breeding opportunities using newly generated polyploid plantlets.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.

Acute immune stress improves cell resistance to chemical poison damage in SP600125-induced polyploidy of fish cells in vitro

Previous research has indicated that the small compound, SP600125, could induce polyploidy of fish cells, and has established a stable tetraploid cell line from diploid fish cells. In order to explore how fish cells maintain homeostasis under SP600125-stress in vitro, this study investigates impacts of SP600125-stress on intracellular pathways, as well as on regulation of the cellular homeostasis feedback in fish cells. Transcriptomes are obtained from the SP600125-treated cells. Compared with unigenes expressed in control group (crucial carp fin cells), a total of 2670 and 1846 unigenes are significantly upregulated and downregulated in these cells, respectively. Differentially expressed genes are found, which are involved in innate defense, inflammatory pathways and cell adhesion molecules-related pathways. The SP600125-stress enhances cell-mediated immunity, characterized by significantly increasing expression of multiple immune genes. These enhanced immune genes include the pro-inflammatory cytokines (IL-1β, TNF-ɑ, IL-6R), the adaptor signal transducers (STAT, IκBɑ), and the integrins (ɑ2β1, ɑMβ2). Furthermore, mitochondria are contributed to the cellular homeostasis regulation upon the SP600125-stress. The results show that acute inflammation is an adaptive and controlled response to the SP600125-stress, which is beneficial for alleviating toxicity by SP600125. They provide a potential way of breeding fish polyploidy induced by SP600125 in the future research.

Abstract 3031: Replication stress and DNA damage promote genomic instability in near-tetraploid colorectal cancer cells

Abstract Aneuploidy represents a hallmark of most solid tumours, and potentially has a causal role in carcinogenesis. Also, cancer cells exhibit high rates of chromosome missegregation, which leads to chromosomal instability (CIN). However, a large amount of tumours present near-triploid karyotypes likely to go through a tetraploid transient stage. Here, by using DLD-1 isogenic diploid and tetraploid cell lines generated by single cell FACS-sorting, we aimed at exploring how polyploidization affects cellular functions and whether tetraploid per se generates CIN in a genomically stable background. First, gene expression and subsequent gene set enrichment analysis revealed that genes involved in the machinery of the DNA synthesis and replication, such as MCM2 and RRM2, and genes involved in cell cycle, were significantly upregulated in near-tetraploid cells compared to their diploid counterparts. Functionally, polyploidy cells exhibited replication stress, as indicated by higher levels of pCHK1, RPA and 53BP1 foci, which resulted in increased DNA damage in S- and M-phase. In addition, tetraploid cells displayed impaired proliferative capabilities as a consequence of a cell cycle delay confirmed by BrdU pulse and flow cytometry. Furthermore, near-tetraploid clones showed a higher amount of intracellular karyotypic heterogeneity due to the higher frequency of micronuclei formation compared to their diploid counterparts. In fact, polyploid cells displayed an increased tendency of abnormal anaphase events, including lagging chromosomes and acentric fragments. Interestingly, these heterogeneous cellular populations showed strengthen migratory capabilities and preliminary experiments suggested an increment in tumor invasiveness too. Taken together, our data suggest that near-tetraploid cells systematically undergo replication stress, which can be responsible for the increased levels of genomic instability. Citation Format: Isabel Quintanilla, Darawalee Wangsa, Markus Brown, Amaia Ercilla, Greg Klus, Maria Vila, Juan José Lozano, Zoltan Szallsi, Neus Agell, Antoni Castells, Thomas Ried, Jordi Camps. Replication stress and DNA damage promote genomic instability in near-tetraploid colorectal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3031. doi:10.1158/1538-7445.AM2015-3031

Polyploidy in myelofibrosis: analysis by cytogenetic and SNP array indicates association with advancing disease

BackgroundMyelofibrosis occurs as primary myelofibrosis or as a late occurrence in the evolution of essential thrombocythaemia and polycythaemia vera. It is the rarest of the three classic myeloproliferative neoplasms (MPN). Polyploidy has only rarely been reported in MPN despite the prominent involvement of abnormal megakaryocytes. The use of peripheral blood samples containing increased numbers of haematopoietic progenitors has improved the output from cytogenetic studies in myelofibrosis and together with the use of single nucleotide polymorphism arrays (SNPa) has contributed to an improved knowledge regarding the diverse genetic landscape of this rare disease.ResultsCytogenetic studies performed on a consecutive cohort of 42 patients with primary or post ET/PV myelofibrosis showed an abnormal karyotype in 24 cases and of these, nine showed a polyploid clone. Six of the nine cases showed a tetraploid (4n) subclone, whereas three showed mixed polyploid subclones with both tetraploid and octoploid (4n/8n) cell lines. The abnormal clone evolved from a near diploid karyotype at the initial investigation to a tetraploid karyotype in follow-up cytogenetic analysis in four cases. In total, six of the nine polyploid cases showed gain of 1q material. The remaining three cases showed polyploid metaphases, but with no detectable structural karyotypic rearrangements. Three of the nine cases showed chromosome abnormalities of 6p, either at diagnosis or later acquired. SNPa analysis on eight polyploid cases showed additional changes not previously recognised by karyotype analysis alone, including recurring changes involving 9p, 14q, 17q and 22q. Except for gain of 1q, SNPa findings from the polyploid group compared to eight non-polyploid cases with myelofibrosis found no significant differences in the type of abnormality detected.ConclusionsThe study showed the use of peripheral blood samples to be suitable for standard karyotyping evaluation and DNA based studies. The overall profile of abnormalities found were comparable with that of post-MPN acute myeloid leukaemia or secondary myelodysplastic syndrome and cases in the polyploidy group were associated with features of high risk disease. The above represents the first documented series of polyploid karyotypes in myelofibrosis and shows a high representation of gain of 1q.

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.

Effects of chromosomal polyploidy on survival of colon cancer cells

Tetraploid cells are frequently observed in the inflamed mucosal epithelial cells of the patients with Barrett's esophagus or chronic ulcerative colitis. Polyploidy often occurs during cell fusion, abortive cell cycle, and endoreplication. Most tetraploid cells are engaged to apoptotic pathway, but some remaining stable tetraploid cells consequently cause aneuploidization and chromosomal instability. We investigated whether tetraploid cells could acquire survival advantage and hold a dominant position for natural selection. We established tetraploid cell line (HCT116GH) from parental diploid colorectal cancer cell line (HCT116) via PEG-mediated cell fusion and compared its cell viability, cell cycle response and apoptotic fractions responded to H₂O₂) with diploid HCT116 and p53 suppressed HCT116/H6 cell lines. Using MTT assay, plating efficiency and clonogenicity, we evaluated the survival of each cell line. Tetraploid cell line HCT116GH demonstrated an 83 fold greater resistance to 100 microM H₂O₂ than the parental diploid HCT116, and 6 fold greater than even the p53 negative diploid HCT116/E6. Cellular sensitivity, G2/M arrests, and apoptotic proportion were observed less in response to H₂O₂ in HCT116GH compared with HCT116 and HCT116/E6. HCT116GH expressed lower level of p53 and p21 than diploid HCT116. Stable tetraploid cell lines showed enhanced viability in comparison to parental diploid cell lines. The enhanced viability observed in tetraploidization surpassed that from downregulation of p53. Frequent appearance of tetraploid cells in stressful condition can be caused by natural selection owing to their enhanced viability and may consequently contribute to cancer cell transformation.

Zebra finch cell lines from naturally occurring tumors

The zebra finch (Taeniopygia guttata) has been intensively studied in many research fields including neuroscience, behavioral neurobiology, and evolution of the genome. Although numerous molecular and genomic resources are available for this model species, immortalized cell lines have been lacking. We have established two zebra finch cell lines derived from spontaneous tumors. ZFTMA is a tetraploid female cell line and G266 as a diploid male cell line. These first zebra finch cell lines should facilitate development of research on this model species.

Abstract A79: Genome-wide RNAi screen for tetraploid-specific lethality in cancer cells

Abstract Cancer cells are often genetically unstable, resulting in aneuploid genomes. The consequences of aneuploidy are poorly understood, but have been suggested to play roles in tumor aggressiveness, chemotherapy resistance, and metastasis. One important route to an aneuploid genome may be through doubling of the genome, leading to unstable tetraploid cells. Many cell division errors are ultimately manifest as a failure of cytokinesis. Especially in cells defective in p53 function, tetraploid cells proliferate, but generate both high rates of whole chromosome aneuploidy and defects in the maintenance of chromosome structural integrity. Our laboratory has demonstrated that, in p53-primary cells, tetraploidy promotes tumor development and the resulting tumors display markedly abnormal genomes (Fujiwara T. et al. Nature 2005, 437: 1043). It is now known that several human cancer-mutations — e.g. Rb loss or loss of the adenomatous polyposis coli tumor suppressor — also predispose cytokinesis failure and tetraploidy. These findings highlight a need to understand in detail the physiological changes that accompany tetraploidy. To gain a comprehensive understanding of physiological alterations associated with tetraploidy in cancer cells, we have established tetraploid cell lines derived from a near diploid human colorectal cancer cell line HCT116. Using these paired tetraploid and diploid cell lines, we have performed a genome-wide RNAi screen to identify genes whose knock down selectively affect the viability of either diploids or tetraploid cells. We are currently validating the result of our ploidy-specific lethality screen and we would like to update our progress at this conference. Citation Information: Cancer Res 2009;69(23 Suppl):A79.

The Probability to Initiate X Chromosome Inactivation Is Determined by the X to Autosomal Ratio and X Chromosome Specific Allelic Properties

BackgroundIn female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this probability, we studied the role of the X to autosome (X∶A) ratio in initiation of XCI, and have used the experimental data in a computer simulation model to study the cellular population dynamics of XCI.Methodology/Principal FindingsTo obtain more insight in the role of the X∶A ratio in initiation of XCI, we generated triploid mouse ES cells by fusion of haploid round spermatids with diploid female and male ES cells. These fusion experiments resulted in only XXY triploid ES cells. XYY and XXX ES lines were absent, suggesting cell death related either to insufficient X-chromosomal gene dosage (XYY) or to inheritance of an epigenetically modified X chromosome (XXX). Analysis of active (Xa) and inactive (Xi) X chromosomes in the obtained triploid XXY lines indicated that the initiation frequency of XCI is low, resulting in a mixed population of XaXiY and XaXaY cells, in which the XaXiY cells have a small proliferative advantage. This result, and findings on XCI in diploid and tetraploid ES cell lines with different X∶A ratios, provides evidence that the X∶A ratio determines the probability for a given X chromosome to be inactivated. Furthermore, we found that the kinetics of the XCI process can be simulated using a probability for an X chromosome to be inactivated that is proportional to the X∶A ratio. These simulation studies re-emphasize our hypothesis that the probability is a function of the concentration of an X-encoded activator of XCI, and of X chromosome specific allelic properties determining the threshold for this activator.ConclusionsThe present findings reveal that the probability for an X chromosome to be inactivated is proportional to the X∶A ratio. This finding supports the presence of an X-encoded activator of the XCI process.

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.

Xist RNA is confined to the nuclear territory of the silenced X chromosome throughout the cell cycle.

In mammalian female cells, one X chromosome is inactivated to prevent a dose difference in the expression of X-encoded proteins between males and females. Xist RNA, required for X chromosome inactivation, is transcribed from the future inactivated X chromosome (Xi), where it spreads in cis, to initiate silencing. We have analyzed Xist RNA transcription and localization throughout the cell cycle. It was found that Xist transcription is constant and that the mature RNA remains attached to the Xi throughout mitosis. Diploid and tetraploid cell lines with an MS2-tagged Xist gene were used to investigate spreading of Xist. Most XXXX(MS2) tetraploid mouse embryonic stem (ES) cells inactivate the X(MS2) chromosome and one other X chromosome. Analysis of cells with two Xi's indicates that Xist RNA is retained by the Xi of its origin and does not spread in trans. Also, in XX(MS2) diploid mouse ES cells with an autosomal Xist transgene, there is no trans exchange of Xist RNA from the Xi to the autosome. We propose that Xist RNA does not dissociate from the Xi of its origin, which precludes a model of diffusion-mediated trans spreading of Xist RNA.

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

Establishment of tetraploid ES cells. Mouse H-1 (ES) cells were polyploidized by demecolcine and released from the drug. 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. A pluripotent tetraploid cell line (4nH1 cells) was established.

Selective Resistance of Tetraploid Cancer Cells against DNA Damage‐Induced Apoptosis

Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system.

Defining Ploidy-Specific Thresholds in Array Comparative Genomic Hybridization to Improve the Sensitivity of Detection of Single Copy Alterations in Cell Lines

Array comparative genomic hybridization (CGH) is being widely used to screen for recurrent genomic copy number alterations in neoplasms, with imbalances typically detected through the application of gain and loss thresholds. Review of array CGH publications for the year 2005 showed that a wide range of thresholds are used. However, the effect of sample ploidy on the sensitivity of these thresholds for single copy alterations (SCAs) has not been evaluated. Here, we describe a method to evaluate the detection accuracy of thresholds for detecting SCAs in cell line array CGH data. By applying a hidden Markov model-based method, we segmented array CGH data from well-karyotyped cell lines and generated ploidy-specific sensitivity-specificity plots, from which we identified optimum thresholds relevant to sample ploidy. We demonstrate that commonly used nonploidy-specific thresholds are suboptimal in their ability to call SCAs, particularly when applied to hypertriploid or tetraploid cell lines. We conclude that the use of ploidy-specific thresholds improves the sensitivity of thres-hold-based array CGH for detecting SCAs in cell lines. Because polyploidy is a common feature of cancer cells, the application of ploidy-specific thresholds to cell lines (and potentially to clinical samples) may improve the detection sensitivity of SCAs of biological significance.

Cell size as a morphological marker to calculate the mitotic index and ploidy level of citrus callus

We have developed a new method for calculating the mitotic index (MI) and ploidy level of citrus callus. The first step was to symbolize the diameters of new daughter cells and metaphase cells with RD and RM, respectively. The formula RD=0.7937 RM was subsequently established. The validity of this hypothesis was verified by morphological, cytological and fluorescent observations as well as by a protoplast electrofusion test. The MI and the ploidy level of a cell population were then calculated according to this formula. As a final step, tetraploid and hexaploid cell lines were produced from a mixoploid cell population, and somatic embryos were regenerated from these cell lines. These results suggest that cell size can be used as a morphological marker to calculate MI and ploidy level of citrus callus.

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.