Telomere and telomerase dynamics in human cells

Accumulating evidence now implicates telomeres and telomerase as critical regulators genomic stability and replicative lifespan in mammalian cells. Disruption of telomere maintenance and/or telomerase expression contributes to the etiology of some degenerative diseases and may participate in the process of aging. Although telomere dysfunction and aberrant telomerase expression clearly play important roles in cancer development, the contribution of telomere biology to cancer is complex and involves both positive and negative influences on tumor development. Indeed, recent work from several laboratories suggests additional roles for telomeres and telomerase in both normal and malignant physiology. Understanding the complexity of telomere biology will provide further insights into chromosome biology in both normal and malignant cells.     

Telomere shortening in human HL60 cells by treatment with 3'-azido-2',3'-dideoxynucleosides and telomerase inhibition by their 5'-triphosphates

Telomerase is thought to play an important role in the mechanism of tumor cell immortalization by maintenance of telomere length. To obtain information on the susceptibility of telomerase to nucleoside analogues, the effects of base-modified 3'-azido-2',3'-dideoxynucleoside triphosphates on the enzyme were investigated. It is suggested that the 2-amino group of the nucleotide purine nucleus is important for the inhibitory activity. Telomere shortening caused by long-term treatment with these nucleosides is also described.     

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.     

Potent inhibition of human telomerase by U-73122

Summary Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC₅₀ value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 μM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC₅₀ values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.     

Porcine endothelial cells,unlike human endothelial cells,can be killed by human CTL via Fas ligand and cannot be protected by Bcl-2

In clinical transplantation host CTL are major effectors of acute rejection, and graft endothelial cells (EC) are major targets of the CTL response. It is unclear what roles CTL will play in pig-into-human xenotransplantation. We compared the mechanisms of killing used by human CTL (huCTL) vs allogeneic and pig xenogeneic EC targets. Both responses show MHC class I restriction of target cell recognition. A granzyme B inhibitor peptide completely blocks anti-human and partially blocks anti-pig responses, while inhibitory Fas ligand Ab only blocks killing of porcine cells despite similar levels of Fas expression in both target cell types. Transduction of Bcl-2 completely protects human EC from huCTL, but has no effect on huCTL-mediated killing of porcine EC despite its efficacy vs drug-induced apoptosis. Bcl-2 effectively protects human EC rendered sensitive to Fas ligand by overexpressing Fas from huCTL, yet fails to protect porcine aortic endothelial cells from huCTL in the presence of anti-Fas ligand Ab. These data reveal differences in the susceptibility of human and porcine targets to huCTL.     

NK4基因转染对人卵巢癌细胞SKOV-3生物学特性的影响

目的 通过在人卵巢癌细胞SKOV-3中转染NK4基因表达片段,研究NK4对人卵巢癌的治疗作用,从而验证NK4可作为卵巢癌潜在的基因治疗新策略.方法 用NK4和荧光霉素表达质粒分别稳定转染人卵巢癌细胞SKOV-3.用West-ern blot检测细胞培养基中NK4蛋白表达,以及经不同培养基(SKOV-3、SKOV-3/LUC和SKOV-3/NK4培养上清)培养后SKOV-3细胞中c-Met和磷酸化-c-Met的表达.用MTT试剂盒绘制细胞生长曲线.用细胞划痕试验检测NK4对细胞转移的作用.结果 在SKOV-3/NK4培养基中有NK4蛋白表达,对照(SKOV-3,SKOV-3/LUC)细胞中无表达.磷酸化-c-Met在SKOV-3/NK4培养基培养的SKOV-3细胞中被抑制,而在对照(SKOV-3,SKOV-3/LUC)细胞培养基培养的SKOV-3细胞中正常表达.c-Met表达在各组差异无统计学意义.三种细胞体外生长曲线差异无统计学意义(P＞0.05).细胞划痕试验表明SKOV-3/NK4划痕区域的细胞个数明显少于SKOV-3和SKOV-3/LUC细胞.结论 NK4蛋白在SKOV-3/NK4细胞培养基中大量分泌,NK4能抑制人卵巢癌细胞c-Met受体磷酸化,并能抑制卵巢癌细胞体外活动能力,为应用NK4作为卵巢癌基因治疗方法提供了研究基础.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Effect of ovarian cancer ascites on SKOV-3 cells proteome: new proteins associated with aggressive phenotype in epithelial ovarian cancer

Background

Epithelial ovarian cancer is the second most lethal gynecological cancer worldwide. Ascites can be found in all clinical stages, however in advanced disease stages IIIC and IV it is more frequent and could be massive, associated with worse prognosis. Due to the above, it was our interest to understanding how the ascites of ovarian cancer patients induces the mechanisms by which the cells present in it acquire a more aggressive phenotype and to know new proteins associated to this process.

Methods

A proteomic analysis of SKOV-3 cells treated with five different EOC ascites was performed by two-dimensional electrophoresis coupled to MALDI-TOF. The level of expression of the proteins of interest was validated by RT-PCR because several of these proteins have only been reported at the messenger level.

Results

Among the proteins identified that increased their expression in ascites-treated SKOV-3 cells, were Ran GTPase, ZNF268, and Synaptotagmin like-3. On the other hand, proteins that were negatively regulated by ascites were HLA-I, HSPB1, ARF1, Synaptotagmin 1, and hnRNPH1, among others. Furthermore, an interactome for every one of these proteins was done in order to identify biological processes, molecular actions, and cellular components in which they may participate.

Conclusions

Identified proteins participate in cellular processes highly relevant to the aggressive phenotype such as nuclear transport, regulation of gene expression, vesicular trafficking, evasion of the immune response, invasion, metastasis, and in resistance to chemotherapy. These proteins may represent a source of information which has the potential to be evaluated for the design of therapies directed against these malignant cells that reside on ovarian cancer ascites.

     

Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.     

Telomere protection without a telomerase; the role of ATM and Mre11 in Drosophila telomere maintenance

The conserved ATM checkpoint kinase and the Mre11 DNA repair complex play essential and overlapping roles in maintaining genomic integrity. We conducted genetic and cytological studies on Drosophila atm and mre11 knockout mutants and discovered a telomere defect that was more severe than in any of the non-Drosophila systems studied. In mutant mitotic cells, an average of 30% of the chromosome ends engaged in telomere fusions. These fusions led to the formation and sometimes breakage of dicentric chromosomes, thus starting a devastating breakage-fusion-bridge cycle. Some of the fusions depended on DNA ligase IV, which suggested that they occurred by a nonhomologous end-joining (NHEJ) mechanism. Epistasis analyses results suggest that ATM and Mre11 might also act in the same telomere maintenance pathway in metazoans. Since Drosophila telomeres are not added by a telomerase, our findings support an additional role for both ATM and Mre11 in telomere maintenance that is independent of telomerase regulation.     

Changes in N-glycosylation of human stromal cells by telomerase expression

It was established that remarkable changes in the N-glycosylation are induced in immortalized cancer cells. Whether changes were induced in human stromal cells immortalized by transfection with the human telomerase catalytic subunit (hTert) cDNA was examined by lectin blot analysis. Morphological appearance and growth rate of the gene-transfected stromal cells were not changed significantly. However, lectin blot analysis of membrane glycoprotein samples showed that bindings of Ricinus communis agglutinin-I (RCA-I) and of leuko-agglutinating phytohemagglutinin to glycoprotein bands increase significantly in the gene-transfected cells. No lectin binding was observed when blotted filters were treated with diplococcal beta-1,4-galactosidase or N-glycanase prior to incubation with RCA-I. In contrast, no changes in Coomassie brilliant blue-staining and in binding of concanavalin A were obtained between the primary and gene-transfected stromal cells. These results indicate that the highly branched N-glycosylation with augmented galactosylation is induced in human stromal cells immortalized by the telomerase expression.     

Genistein增加顺铂诱导的SKOV-3细胞的凋亡与活性氧的关系

目的:探讨Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV-3凋亡的可能作用机制.方法:倒置相差显微镜下观察药物处理后细胞形态学的变化;MTT比色法检测不同药物处理后对SKOV-3细胞增殖的影响;流式细胞仪检测药物处理后细胞的凋亡情况;流式细胞仪和荧光显微镜检测细胞内活性氧(ROS)的水平.结果:10ug/ml的Genistein和2.5ug/ml的顺铂联用24h后,引起了细胞内ROS的增加,细胞的凋亡率也显著增高,与单用顺铂组相比差异有显著性(P＜0.05);用NAC预处理细胞2h后,有效抑制了ROS的产生,并增加了细胞的活性,降低了细胞的凋亡率,与未加NAC组相比差异有显著性(P＜0.05).结论:Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV一3的凋亡与细胞内ROS水平的升高有关,这可能是Genistein增加顺铂诱导的耐药卵巢癌细胞SKOV-3凋亡的作用机制之一.     

Short telomeres are preferentially elongated by telomerase in human cells

Short telomeres have been shown to be preferentially elongated in both yeast and mouse models. We examined this in human cells, by utilising cells with large allelic telomere length differentials and observing the relative rates of elongation following the expression of hTERT. We observed that short telomeres are gradually elongated in the first 26 PDs of growth, whereas the longer telomeres displayed limited elongation in this period. Telomeres coalesced at similar lengths irrespective of their length prior to the expression of hTERT. These data indicate that short telomeres are marked for gradual elongation to a cell strain specific length threshold.     

Telomere length analysis of human mesenchymal stem cells by quantitative PCR

Human mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells are required, which necessitates extensive sub-culturing and expansion of hMSCs. Throughout ex vivo expansion, the cells undergo telomere shortening, and critically short telomeres can trigger loss of cell viability. Telomeres are nucleoprotein structures that cap the ends of chromosomes, and serve to protect the DNA from the degradation which occurs due to the end-replication problem in all eukaryotes. As hMSCs have only a finite ability for self-renewal like most somatic cells, assaying for telomere length in hMSCs provides critical information on the replicative capacity of the cells, an important criterion in the selection of hMSCs for therapy. Telomere length is generally quantified by Southern blotting and fluorescence in situ hybridization, and more recently by PCR-based methods. Here we describe the quantification of hMSC telomere length by real-time PCR; our results demonstrate the effect of telomere shortening on the proliferation and clonogenicity of hMSCs. Thus, this assay constitutes a useful tool for the determination of relative telomere length in hMSCs.     

Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase

Somatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme "skewing" toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in?vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines.