Effect of cell cycle inhibitor p19ARF on senescence of human diploid cell

To investigate the effect of cell cycle inhibitor p19ARF on replicative senescence of human diploid cell, recombinant p19ARF eukaryotic expression vector was constructed and p19ARF gene was transfected into human diploid fibroblasts (WI-38 cells) by liposome-mediated transfection for overexpression. Then, the effects of p19ARF on replicative senescence of WI-38 cells were observed. The results re- vealed that, compared with control cells, the WI-38 cells in which p19ARF gene was introduced showed significant up-regulation of p53 and p21 expression level, decrease of cell generation by 10 12 generations, decline of cell growth rate with cell cycle being arrested at G1 phase, increase of positive rate of senescent marker SA-β-gal staining, and decrease of mitochondrial membrane potential. The morphology of the transfected fibroblasts presented the characteristics changes similar to senescent cells. These results indicated that high expression of p19ARF may promote the senescent process of human diploid cells.     

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.     

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.     

Screening of senescence-associated genes with specific DNA array reveals the role of IGFBP-3 in premature senescence of human diploid fibroblasts

Repeated exposures to sublethal concentrations of tert-butylhydroperoxide and ethanol trigger premature senescence of WI-38 human diploid fibroblasts. We found 16 replicative senescence-related genes with similar alterations in expression level in replicative senescence and two models of stress-induced premature senescence. Among these genes was IGFBP-3. Using a siRNA approach, we showed that IGFBP-3 regulates the appearance of several biomarkers of senescence after repeated exposures of WI-38 fibroblasts to tert-butylhydroperoxide and ethanol.     

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.     

Extension of replicative lifespan in WI-38 human fibroblasts by dexamethasone treatment is accompanied by suppression of p21 Waf1/Cip1/Sdi1 levels

Numerous studies have shown that supplementation of the growth medium of human fibroblasts with dexamethasone at physiologic concentrations extends replicative lifespan up to 30%. While this extension of lifespan has been used to probe various aspects of the senescent phenotype, no mechanism for the increased lifespan of human fibroblasts grown in the presence of dexamethasone has ever been identified. In the present study we present evidence that the extended lifespan of human lung fibroblasts (WI-38 cells) that occurs when these cells are maintained in culture medium supplemented with dexamethasone is accompanied by a suppression of p21(Waf1/Cip1/Sdi1) levels, which normally increase as these cells enter senescence, while p16(INK4a) levels are unaffected. These results suggest that the delay of senescence in cultures grown in the presence of dexamethasone is due to a suppression of the senescence related increase in p21(Waf1/Cip1/Sdi1). These results are consistent with models of replicative senescence in which p53 and p21(Waf1/Cip1/Sdi1) play a role in the establishment of the senescent arrest.     

Phospholipase D modulation by ceramide in senescence

Phospholipase D (PLD) has been implicated in mitotic regulation and has been shown to be defective in cells following replicative senescence. We examined the source of changes in PLD activity in senescent human umbilical vein endothelial cells and in human diploid fibroblasts. Using fractionated cell components we found that the cytosolic components were necessary for maximum PLD activation. In comparison to low-passage cells, senescent cells showed a profound lack of PLD activatability. By recombining fractionated components from senescent and low-passage cells, we found that in senescence the membrane component is defective in activating PLD implicating either the PLD enzyme itself or its interaction with PKC and/or ARF. The sphingolipid ceramide has been implicated in mediating senescence. Treatment with ceramide resulted in a decrease in PLD activity, implicating ceramide as the mediator of the inhibition.     

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.     

Mammalian SIRT1 limits replicative life span in response to chronic genotoxic stress

The Saccharomyces cerevisiae chromatin silencing factor Sir2 suppresses genomic instability and extends replicative life span. In contrast, we find that mouse embryonic fibroblasts (MEFs) deficient for SIRT1, a mammalian Sir2 homolog, have dramatically increased resistance to replicative senescence. Extended replicative life span of SIRT1-deficient MEFs correlates with enhanced proliferative capacity under conditions of chronic, sublethal oxidative stress. In this context, SIRT1-deficient cells fail to normally upregulate either the p19(ARF) senescence regulator or its downstream target p53. However, upon acute DNA damage or oncogene expression, SIRT1-deficient cells show normal p19(ARF) induction and cell cycle arrest. Together, our findings demonstrate an unexpected SIRT1 function in promoting replicative senescence in response to chronic cellular stress and implicate p19(ARF) as a downstream effector in this pathway.     

Down-regulation of asymmetric arginine methylation during replicative and H2O2-induced premature senescence in WI-38 human diploid fibroblasts

Protein arginine methylation is one of the post-translational modifications which yield monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. In the present study, we investigated the status of protein arginine methylation during human diploid fibroblast senescence. When the expression of protein arginine methyltransferases (PRMTs), namely PRMT1, PRMT4, PRMT5 and PRMT6 was examined, a significant reduction was found in replicatively senescent cells as well as their catalytic activities against histone mixtures compared with the young cells. Furthermore, when the endogenous level of arginine-dimethylated proteins was determined, asymmetric modification (the product of type I PRMTs including PRMT1, PRMT4 and PRMT6) was markedly down-regulated. In contrast, both up- and down-regulations of symmetrically arginine-methylated proteins (the product of type II PRMTs including PRMT5) during replicative senescence were found. Furthermore, when young fibroblasts were induced to premature senescence by sub-cytotoxic H2O2 treatment, results similar to replicative senescence were obtained. Finally, we found that SV40-mediated immortalized WI-38 and HeLa cell lines maintained a higher level of asymmetrically modified proteins as well as type I PRMTs than young fibroblasts. These results suggest that the maintenance of asymmetric modification in the expressed target proteins of type I PRMTs might be critical for cellular proliferation.     

Paradoxical microRNAs

In mammalian cells, microRNAs regulate the expression of target mRNAs generally by reducing their stability and/or translation, and thereby control diverse cellular processes such as senescence. We recently reported the differential abundance of microRNAs in young (early-passage, proliferating) relative to senescent (late-passage, non-proliferating) WI-38 human diploid fibroblasts. Here we report that the levels of the vast majority of mRNAs were unaltered in senescent compared to young WI-38 cells, while overall mRNA translation was potently reduced in senescent cells. Downregulation of Dicer or Drosha, two major enzymes in microRNA biogenesis, lowered microRNA levels, but, unexpectedly, it also reduced global translation. While a reduction in Dicer levels markedly enhanced cellular senescence, reduction of Drosha levels did not, suggesting that the Drosha/Dicer effects on translation may be independent of senescence, and further suggesting that microRNAs may directly or indirectly enhance mRNA translation in WI-38 cells. We discuss possible scenarios through which Dicer/Drosha/microRNAs could enhance translation.     

The peptide methionine sulfoxide reductases, MsrA and MsrB (hCBS-1), are downregulated during replicative senescence of human WI-38 fibroblasts

In contrast to other oxidative modifications of amino acids, methionine sulfoxide can be enzymatically reduced back to methionine in proteins by the peptide methionine sulfoxide reductase system, composed of MsrA and MsrB. The expression of MsrA and one member of the MsrB family, hCBS-1, was analyzed during replicative senescence of WI-38 human fibroblasts. Gene expression decreased for both enzymes in senescent cells compared to young cells, and this decline was associated with an alteration in catalytic activity and the accumulation of oxidized proteins during senescence. These results suggest that downregulation of MsrA and hCBS-1 can alter the ability of senescent cells to cope with oxidative stress, hence contributing to the age-related accumulation of oxidative damage.     

Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O(2) concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.     

有丝分裂诱导基因6(MIG-6)可诱导人成纤维细胞发生提前衰老

年老细胞许多基因表达发生变化,其中有些基因的表达可以诱导成纤维细胞早衰.癌基因诱导的衰老(oncogene-induced senescence,OIS)是由一些连续癌症基因的信号,以阻止细胞增殖造成的反应.有丝分裂诱导基因6(mitogen-inducible gene-6,MIG-6)是一个抑癌基因,是ErbB RTK通路的负调控因子,抑制肿瘤细胞增殖.本文以人胚肺二倍体成纤维细胞为对象,研究MIG-6蛋白在细胞衰老中的作用.通过Western印迹发现,在老年细胞中MIG-6表达升高.利用逆转录病毒载体将MIG-6基因转入年轻的人胚肺二倍体成纤维细胞中,通过Western印迹方法检测是否过表达MIG-6,然后通过SA-β-gal染色检测其阳性率,发现转染MIG-6基因后的成纤维细胞SA-β-gal染色率明显高于对照组,生长缓慢.实验结果证实,MIG-6蛋白可以诱导人二倍体成纤维细胞提前衰老.