Increase In Ploidy Research Articles

Novel antagonistic interactions associated with plant polyploidization influence trait selection and habitat preference

Polyploidization is an important mechanism for sympatric speciation in plants. Still, we know little about whether plant polyploidization leads to insect host shifts, and if novel interactions influence habitat and trait selection in plants. We investigated herbivory by the flower bud gall-forming midge Dasineura cardaminis on tetraploids and octoploids of the herb Cardamine pratensis. Gall midges attacked only octoploid plant populations, and a transplantation experiment confirmed this preference. Attack rates were higher in populations that were shaded, highly connected or occurred along stream margins. Within populations, late-flowering individuals with many flowers were most attacked. Galling reduced seed production and significantly influenced phenotypic selection on flower number. Our results suggest that an increase in ploidy may lead to insect host shifts and that plant ploidy explains insect host use. In newly formed plant polyploids, novel interactions may alter habitat preferences and trait selection, and influence the further evolution of cytotypes.

Origin of the complex karyotype of the polyploid parthenogenetic grasshopper Saga pedo (Orthoptera: Tettigoniidae)

Karyotypes of the polyploid parthenogenetic species Saga pedo from four localities in France and the Republic of Mace- donia were constructed and compared. All these karyotypes consist of 70 chromosomes, which is more than twice that in other spe- cies of the genus. The chromosomes differ from each other, making the matching of homologues difficult. Karyotypes of French specimens are similar, except for differences in the heterochromatin. Compared to that of the Macedonian specimens those from French specimens differ by the shortening of a single chromosome. The difficulty experienced in identifying tetrads and even pairs of chromosomes indicates that either many chromosome rearrangements have occurred since the polyploidisation event(s) or that the addition of quite different genomes is the cause. On the other hand, that the karyotypes are similar indicates a common origin of both the Macedonian and French populations. Thus, most chromosome changes preceded the separation from their common ancestor. Both the DNA content and chromosome analyses suggest that the S. pedo karyotype is pentaploid and not tetraploid as previously proposed. This odd ploidy number rules out the hypothesis that it could only have originated by endoreduplication. It is more likely that it originated by the association of five copies of the 14,X haploid karyotype, which exists in the gametes of the closely related species, S. campbelli and S. rammei (male / female 2n = 27, X / 28, XX). Fertilization of a parthenogenetic 56, XXXX female by a 14, X spermatozoa could have resulted in the last increase in ploidy.

Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD + levels and SIRT inhibition

Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood CD34(+) cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy. We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mobilized peripheral blood CD34(+) cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD(+) precursors. We tested additional inhibitors of the sirtuin (or SIRT) 1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets. NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure; 6.25 mM NIC increased NAD(+) levels fivefold. Quinolinic acid increased NAD(+) similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. NIC greatly increases Mk ploidy under a wide range of conditions without altering Mk morphology. Inhibition of SIRT1 and/or SIRT2 is primarily responsible for NIC effects on Mk maturation.

The frequency of polyploid speciation in vascular plants.

Since its discovery in 1907, polyploidy has been recognized as an important phenomenon in vascular plants, and several lines of evidence indicate that most, if not all, plant species ultimately have a polyploid ancestry. However, previous estimates of the frequency of polyploid speciation suggest that the formation and establishment of neopolyploid species is rare. By combining information from the botanical community's vast cytogenetic and phylogenetic databases, we establish that 15% of angiosperm and 31% of fern speciation events are accompanied by ploidy increase. These frequency estimates are higher by a factor of four than earlier estimates and lead to a standing incidence of polyploid species within genera of 35% (n = 1,506). Despite this high incidence, we find no direct evidence that polyploid lines, once established, enjoy greater net species diversification. Thus, the widespread occurrence of polyploid taxa appears to result from the substantial contribution of polyploidy to cladogenesis, but not from subsequent increases in diversification rates of polyploid lines.

Role for MKL1 in megakaryocytic maturation

AbstractMegakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation. When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34+ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

Body shape evolution among ploidy levels of the Squalius alburnoides hybrid complex (Teleostei, Cyprinidae)

Hybridization, ploidy level and genomic constitution may be important to respond to different environments, by producing different phenotypes and thus reducing competitive interaction. Through geometric morphometrics, we examined variation in body size and shape among biotypes of the Squalius alburnoides hybrid complex and their sperm donor (Squalius carolitertii). Results showed that S. carolitertii is significantly larger in size than the biotypes of the complex. No significant relationship was observed between ploidy and body size among S. alburnoides biotypes. Significant variation in body shape was found between S. carolitertii and S. alburnoides, and between tetraploids and the other biotypes. These differences in biotypes may reduce resource competition, highlighting the potential importance of resource availability favouring one biotype over another. In S. alburnoides, the adaptation to different trophic niches through modification of trophic morphology, body shapes, and feeding behaviour, may result from an increase in ploidy and genomic constitution. This adaptation may account also for the formation and maintenance of this nonsexual complex.

The Chemotherapeutic Agents XK469 (2-{4-[(7-Chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-Bromo-2-quinolinyl)oxy]phenoxy}propionic acid) Inhibit Cytokinesis and Promote Polyploidy and Induce Senescence

The therapeutic usefulness of the quinoxaline derivatives XK469 (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid) and SH80 (2-{4-[(7-bromo-2-quinolinyl)oxy]phenoxy}propionic acid) has been attributed to their abilities to induce G(2)/M arrest and apoptotic or autophagic cell death. Concentrations of XK469 or SH80 > or = 5 microM were cytostatic to cultures of the normal murine melanocyte cell line Melan-a. Higher concentrations caused dose-dependent cytotoxicity. Concentrations > or =10 microM provoked dramatic morphological changes typified by marked increases in cell size and granularity. XK469/SH80-treated cultures accumulated tetraploid (4N) DNA-containing cells within 24 h of treatment, an 8N population within 3 days, and a 16N population within 5 days. Increases in ploidy correlated with the appearance of multinucleated cells. Under no circumstances did cells exhibit evidence of furrow formation. Both drugs suppressed cytokinesis in additional mammalian cell lines. Cytotoxic concentrations of XK469 elevated DEVDase activities (a measure of procaspase-3/7 activation) and enhanced cellular staining by a fluorescent analog of the pan caspase inhibitor valine-alanine-aspartic acid-fluoromethyl ketone within 48 to 96 h of treatment. Within 48 h of treatment, cytostatic and cytotoxic concentrations of XK469 elevated p21 contents, reduced Bcl-2 and Bcl-XL contents, and induced autophagy, as monitored by the accumulation of phosphatidylethanolamine-modified cleavage product of microtubule-associated protein light chain 3 (LC3-II). Cultures treated with > or =10 microM XK469 or SH80 for 5 days could not be induced to divide upon removal of drugs. Such cultures maintained high LC3-II contents, exhibited reduced cyclin E and D1 contents, and extensively expressed senescence-associated beta-galactosidase within 14 to 17 days of cessation of drug treatment. Hence, XK469 and SH80 inhibit cytokinesis, promote polyploidy, and induce senescence in Melan-a cells.

Nicotinamide Increases the Megakaryocytic Maturation of Human Hematopoietic Stem Cells Primarily Due to SIRT Inhibition

Introduction: Megakaryocytic cells (Mks), the precursors to platelets, are among the least understood blood cell types. A primary aspect of Mk differentiation is endomitosis, whereby Mks duplicate their DNA content without undergoing cytokinesis and form cells with 4N, 8N, 16N, etc. Mk ploidy strongly correlates with platelet production. Thrombocytopenia accompanies several hematologic malignancies including myelodysplastic syndromes and is often associated with low in vivo Mk ploidy. Elucidation of the factors that regulate Mk endomitosis will aid in developing treatments for Mk-related disorders. We have previously shown that the B3 vitamin nicotinamide (NIC) causes a dose-dependent increase in Mk size and the fraction of high-ploidy (≥ 8N) Mks and leads to more complex proplatelet formation without affecting Mk commitment, ultrastructure, apoptosis, or viability in cultures of CD34+ cells (Giammona LM, et al. Br J Haem 135 (2006): 554). We examined whether NIC's roles as an inhibitor of the sirtuin family of histone/protein deacetylases (SIRTs) and as a precursor for NAD+ were responsible for its effects on Mk ploidy.Methods: CD34+ cells, isolated from healthy G-CSF-mobilized peripheral blood donors, were maintained in serum-free X-VIVO 20 media supplemented with 100 ng/mL thrombopoietin (Tpo). On day 5, cells were treated with 6.25 mM NIC, 10 μM cambinol (SIRT1/2 inhibitor), or 10 μM AGK2 (SIRT2 inhibitor) or maintained with Tpo alone. Flow cytometry was used to determine Mk commitment (CD41+), viability, apoptosis, ploidy, and intracellular levels of total and acetylated p53. The intracellular concentration of NAD(H) (NAD+ plus NADH) was determined using an enzymatic assay. Immunoblots were used to detect acetylated and total nucleosomes, as well as the NAD processing enzyme Nmnat1. p53 DNA-binding activity was determined using EMSA analysis.Results: Adding NIC to CD34+ cell cultures increased the percentage of high-ploidy Mks by 3-fold. The SIRT1/2 inhibitor cambinol increased Mk ploidy to a similar extent as NIC, while the SIRT2 inhibitor AGK2 was only 30% as effective. NIC and cambinol more than tripled the fractions of 16N and 32N Mks (Figure). None of the additives affected Mk commitment, viability, or apoptosis. Functional inhibition of SIRT1/2 by NIC was confirmed by increased acetylation of several SIRT1/2 target proteins. Both SIRTs deacetylate histones and we observed up to 3-fold greater nucleosome acetylation in cells treated with NIC. Flow cytometry showed that the ratio of AcK382p53 to total p53 was 3-fold higher in cells treated with NIC as compared to Tpo alone. Consistent with reports that acetylation increases p53 DNA-binding activity, EMSA analysis showed that p53 binding to the p53 consensus sequence was 50% greater in NIC-treated Mks. We have previously shown that p53 knockdown increases Mk ploidy in culture (Fuhrken PG, et al. J Biol Chem 283 (2008): 15589). These results suggest that increased p53 acetylation differentially affects different p53 target genes. NIC increased intracellular levels of NAD(H) by 5-fold. In contrast, an NAD+de novo pathway precursor had minimal impact on ploidy. NIC is incorporated into NAD+ via the salvage pathway, which is localized to the nucleus in yeast, whereas the de novo pathway is distributed throughout the cell. This suggests that NAD+ production in the nucleus may also play a role in NIC-mediated increases in Mk ploidy, and is consistent with higher nuclear levels of the NAD+ salvage pathway enzyme Nmnat1 detected in cells treated with NIC.Conclusions: Inhibition of SIRT1 and SIRT2 appears to be the primary mechanism for NIC-mediated increases in Mk ploidy, and increased p53 acetylation is likely to play an important role in this process. Further study of SIRT targets associated with DNA repair, apoptosis, and cell cycle regulation may provide additional insight into Mk polyploidization. [Display omitted]

The CXCR4 Antagonist AMD3100 Impairs Survival of AML Cells and Induces Their Differentiation.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, participate in the retention of AML cells within the bone marrow microenvironment and their release into the circulation. We previously showed that the expression of cell surface elastase in AML cells is dependent on the SDF-1/CXCR4 axis, giving a first evidence for the association between the SDF-1 and elastase pathways. In the present study, we hypothesized that inhibition of the SDF-1/CXCR4 axis or elastase may regulate similar pathways and genes in AML cells. In order to test our hypothesis, we compared gene expression profiles of U937 AML cells that express high level of membrane CXCR4, treated with neutralizing CXCR4 mAb or elastase inhibitor (EI), with non-treated cells, by employing gene-array technology. Unsupervised hierarchical clustering analysis demonstrated similar gene expression profiles of anti-CXCR4 antibody or EI treated cells, as compared to control. Analysis of the 364 genes that were differentially expressed in both anti CXCR4 mAb or EI treated cells under both treatment agents identified genes that correspond to the transcriptional profiles of differentiated myeloid cells in a significantly high prevalence. Quantitative Real-time RT-PCR for 4 selected genes C/EBPe, FLT3, HOXA9 and G-CSFR, demonstrated gene expression profiles identical to the microarray expression profiles. Given thus, we further analyzed the effect of CXCR4 inhibition on AML cell growth and differentiation using the antagonist AMD3100. AMD3100 arrested proliferation in five AML cell lines as was suggested by proliferation assay, cell cycle and caspase 3 activity analyses. Moreover, cell differentiation was demonstrated by:Modifications in cell morphology,Increased expression of myeloid differentiation antigensAcquisition of the ability to produce oxidative burstsAn increase in DNA ploidy in megakaryoblasts. Our study defines a new role for the SDF-1/CXCR4 axis in the regulation of leukemic cell survival and differentiation.

Dosage Compensation by Gene-Copy Silencing in a Triploid Hybrid Fish

In mammals, the increase in gene dosage, in the form of polyploidy or involving chromosomal fragments, has deleterious effects [1]. Regulation of appropriate gene product amounts has to be warranted by complex dosage-compensation mechanisms. Lower vertebrates, on the other hand, cope very well with ploidy increase [2-4], implying either effective compensation or a lack of necessity for such mechanisms. Unfortunately, nothing is known about the genetic and molecular mechanisms underlying this phenomenon. For an experimental approach, we have studied gene expression in the allotriploid form of Squalius alburnoides. In these organisms, different genomes are joined through hybridization; thus, sequence differences can be used to follow expression of different alleles [5, 6]. We found that a compensation mechanism exists, reducing transcript levels to the diploid state. Our data suggest a silencing of one of the three alleles. Unexpectedly, it is not a whole haplome that is inactivated. The allelic expression patterns differ between genes and between different tissues for one and the same gene. Our data provide the first evidence of a regulation mechanism involving gene-copy silencing in a triploid vertebrate.

Transgene-Induced Gene Silencing Is Not Affected by a Change in Ploidy Level

BackgroundWhole genome duplication, which results in polyploidy, is a common feature of plant populations and a recurring event in the evolution of flowering plants. Polyploidy can result in changes to gene expression and epigenetic instability. Several epigenetic phenomena, occurring at the transcriptional or post-transcriptional level, have been documented in allopolyploids (polyploids derived from species hybrids) of Arabidopsis thaliana, yet findings in autopolyploids (polyploids derived from the duplication of the genome of a single species) are limited. Here, we tested the hypothesis that an increase in ploidy enhances transgene-induced post-transcriptional gene silencing using autopolyploids of A. thaliana.Methodology/Principal FindingsDiploid and tetraploid individuals of four independent homozygous transgenic lines of A. thaliana transformed with chalcone synthase (CHS) inverted repeat (hairpin) constructs were generated. For each line diploids and tetraploids were compared for efficiency in post-transcriptional silencing of the endogenous CHS gene. The four lines differed substantially in their silencing efficiency. Yet, diploid and tetraploid plants derived from these plants and containing therefore identical transgene insertions showed no difference in the efficiency silencing CHS as assayed by visual scoring, anthocyanin assays and quantification of CHS mRNA.Conclusions/SignificanceOur results in A. thaliana indicated that there is no effect of ploidy level on transgene-induced post-transcriptional gene silencing. Our findings that post-transcriptional mechanisms were equally effective in diploids and tetraploids supports the use of transgene-driven post-transcriptional gene silencing as a useful mechanism to modify gene expression in polyploid species.

Controlling Size in Multicellular Organs: Focus on the Leaf

In leaves, cells get larger as cell division decreases or the ploidy increases. This might seem a logical response, but the controls are more complicated.

Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening

Tomato fruit cells are characterized by a strong increase in nuclear ploidy during fruit development. Average ploidy levels increased to similar levels (above 50C) in two distinct fruit tissues, pericarp and locular tissue. However, ploidy profiles differed significantly between these two tissues suggesting a tissue-specific control of endoreduplication in tomato fruit. To determine possible relationships between endoreduplication and epigenetic mechanisms, the methylation status of genomic DNA from pericarp and locular tissue of tomato fruit was analysed. Pericarp genomic DNA was characterized by an increase of CG and/or CNG methylation at the 5S and 18S rDNA loci and at gyspsy-like retrotransposon sequences during fruit growth. A sharp decrease of the global DNA methylation level together with a reduction of methylation at the rDNA loci was also observed in pericarp during fruit ripening. Inversely, no major variation of DNA methylation either global or locus-specific, was observed in locular tissue. Thus, tissue-specific variations of DNA methylation are unlikely to be triggered by the induction of endoreduplication in fruit tissues, but may reflect tissue-specific ploidy profiles. Expression analysis of eight putative tomato DNA methyltransferases encoding genes showed that one chromomethylase (CMT) and two rearranged methyltransferases (DRMs) are preferentially expressed in the pericarp during fruit growth and could be involved in the locus-specific increase of methylation observed at this developmental phase in the pericarp.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1

AbstractThe molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34+ cells, we observed that p19INK4D expression was increased both at the mRNA and protein levels during ploidization. p19INK4D knockdown led to a moderate increase (31.7% ± 5%) in the mean ploidy of MKs suggesting a role of p19INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41highCD42high) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19INK4D overexpression in CD34+ cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19INK4D KO mice exhibited an increase in mean ploidy level from 18.7N (± 0.58N) to 52.7N (± 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19INK4D promoter. Moreover, AML-1 inhibition led to the p19INK4D down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.

Aurora kinases

Aurora kinases

Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage-specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK-/- megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)-mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

FLT3 Mediated MAPK Activation Participates in the Control of Megakaryopoiesis in Primary Myelofibrosis

Myeloproliferation, myelofibrosis, osteosclerosis and neo-angiogenesis are the major intrinsic pathophysiological features of Primary Myelofibrosis (PMF). The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of “dystrophic” megakaryocytes (MK) through to be responsible for myelofibrosis thought fibrogenic factor release. Comparison of CD34+ and MK cell gene expression profiling between PMF patients and healthy donors revealed a global deregulation of the MAPK pathway genes. This alteration is associated with a modulation of the FLT3 tyrosine kinase gene expression in CD34+ and MK cells from patients, independently of the JAK2V617F mutation presence. Quantification of the FLT3 transcript in mononuclear cells from patients with Polycythemia Vera and Essential Thrombocythemia showed that this over expression is mainly observed in JAK2WT PMF patients. This is associated with a higher proportion of FLT3+CD34+CD41+ cells in the blood of patients. Analysis of FLT3 membrane expression in MK-derived CD34+ cultures revealed that its expression was maintained all along MK differentiation in patients in contrast to healthy donors. Such a higher expression of FLT3 is associated with an increased concentration of its ligand in the platelet rich plasma from patients, independently of their JAK2 mutational status. The role of FLT3 in the regulation of hematopoiesis incited us to analyse whether its alteration could take part in the myeloproliferation and dysmegakaryopoiesis that characterizes PMF. A flow cytometry analysis of FLT3-downstream MAPK activation in PMF CD34+ cells showed a hyperphosphorylation of p38 and JNK as compared to CD34+ cells from normal blood. This phosphorylation was maintained in PMF MK-derived CD34+ cells at day 10. Addition of PD98059, a MAPK inhibitor, induced a dose dependent restoration of the in vitro megakaryopoiesis in PMF as shown by an increase in MK ploidy with apparition of 32N cells associated with a mature cytological aspect and an increase in CD41, CD42a and CD9 MK differentiation marker expression. PD98059 also increased the MK clonogenicity of CD34+ cells from all patients tested (5/5) as compared to healthy donors. Preliminary results using a specific chemical inhibitor of FLT3 in MK-derived CD34+ cell cultures reinforced the involvement of FLT3 in PMF MK differentiation. In presence of FLT3 ligand, the FLT3 mediated MAPK hyperphosphorylation in PMF MK cultures (D6) is reversed by either PD98059 or UO126, another ERK inhibitor and is accompanied by a slight increase in proliferative MK. This effect is not observed in MK cultures from normal CD34+ cells. Surprisingly, ligation of FLT3 by a monoclonal anti-FLT3 antibody in CD34+ cell cultures resulted in an increase MK proliferation. In conclusion, this work shows a deregulation of FLT3 and MAPK pathway in the PMF CD34+ cells and suggests that the persistence of the FLT3 mediated MAPK activation participates in the dysmegakaryopoiesis of PMF patients.

RB Loss Promotes Aberrant Ploidy by Deregulating Levels and Activity of DNA Replication Factors

The retinoblastoma tumor suppressor (RB) is functionally inactivated in many human cancers. Classically, RB functions to repress E2F-mediated transcription and inhibit cell cycle progression. Consequently, RB ablation leads to loss of cell cycle control and aberrant expression of E2F target genes. Emerging evidence indicates a role for RB in maintenance of genomic stability. Here, mouse adult fibroblasts were utilized to demonstrate that aberrant DNA content in RB-deficient cells occurs concomitantly with an increase in levels and chromatin association of DNA replication factors. Furthermore, following exposure to nocodazole, RB-proficient cells arrest with 4 n DNA content, whereas RB-deficient cells bypass the mitotic block, continue DNA synthesis, and accumulate cells with higher ploidy and micronuclei. Under this condition, RB-deficient cells also retain high levels of tethered replication factors, MCM7 and PCNA, indicating that DNA replication occurs in these cells under nonpermissive conditions. Exogenous expression of replication factors Cdc6 or Cdt1 in RB-proficient cells does not recapitulate the RB-deficient cell phenotype. However, ectopic E2F expression in RB-proficient cells elevated ploidy and bypassed the response to nocodazole-induced cessation of DNA replication in a manner analogous to RB loss. Collectively, these results demonstrate that deregulated S phase control is a key mechanism by which RB-deficient cells acquire elevated ploidy.

The cell cycle‐associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.

Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence

Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.