Tetraploid State Research Articles

Chromosome Study of Six Populations of Trifolium tumens Stev. ex M. B. in IRAN

Trifolium tumens Stev. ex M. B is a perennial legume with long‐lived and deep rooting from the Vesicaria section in Trifolium genus. It is distributed in Iran. The base chromosome number (x = 8) in the population, confirmed the views of the previous researchers. In this study, the number of chromosomes in the mitotic metaphase stage in the meristematic cells of the root ends were counted in six populations of Trifolium tumens. Also, chloroplast count of stomata guard cells, morphological studies, pollen grain size and flowcytometry were studied in six accessions. Our results showed that this species has ploidy level (2n=16, 2n=32). and the tetraploid state in the species T. tumens is reported. Through flowcytometry, chloroplast count of stomata guard cells and compare the pollen grain, different characters were showed between diploid and tetraploid populations, too. Based on the results, this new cytotype is reported from Iran.

To be or not to be tetraploid-the impact of marker ploidy on genomic prediction and GWAS of potato.

Cultivated potato, Solanum tuberosum L., is considered an autotetraploid with 12 chromosomes with four homologous phases. However, recent evidence found that, due to frequent large phase deletions in the genome, gene ploidy is not constant across the genome. The elite cultivar "Otava" was found to have an average gene copy number of 3.2 across all loci. Breeding programs for elite potato cultivars rely increasingly on genomic prediction tools for selection breeding and elucidation of quantitative trait loci underpinning trait genetic variance. These are typically based on anonymous single nucleotide polymorphism (SNP) markers, which are usually called from, for example, SNP array or sequencing data using a tetraploid model. In this study, we analyzed the impact of using whole genome markers genotyped as either tetraploid or observed allele frequencies from genotype-by-sequencing data on single-trait additive genomic best linear unbiased prediction (GBLUP) genomic prediction (GP) models and single-marker regression genome-wide association studies of potato to evaluate the implications of capturing varying ploidy on the statistical models employed in genomic breeding. A panel of 762 offspring of a diallel cross of 18 parents of elite breeding material was used for modeling. These were genotyped by sequencing and phenotyped for five key performance traits: chipping quality, length/width ratio, senescence, dry matter content, and yield. We also estimated the read coverage required to confidently discriminate between a heterozygous triploid and tetraploid state from simulated data. It was found that using a tetraploid model neither impaired nor improved genomic predictions compared to using the observed allele frequencies that account for true marker ploidy. In genome-wide associations studies (GWAS), very minor variations of both signal amplitude and number of SNPs supporting both minor and major quantitative trait loci (QTLs) were observed between the two data sets. However, all major QTLs were reproducible using both data sets.

Cell shape instability during cytokinesis in tetraploid HCT116 cells

Tetraploidy is a hallmark of broad cancer types, but it remains largely unknown which aspects of cellular processes are influenced by tetraploidization in human cells. Here, we found that tetraploid HCT116 cells manifested severe cell shape instability during cytokinesis, unlike their diploid counterparts. The cell shape instability accompanied the formation of protrusive deformation at the cell poles, indicating ectopic contractile activity of the cell cortex. While cytokinesis regulators such as RhoA and anillin correctly accumulated at the equatorial cortex, myosin II was over-accumulated at the cell poles, specifically in tetraploid cells. Suppression of myosin II activity by Y27632 treatment restored smooth cell shape in tetraploids during cytokinesis, indicating dysregulation of myosin II as a primary cause of the cell shape instability in the tetraploid state. Our results demonstrate a new aspect of the dynamic cellular process profoundly affected by tetraploidization in human cells, which provides a clue to molecular mechanisms of tetraploidy-driven pathogenic processes.

Cultivar-dependent phenotypic and chemotypic responses of drug-type Cannabis sativa L. to polyploidization.

Cannabis sativa L. is a plant with a wide range of potential medicinal applications. In recent years, polyploidy has gained attention as a potential strategy for rapidly improving C. sativa, which, unlike other modern crops, has not yet benefitted from this established biotechnological application. Currently, no reports on high THCA and CBDA drug-type polyploid cultivars have been published. Moreover, it still needs to be clarified if different cultivars react similarly to polyploidization. For these reasons, we set out to evaluate and compare the phenotype and chemotype of three high Δ9-tetrahydrocannabinolic acid (THCA) and one high cannabidiolic acid (CBDA) drug-type cultivars in their diploid, triploid and tetraploid state through agronomic and metabolomic approaches. Our observations on plant morphology revealed a significant increase in plant height and leaf size with increasing ploidy levels in a cultivar-dependent manner. In contrast, cannabinoids were negatively affected by polyploidization, with the concentration of total cannabinoids, THCA, CBDA and cannabigerolic acid (CBGA) decreasing significantly in higher ploidy levels across all four cultivars. Headspace analysis of volatiles revealed that total volatile content decreased in triploids. On the other hand, tetraploids reacted differently depending on the cultivars. Two THCA dominant cultivars showed an increase in concentrations, while in the other two cultivars, concentrations decreased. Additionally, several rare compounds not present in diploids appeared in higher ploidy levels. Moreover, in one high THCA cultivar, a couple of elite tetraploid genotypes for cannabinoid and volatile production were identified, highlighting the role of cultivar and genotypic variability as an important factor in Cannabis sativa L. polyploids. Overall, our observations on plant morphology align with the giga phenotype observed in polyploids of other plant species. The decrease in cannabinoids and volatiles production in triploids have relevant implications regarding their commercial use. On the other hand, this study found that tetraploidization is a suitable approach to improve Cannabis sativa L. medicinal potential, although the response is cultivar and genotype-dependent. This work lays the ground for further improving, evaluating and harnessing Cannabis sativa L. chemical diversity by the breeding, biotechnological and pharmaceutical sectors.

Tetraploidy accelerates adaptation under drug selection in a fungal pathogen.

Baseline ploidy significantly impacts evolutionary trajectories and, specifically, tetraploidy is associated with higher rates of adaptation relative to haploidy and diploidy. While the majority of experimental evolution studies investigating ploidy use the budding yeast Saccharomyces cerivisiae, the fungal pathogen Candida albicans is a powerful system to investigate ploidy dynamics, particularly in the context of acquiring antifungal drug resistance. C. albicans laboratory and clinical strains are predominantly diploid, but have been isolated as haploid and polyploid. Here, we evolved diploid and tetraploid C. albicans for ~60 days in the antifungal drug caspofungin. Tetraploid-evolved lines adapted faster than diploid-evolved lines and reached higher levels of caspofungin resistance. While diploid-evolved lines generally maintained their initial genome size, tetraploid-evolved lines rapidly underwent genome-size reductions and did so prior to caspofungin adaptation. While clinical resistance was largely due to mutations in FKS1, these mutations were caused by substitutions in diploid, and indels in tetraploid isolates. Furthermore, fitness costs in the absence of drug selection were significantly less in tetraploid-evolved lines compared to the diploid-evolved lines. Taken together, this work supports a model of adaptation in which the tetraploid state is transient but its ability to rapidly transition ploidy states improves adaptive outcomes and may drive drug resistance in fungal pathogens.

Whole-genome doubling confers unique genetic vulnerabilities on tumour cells.

SummaryWhole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development1,2. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD– cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD– cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD– cells that comprise human tissue.

Abstract 3591: Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells

Abstract In normal lung tissue the expression levels of 14-3-3 gamma are very low and tightly regulated, however, in cancer it is evident that 14-3-3 gamma's expression patterns become deregulated and significantly elevated. In patients with advanced non-small cell lung cancer (NSCLC), increased expression of this isoform is associated with poorer survival and significantly correlates with lymph node and distant metastasis. These data prompted us to analyze TCGA's NSCLC gene expression dataset, and it is clear that 14-3-3 gamma's expression continues to increase in the progression from early to late stage cancers. Taken together, these data suggest that overexpression of 14-3-3 gamma is correlated with a more aggressive tumor phenotype, an observation also seen with breast and hepatocellular carcinoma. The focus of this study is to elucidate the mechanism(s) causing these more aggressive cancer phenotypes. We have previously shown that overexpressing 14-3-3 gamma in human lung cancer cells harboring very low levels of endogenous 14-3-3 gamma and no wildtype p53 results in a stable subpopulation of cells with polyploid DNA content. Interestingly, approximately 40% of lung adenocarcinomas present with hyper-triploid karyotypes, and even a higher percentage, 40-60%, have inactivation of p53. It is well established that polyploid tumors have the capacity to increase tumorigenicity by allowing resistance to conventional therapies and also permitting elevated tolerance to chromosomal instability (CIN). This leads us to hypothesize that in the absence of p53, NSCLC tumors overexpressing 14-3-3 gamma result in a polyploid population of cells that may be influencing the aggressiveness of the tumor. Further in vitro analysis of these 14-3-3 gamma induced tetraploid cells show that they have a prolonged mitosis with significantly more lagging chromosomes in anaphase than their diploid counterparts, indicating an increase in chromosomal instability (CIN). After isolating isogenic tetraploid clones with or without 14-3-3 gamma expression, the stability of the tetraploid cell state was assessed. Clones not expressing 14-3-3 gamma quickly reverted back to a pseudo-diploid state, whereas overexpression of 14-3-3 gamma significantly prolonged the tolerance of tetraploidy and eventually resulted in an aneuploid cell state. Our data suggests that overexpression of 14-3-3 gamma increases tumorigenicity through the stabilization of a polyploid cell state. Citation Format: Cecil J. Gomes, Michael Harman, Sara Centuori, Charles Wolgemuth, Jesse Martinez. Expression of 14-3-3 gamma stabilizes polyploidization in NSCLC cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3591.

Abstract 2066: Aberrant upregulation of 14-3-3 gamma promotes mononucleated polyploidization in human lung cancers

Abstract Advanced non-small cell lung cancers (NSCLC), frequently display increased expression of 14-3-3 gamma (14-3-3γ); a scaffolding protein shown to have oncogenic capacity. This aberrant increase in expression has also been correlated with poorer survival, indicating that upregulation of 14-3-3γ results in a more aggressive tumor phenotype. Our lab is in pursuit of characterizing the mechanisms driving this more advanced cancer phenotype. One such mechanism currently being investigated is 14-3-3γ's ability to promote polyploidization in lung cancer cells. Polyploidization is a known mechanism observed to increase tumorigenicity, resistance to conventional therapies, and theorized to promote chromosomal instability. Our data shows that overexpression of 14-3-3γ results in a subpopulation of cells that harbor mono-nucleated tetraploid DNA content. This suggests that elevated expression of 14-3-3γ promotes aberrant cell cycle progression, either by mechanisms of an abortive mitosis known as endomitosis, or by means of endoreplication, a re-replication event occurring in the absence of mitosis. Further analyses have confirmed that these cells, in the absence of tumor suppressor p53, remain viable and undergo cellular division; supporting our hypothesis that upregulation of 14-3-3γ is resulting in an unstable, tumorigenic tetraploid cell state. Taken together, our data elucidates one role by which 14-3-3γ may be contributing to a more aggressive tumor phenotype in patients with advanced NSCLC. Citation Format: Cecil J. Gomes, Michael Harman, Jesse Martinez, Sara Centuori. Aberrant upregulation of 14-3-3 gamma promotes mononucleated polyploidization in human lung cancers. [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 2066. doi:10.1158/1538-7445.AM2015-2066

Parasexual Ploidy Reduction Drives Population Heterogeneity Through Random and Transient Aneuploidy in Candida albicans.

The opportunistic pathogen Candida albicans has a large repertoire of mechanisms to generate genetic and phenotypic diversity despite the lack of meiosis in its life cycle. Its parasexual cycle enables shifts in ploidy, which in turn facilitate recombination, aneuploidy, and homozygosis of whole chromosomes to fuel rapid adaptation. Here we show that the tetraploid state potentiates ploidy variation and drives population heterogeneity. In tetraploids, the rate of losing a single heterozygous marker [loss of heterozygosity (LOH)] is elevated ∼30-fold higher than the rate in diploid cells. Furthermore, isolates recovered after selection for LOH of one, two, or three markers were highly aneuploid, with a broad range of karyotypes including strains with a combination of di-, tri-, and tetrasomic chromosomes. We followed the ploidy trajectories for these tetraploid- and aneuploid-derived isolates, using a combination of flow cytometry and double-digestion restriction-site-associated DNA analyzed with next-generation sequencing. Isolates derived from either tetraploid or aneuploid isolates predominately resolved to a stable euploid state. The majority of isolates reduced to the conventional diploid state; however, stable triploid and tetraploid states were observed in ∼30% of the isolates. Notably, aneuploid isolates were more transient than tetraploid isolates, resolving to a euploid state within a few passages. Furthermore, the likelihood that a particular isolate will resolve to the same ploidy state in replicate evolution experiments is only ∼50%, supporting the idea that the chromosome loss process of the parasexual cycle is random and does not follow trajectories involving specific combinations of chromosomes. Together, our results indicate that tetraploid progenitors can produce populations of progeny cells with a high degree of genomic diversity, from altered ploidy to homozygosis, providing an excellent source of genetic variation upon which selection can act.

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.

Emerging trends in polyploidy research

The Key Word ‘Polyploidy’ (i.e., the presence of more than two chromosome sets/more than two complete genomes in a cell) coined by Winkler in 1916 [25] has lived nearly a Century. Genome sizes in plants are remarkably diverse, with a 2350-fold range from the smallest 63 Mb (in two species of carnivorous Genlisea, G. aurea (2n=approximately 52) and G. margaretae (2n=approximately 40)), to the largest 149 000 MB (in Paris japonica, 2n=8x=40), divided into n=2 to n=aprox. 600 chromosomes [1]. The polyploidy phenomenon has intrigued biologists over the years and many attractive hypotheses have been proposed in an attempt to assign functionality to the increased content of a duplicated genome [14, 19–21].With the advent of newer tools of genetic analysis there has been tremendous resurgence of interest in polyploidy research, and several publishers have brought out special issues of the journals / reference books [4, 6, 12, 17, 18, 22, 24]. Ch romosome doub l i n g i s b r ough t abou t by endoreduplication/2n gametes/inter-genomic hybridization— all constraining genetic stability. That polyploidy can be induced with chemicals such as Colchicine, was first demonstrated by OJ Eigsti in 1935 cited in Blakeslee and Avery [2]. Genome duplication impedes sexual reproduction and may impact growth and development. A change to tetraploid state nearly doubles the cell volume and enhances cell surface area by about 1.5 times, often facilitating development of larger organs in plants. But in animals there is a tendency to preserve the same body size by reducing the overall number of cells, thus indicating differential effect to polyploidy change across the taxonomic groups [13].

Recurrent micronucleation through cell cycle progression in the presence of microtubule inhibitors.

Although most cell lines undergo mitotic arrest after prolonged exposure to microtubule inhibitors, some cells subsequently exit this state and become tetraploid. Among these cells, limited numbers of rodent cells are known to undergo multinucleation to generate multiple small independent nuclei, or micronuclei by prolonged colcemid treatment. Micronuclei are thought to be formed when cells shift to a pseudo G1 phase, during which the onset of chromosomal decondensation allows individual chromosomes distributed throughout the cell to serve as sites for the reassembly of nuclear membranes. To better define this process, we used long-term live cell imaging to observe micronucleation induced in mouse A9 cells by treating with the microtubule inhibitor colcemid. Our observations confirm that nuclear envelope formation occurs when mitotic-arrested cells shift to a pseudo G1 phase and adopt a tetraploid state, accompanied by chromosome decondensation. Unexpectedly, only a small number of cells containing large micronuclei were formed. We found that tetraploid micronucleated cells proceeded through an additional cell cycle, shifting to a pseudo G1 phase and forming octoploid micronucleated cells that were smaller and more numerous compared with the tetraploid micronucleated cells. Our data suggest that micronucleation occur when cells shift from mitotic arrest to a pseudo G1 phase, and demonstrate that, rather than being a single event, micronucleation is an inducible recurrent process that leads to the formation of progressively smaller and more numerous micronuclei.

Deacetylation of the mitotic checkpoint protein BubR1 at lysine 250 by SIRT2 and subsequent effects on BubR1 degradation during the prometaphase/anaphase transition

Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays an important role in the efficacy of cancer cell killing by microtubule inhibitors. Prolonged mitotic arrest at the spindle assembly checkpoint (SAC) is a well-known requirement for mitotic catastrophe and, thus, for conferring sensitivity to microtubule inhibitors. We previously reported that downregulation of SIRT2, a member of the sirtuin family of NAD+-dependent deacetylases, confers resistance to microtubule inhibitors by abnormally prolonging mitotic arrest and thus compromising the cell death pathway after mitotic slippage. Thus, turning off SAC activation after a defined period is an additional requirement for efficient post-slippage death. Here, we investigated whether SIRT2 deacetylates BubR1, which is a core component of the SAC; acetylation of BubR1 at lysine 250 (K250) during prometaphase inhibits its APC/C-dependent proteolysis and thus regulates timing in anaphase entry. We showed that SIRT2 deacetylates BubR1 K250 both in vitro and in vivo. We also found that SIRT2 knockdown leads to increased levels of BubR1 acetylation at prometaphase; however, this increase is not substantial to elevate the levels of total BubR1 or delay the transition from prometaphase to anaphase. The present study shows that SIRT2 is a deacetylase for BubR1 K250, although the abnormally prolonged SAC activation observed in SIRT2 knockdown cells is not accompanied by a change in BubR1 levels or by delayed progression from prometaphase to anaphase.

Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of chromosomes may contribute to cancer induction and perhaps be involved in the induction of adverse pregnancy outcomes associated with long-term consumption of disinfected water. Here we present the first observation of the induction of hyperploidy by a class of DBPs.

Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

SIRT2 knockdown increases basal autophagy and prevents postslippage death by abnormally prolonging the mitotic arrest that is induced by microtubule inhibitors

Mitotic catastrophe, a form of cell death that occurs during mitosis and after mitotic slippage to a tetraploid state, plays important roles in the efficacy of cancer cell killing by microtubule inhibitors (MTIs). Prolonged mitotic arrest by the spindle assembly checkpoint is a well-known requirement for mitotic catastrophe, and thus for conferring sensitivity to MTIs. We previously reported that turning off spindle assembly checkpoint activation after a defined period of time is another requirement for efficient postslippage death from a tetraploid state, and we identified SIRT2, a member of the sirtuin protein family, as a regulator of this process. Here, we investigated whether SIRT2 regulates basal autophagy and whether, in that case, autophagy regulation by SIRT2 is required for postslippage death, by analogy with previous insights into SIRT1 functions in autophagy. We show, by combined knockdown of autophagy genes and SIRT2, that SIRT2 serves this function at least partially by suppressing basal autophagy levels. Notably, increased autophagy induced by rapamycin and mild starvation caused mitotic arrest for an abnormally long period of time in the presence of MTIs, and this was followed by delayed postslippage death, which was also observed in cells with SIRT2 knockdown. These results underscore a causal association among increased autophagy levels, mitotic arrest for an abnormally long period of time after exposure to MTIs, and resistance to MTIs. Although autophagy acts as a tumor suppressor mechanism, this study highlights its negative aspects, as increased autophagy may cause mitotic catastrophe malfunction. Thus, SIRT2 offers a novel target for tumor therapy.

Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells

The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed "reprogramming." This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes.

Intratumour Diversity of Chromosome Copy Numbers in Neuroblastoma Mediated by On-Going Chromosome Loss from a Polyploid State

Neuroblastomas (NBs) are tumours of the sympathetic nervous system accounting for 8–10% of paediatric cancers. NBs exhibit extensive intertumour genetic heterogeneity, but their extent of intratumour genetic diversity has remained unexplored. We aimed to assess intratumour genetic variation in NBs with a focus on whole chromosome changes and their underlying mechanism. Allelic ratios obtained by SNP-array data from 30 aneuploid primary NBs and NB cell lines were used to quantify the size of clones harbouring specific genomic imbalances. In 13 cases, this was supplemented by fluorescence in situ hybridisation to assess copy number diversity in detail. Computer simulations of different mitotic segregation errors, single cell cloning, analysis of mitotic figures, and time lapse imaging of dividing NB cells were used to infer the most likely mechanism behind intratumour variation in chromosome number. Combined SNP array and FISH analyses showed that all cases exhibited higher inter-cellular copy number variation than non-neoplastic control tissue, with up to 75% of tumour cells showing non-modal chromosome copy numbers. Comparisons of copy number profiles, resulting from simulations of different segregation errors to genomic profiles of 120 NBs indicated that loss of chromosomes from a tetraploid state was more likely than other mechanisms to explain numerical aberrations in NB. This was supported by a high frequency of lagging chromosomes at anaphase and polyploidisation events in growing NB cells. The dynamic nature of numerical aberrations was corroborated further by detecting substantial copy number diversity in cell populations grown from single NB cells. We conclude that aneuploid NBs typically show extensive intratumour chromosome copy number diversity, and that this phenomenon is most likely explained by continuous loss of chromosomes from a polyploid state.

The effect of tetraploidization of wild Arachis on leaf morphology and other drought-related traits

Cultivated peanut is an allotetraploid (genome type AABB) with a very narrow genetic base, therefore wild species are an attractive source of new variability and traits. Because most wild species are diploid, the first step of introgression usually involves hybridization of wild species and polyploidization to produce a synthetic allotetraploid (AABB) that is sexually compatible with peanut. This study investigates drought-related traits such as leaf morphology, transpiration profile, chlorophyll meter readings (SCMR), specific leaf area (SLA) and transpiration rate per leaf area for two wild diploids (Arachis duranensis and Arachis ipaënsis) that could be of interest for improvement of the peanut crop. Furthermore, the inheritance of the traits from the diploid to the tetraploid state was investigated. Results showed that whilst some diploid traits such as SCMR, are maintained through hybridization and polyploidization, most characters, such as the leaf area, stomata size, trichome density and transpiration profile, are substantially modified. The study concludes that direct evaluations of drought-related traits in wild diploids may be useful for evaluation of wild species to be used in introgression. However, evaluations on wild-derived synthetic tetraploids are likely to be more informative.

Study of Some Characteristics Related to the Sexual Reproduction in Autotetraploid Vicia narbonensis

Some parameters such as frequency of pod formation, average number of seeds per pod and percentage of emptypods were used to evaluate the variation of fertility in autotetraploid Vicia narbonensis. The results showed thatthe autotetraploid and diploid parents had the same number of ova per gynaecium. However, the tetraploidseedling produced fewer seeds per pod and per seedling than their corresponding diploids; the poor fertilizationof many ovum was probably behind the decline of fertility in the autotetraploids. Several factors may beinvolved: the low efficiency of diploid pollen (related to a relatively slow growth of the pollen tubes), anunbalanced meiosis for a great number of mother cells of pollen grains and a significant morphologicalmodification of the reproductive system due to the passage from the diploid to the tetraploid state.