Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.     

Vascular smooth muscle cell polyploidy: An adaptive or maladaptive response?

Polyploidy is a state in which a cell contains multiple copies of its entire genome, while a normal diploid cell contains only two sets of homologous chromosomes. Although widely studied and pervasive in nature, the signals and mechanisms of polyploidization and its accompanying operational consequences are still unclear. This review focuses on relevant questions in deciphering the regulation of polyploidization of vascular smooth muscle cells (VSMC) in mammals and the role of polyploidy in various vascular pathologies, such as hypertension and aging. Additionally, we will explore new investigations in polyploidization of VSMCs involving the rapidly expanding fields of oxidative stress and senescence. J. Cell. Physiol. 215: 588-592, 2008. (c) 2008 Wiley-Liss, Inc.     

A decrease in cyclin B1 levels leads to polyploidization in DNA damage‐induced senescence

Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration‐dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin‐induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated β‐galactosidase activity. In DNA damage‐induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage‐induced senescence.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Vascular smooth muscle polyploidization--from mitotic checkpoints to hypertension

Aging and hypertension are accompanied by an increase in mass and rigidity of arterial walls. At capacitance arteries, the enlargement and stiffness of the medial smooth muscle layer promote systolic hypertension and contribute to left ventricular hypertrophy and cardiovascular morbidity. Morphological studies have demonstrated that vascular smooth muscle cell (VSMC) hypertrophy, with minimal hyperplasia, causes the enlargement of vascular smooth muscle at capacitance arteries, and that VSMC hypertrophy is strongly associated with VSMC polyploidization. Recent studies demonstrate that hypertrophic signals, such as those elicited by Angiotensin II, abrogate the mechanisms of control of M phase in VSMC and induce cell cycle re-entry and polyploidization. These polyploid VSMC have a lower replicative rate, but a higher mass, protein content and matrix production than their diploid counterparts. Both, the protein kinase Aktl and the cyclin kinase-associated protein CKsl, have been implicated in the mechanism of VSMC polyploidization during hypertension. Here, we review the function of these proteins at the mitotic spindle cell cycle checkpoint and their role in the process of VSMC polyploidization.     

多倍体植物混合倍性种群的建立机制研究进展

基因组多倍化是物种形成和进化的重要驱动力, 几乎所有植物都经历过至少一次基因组加倍。然而, 由于多倍体植株比二倍体表现出更高的死亡率, 多倍化机制被认为是植物进化的“死胡同”。一些植物物种具有自然混合倍性种群, 即同一物种具有不同倍性, 这为揭示多倍体的进化机制提供了最佳途径。本文从基因组加倍形成多倍体植物开始, 综述了混合倍性种群的形成、建立与维持的研究进展, 探讨了多倍体适应自然环境的种群分化而形成多倍体物种的机制。研究自然混合倍性种群的倍性组成、重复基因的功能分化以及多倍体的生态位分化, 有利于明确混合倍性自然种群的生态适应与维持机理, 以及多倍体植物的进化机制。     

Association of ploidy and sexual system in Lycium californicum (Solanaceae)

In North American Lycium (Solanaceae), the evolution of gender dimorphism has been proposed as a means of restoring outcrossing after polyploidization causes the loss of self-incompatibility. Previous studies of this process in Lycium focused on comparisons between species that differ in ploidy. We examined intraspecific variation in floral morphology and DNA content in populations of L. californicum to determine correlations between sexual system and cytotype. We also used nuclear ITS and GBSSI sequence data to determine whether diploid and polyploid forms represent the same phylogenetic species, and the phylogeographic relationships among populations and ploidy levels. Within populations, no variation in ploidy was found, although among populations there was a perfect correspondence between sexual system and cytotype. Diploid populations were all hermaphroditic, whereas tetraploid populations were all gender dimorphic. There was no clear geographic pattern to the occurrence of diploid and tetraploid forms. Phylogenetic analysis confirms that L. californicum, regardless of ploidy, forms a monophyletic group within the genus Lycium. Sequences from diploid and polyploid individuals did not form reciprocally monophyletic clades, indicating either multiple gains of polyploidy, ongoing gene flow between cytotypes, or lack of lineage sorting since the evolution of polyploidy. The correspondence between ploidy and sex expression is consistent with the hypothesis that polyploidization triggers the evolution of gender dimorphism in this and other Lycium species.     

Polyploidizing mitoses and the biological meaning of polyploidy in liver cells]

The ontogenetic polyploidization of hepatocytes is regarded, within which normal mitoses are changed to polyploidizing mitoses, and diploid hepatocytes transform into polyploid mono- and binuclear cells. A new hypothesis is put forward of the biological significance of the liver cell polyploidy. The hypothesis takes into account a high level of spontaneous chromosomal aberrations in mitotic hepatocytes. The chromosome structural changes interfere with mitosis resulting in the chromosomal imbalance. Polyploidy bestows for hepatocytes a tolerance towards a chromosomal imbalance. Some implications of the hypothesis are discussed: unbalanced genome of hepatocytes after the treatment with mutagens and mitotic stimulators; the reasons of liver cell polyploidy differences in mammalian species; mechanisms of radioresistance of hepatocytes. Chromosomal imbalance of polyploid hepatocytes is assumed to be the basis for wome chronic liver diseases in man.     

广藿香毛状根多倍体诱导及其植株再生

为了提高药用植物广藿香的次生物质广藿香醇含量,采用秋水仙素人工诱导染色体加倍技术,进行了广藿香毛状根多倍体诱导及其植株再生、倍性鉴定和挥发油组分广藿香醇含量的测定。结果表明,广藿香毛状根多倍体诱导的最佳条件为0.05%秋水仙素处理36 h,其多倍体诱导率可达40%以上;经秋水仙素加倍的广藿香毛状根在MS+6-BA 0.2 mg/L+NAA 0.1 mg/L培养基中培养60 d后可获得毛状根多倍体再生植株。与对照(二倍体植株)相比,广藿香毛状根多倍体再生植株根系更发达、茎更粗、节间变短、叶片的长度、宽度和厚度均较二倍体明显增大。根尖细胞染色体压片观察证实,所获得的广藿香毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为128;同时,其叶片的气孔保卫细胞体积及其叶绿体数目均约为对照的两倍;但其气孔密度则随着倍性增加而下降,二倍体植株叶片的气孔密度约为四倍体植株叶片的1.67倍。GC-MS测定结果表明,广藿香毛状根多倍体再生植株的广藿香挥发油组分广藿香醇的含量为4.25 mg/g干重,约为二倍体植株的2.30倍。该结果证实毛状根多倍体化可提高药用植物广藿香的广藿香醇含量。     

mTOR／4E—BP1参与诺考达唑诱导的Dami细胞多倍体化调控

目的：多倍性是物种形成的重要机制,决定一些重要器官细胞产生的数量和功能,而且与某些病理过程（如恶性肿瘤）的发生有密切关系。我们通过建立相对同步化的多倍体细胞模型,已经证实mTOR／S6K1参与多倍体细胞周期的调控。本课题主要研究roTOR下游的另一个重要信号分子4E-BP1是否也参与细胞的倍体化调控。方法：诺考达唑诱导Dami细胞建立相对同步化的多倍体细胞模型,Western-blot分析多倍体细胞模型中mTOR／4E—BP1通路信号分子表达和磷酸化修饰位点的变化,流式细胞仪双荧光分析4E—BP1不同结构域磷酸化位点修饰与细胞周期各时相的关系。结果：诺考达唑诱导的Dami细胞可作为相对同步化的多倍体细胞周期模型,在二倍体和多倍体细胞周期中,mTOR表达增加及第2448位丝氨酸位点磷酸化发生在G1期进入S期,4E—BP1的第37,46位苏氨酸和第65位丝氨酸位点磷酸化发生在G2／M期。结论：mTOR／4E-BP1通路参与多倍体细胞周期的调控。     

Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo.

In different cardiovascular disease states, oxidative stress decreases the bioavailability of endothelial NO, resulting in endothelial dysfunction. An important molecular source of reactive oxygen species is the enzyme family of NAD(P)H oxidases (Nox). Here we provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) line A7r5 and aortas and kidneys of rats. Upon exposure of A7r5 cells to Ang II (1 microM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR(mRen2)27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.     

Stathmin Prevents the Transition from a Normal to an Endomitotic Cell Cycle during Megakaryocytic Differentiation

Physiological polyploidy is a characteristic of several cell types including themegakaryocytes (MK) that give rise to circulating blood platelets. MK achieve polyploidy byswitching from a normal to an endomitotic cell cycle characterized by the absence of late mitoticstages. During an endomitotic cycle, the cells enter into mitosis and proceed normally throughmetaphase and early anaphase. However, late anaphase, telophase and cytokinesis are aborted. Thisabortive mitosis is associated with atypical multipolar mitotic spindles and limited chromosomesegregation. Stathmin is a microtubule-depolymerizing protein that is important for the regulation ofthe mitotic spindle and interfering with its expression disrupts the normal mitotic spindle and leadsto aberrant mitotic exit. As cells enter mitosis, the microtubule depolymerizing-activity of stathminis switched-off, allowing microtubules to polymerize and assemble into a mitotic spindle.Reactivation of stathmin in the later stages of mitosis is necessary for the disassembly of the mitoticspindle and the exit from mitosis. Previous studies had shown that stathmin expression isdownregulated as MK become polyploid and inhibition of its expression in K562 cells increasestheir propensity to become polyploid. In this report, we describe our studies of the mechanism bywhich stathmin plays its role in MK polyploidization. We show that stathmin overexpressionprevents the transition from a mitotic cycle to an endomitotic cycle as determined by a decrease inthe number of multipolar mitotic spindles. These observations support a model in whichdownregulation of stathmin expression in megakaryocytes and other polyploid cells may be acritically important factor in endomitosis and polyploidy.     

Identification of Regulators of Polyploidization Presents Therapeutic Targets for Treatment of AMKL

The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our?study implicates five networks of kinases that?regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in?vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.     

Polyploidization of the BTC-32 cell line from Triatoma infestans (Hemiptera,Reduviidae)

The cytophotometric evaluation of the Feulgen-DNA content of the BTC-32 cells at passage 160 after 6 days of growth demonstrated that polyploidy is relatively frequent in this cell line. 4C values were assumed to pertain to diploid nuclei at the S or G2 phases but also to polyploid nuclei at the G1 phase. Polyploidy in 4C nuclei is assumed to be attained by endomitosis. However, there are morphological indications that polyploidization in cells with an 8C–128C Feulgen-DNA content could result from cellular and nuclear fusions, possibly mediated by viral particles present in the cell culture. Micronucleation was also frequent; it was assumed to be promoted by viral action or deficiency in some culture medium nutrient. These nuclear characteristics should be considered when using the BTC-32 cell line for monitoring the action of infective agents or their products.     

Biological significance of liver cell polyploidy: an hypothesis

The liver cell polyploidy phenomenon, a characteristic of many species of mammals, is reviewed. The liver parenchyma of adult animals represents a mixed population of mononuclear and binuclear cells with different number of chromosome sets and, therefore DNA content per nucleus. The polyploid hepatocytes are formed during postnatal liver growth as a result of a change from normal mitoses to polyploidizing ones. Hence, the polyploidization of hepatocytes is regarded as an equivalent of cell multiplication.An hypothesis of the biological significance of liver cell polyploidy is based on the fact of a high level of spontaneous chromosome aberrations in mitotic hepatocytes. Ploidy increase is known to give resistance against different kinds of genome alteration. Polyploidization of the liver cells ensures protection against deleterious consequences of the aberrant genome formation resulting from aberrant mitoses.Some implications of the hypothesis are discussed: the reasons for species-specific differences of liver cell polyploidy; the mechanisms of hepatocyte radioresistance; the relation of polyploidy to liver cell aging. The prerequisite factors for unbalanced cell genome formation are adduced: DNA and chromosome damage as the first step in the process, stimulation of mitosis as the second one. The aberrant polyploid genome of hepatocytes is assumed to be the cytogenetic basis for some chronic liver diseases in man.     

Polyploid speciation did not confer instant reproductive isolation in Capsella (Brassicaceae)

Polyploid formation is a major mode of sympatric speciation in flowering plants. Unlike other speciation processes, polyploidization is often assumed to confer instant reproductive isolation. Shared polymorphism across ploidy levels has therefore often been attributed to multiple polyploid origins, whereas the alternative hypothesis of introgressive hybridization has rarely been rigorously tested. Here, we sequence 12 nuclear loci representing 6 genes duplicated by polyploidy in 92 accessions of the tetraploid Capsella bursa-pastoris together with the corresponding loci in 21 accessions of its close diploid relative Capsella rubella. In C. bursa-pastoris accessions from western Eurasia, where the 2 species occur in partial sympatry, we find higher levels of nucleotide diversity than in accessions from eastern Eurasia, where C. rubella does not grow. Furthermore, haplotypes are shared across ploidy levels at 4 loci in western but not in eastern Eurasia. We test whether haplotype sharing is due to retention of ancestral polymorphism or due to hybridization and introgression using a coalescent-based isolation-with-migration model. In western but not in eastern Eurasia, there is evidence for unidirectional gene flow from C. rubella to C. bursa-pastoris. An independent estimate of the timing of dispersal of C. bursa-pastoris to eastern Eurasia indicates that it probably predated introgression. Our results show that polyploid speciation need not result in immediate and complete reproductive isolation, that postpolyploidization hybridization and introgression can contribute significantly to genetic variation in a newly formed polyploid, and that divergence population genetic analysis constitutes a powerful way of testing hypotheses on polyploid speciation.     

烟草毛状根多倍体诱导及其植株再生

为了提高烟草的烟碱含量,采用发根农杆菌遗传转化和人工染色体加倍技术,进行了烟草毛状根及其多倍体诱导、植株再生及其烟碱含量测定。结果表明,发根农杆菌ATCC15834感染烟草叶片外植体8 d后产生白色毛状根,15 d后所有叶片外植体产生毛状根。毛状根能在无外源激素的MS固体和液体培养基上自主生长。PCR扩增结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在烟草毛状根基因组中整合并得到表达。烟草毛状根多倍体诱导的最适条件为0.1%的秋水仙素溶液处理36 h,其多倍体诱导率为64.71%。经秋水仙素加倍的烟草毛状根多倍体植株再生的最适宜培养基为MS+6-BA 2.0 mg/L+NAA0.2 mg/L。与对照(二倍体非转化植株)相比,烟草二倍体毛状根再生植株的顶端优势减弱,腋芽增多,叶片变窄;而烟草毛状根多倍体再生植株茎更粗,节间变短,叶色更深,叶片的宽度和厚度均较对照明显增大。根尖细胞染色体压片观察证实,所获得的烟草毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为4n=96。盆栽实验表明,烟草二倍体毛状根植株和多倍体毛状根再生植株比对照植株延迟约21 d开花。GC-MS检测表明,烟草毛状根多倍体再生植株的烟碱含量比对照及二倍体毛状根再生植株显著提高,分别约为对照及二倍体毛状根再生植株的6.90倍和4.57倍。     

A role for polyploidy in the tumorigenicity of Pim-1-expressing human prostate and mammary epithelial cells

Background

Polyploidy is a prominent feature of many human cancers, and it has long been hypothesized that polyploidy may contribute to tumorigenesis by promoting genomic instability. In this study, we investigated whether polyploidy per se induced by a relevant oncogene can promote genomic instability and tumorigenicity in human epithelial cells.

Principal Findings

When the oncogenic serine-threonine kinase Pim-1 is overexpressed in immortalized, non-tumorigenic human prostate and mammary epithelial cells, these cells gradually converted to polyploidy and became tumorigenic. To assess the contribution of polyploidy to tumorigenicity, we obtained sorted, matched populations of diploid and polyploid cells expressing equivalent levels of the Pim-1 protein. Spectral karyotyping revealed evidence of emerging numerical and structural chromosomal abnormalities in polyploid cells, supporting the proposition that polyploidy promotes chromosomal instability. Polyploid cells displayed an intact p53/p21 pathway, indicating that the viability of polyploid cells in this system is not dependent on the inactivation of the p53 signaling pathway. Remarkably, only the sorted polyploid cells were tumorigenic in vitro and in vivo.

Conclusions

Our results support the notion that polyploidy can promote chromosomal instability and the initiation of tumorigenesis in human epithelial cells.