Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

Overexpression of the p21 sdi1 gene induces senescence-like state in human cancer cells: implication for senescence-directed molecular therapy for cancer.

Normal cells in a culture enter a nondividing state after a finite number of population doubling, which is termed replicative senescence, whereas cancer cells have unlimited proliferative potential and are thought to exhibit an immmortal phenotype by escaping from senescence. The p21 gene (also known as sdi1), which encodes the cyclin-dependent kinase inhibitor, is expressed at high levels in senescent cells and contributes to the growth arrest. To examine if the p21sdi1 gene transfer could induce senescence in human cancer cells, we utilized an adenoviral vector-based expression system and four human cancer cell lines differing in their p53 status. Transient overexpression of p21sdi1 on cancer cells induced quiescence by arresting the cell cycle at the G1 phase and exhibited morphological changes, such as enlarged nuclei as well as a flattened cellular shape, specific to the senescence phenotype. We also showed that p21sdi1-transduced cancer cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Moreover, the polymerase chain reaction-based assay demonstrated that levels of telomerase activity were significantly lower in p21sdi1-expressing cells compared to parental cancer cells. These observations provide the evidence that p21sdi1 overexpression could induce a senescence-like state and reduce telomerase activity in human cancer cells, suggesting that these novel p21sdi1 functions may have important implications for anticancer therapy.     

Downregulation of caveolin-1 affects bleomycin-induced growth arrest and cellular senescence in A549 cells

Bleomycin is an anti-cancer drug that induces both apoptosis and senescence, two processes thought to involve caveolin-1. Here we investigate the role of caveolin-1 in bleomycin-induced senescence. We show that bleomycin-treated A549 cells exhibit: senescence-like cell morphology; a senescence-associated increase in SA-beta-galactosidase activity; cell cycle arrest; and upregulation of p53 and p21. As predicted, we find that caveolin-1 amount increases in response to bleomycin-treatment and that modulation of caveolin-1 affects p21 and p53 levels, cell cycling, and senescence (SA-beta-galactosidase activity). Interestingly, senescence-associated cell cycle arrest via p53 and p21 and SA-beta-galactosidase activity is reduced in young A549 cells when short hairpin RNA specific for caveolin-1 was applied before bleomycin-treatment. Our results support the hypothesis that downregulation of caveolin-1 expression affects bleomycin-induced cell cycle arrest and subsequent cellular senescence that is driven by p53 and p21.     

Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1

ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus. To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library. Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A). The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy. Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle. In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone. In addition, the interaction of ECRG1 and Miz-1 could induce expression of P15(INK4b) gene in esophageal cancer 9706 (EC9706) cells. However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15(INK4b) gene. When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/c-3T3 cells, Transfection of Miz-1 couldn't induce P15(INK4b) expression. The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15(INK4b) expression. It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15(INK4b) may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1. The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC.     

Expression of caveolin-1 induces premature cellular senescence in primary cultures of murine fibroblasts

Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a "transformation suppressor" protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G(0)/G(1) phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphology; and 3) a senescence-associated increase in beta-galactosidase activity. These results indicate for the first time that the expression of caveolin-1 in vivo is sufficient to promote and maintain the senescent phenotype. Subcytotoxic oxidative stress is known to induce premature senescence in diploid fibroblasts. Interestingly, we show that subcytotoxic level of hydrogen peroxide induces premature senescence in NIH 3T3 cells and increases endogenous caveolin-1 expression. Importantly, quercetin and vitamin E, two antioxidant agents, successfully prevent the premature senescent phenotype and the up-regulation of caveolin-1 induced by hydrogen peroxide. Also, we demonstrate that hydrogen peroxide alone, but not in combination with quercetin, stimulates the caveolin-1 promoter activity. Interestingly, premature senescence induced by hydrogen peroxide is greatly reduced in NIH 3T3 cells harboring antisense caveolin-1. Importantly, induction of premature senescence is recovered when caveolin-1 levels are restored. Taken together, these results clearly indicate a central role for caveolin-1 in promoting cellular senescence and they suggest the hypothesis that premature senescence may represent a tumor suppressor function mediated by caveolin-1 in vivo.     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

8-Hydroxydeoxyguanosine induces senescence-like changes in KG-1, human acute myelocytic leukemia cell line

8-Hydroxydeoxyguanosine (oh⁸dG) treatment induced senescence-like changes in KG-1 cells, a human acute myelocytic leukemia cell line. The oh⁸dG-treated cells stained positive for senescence associated β-galactosidase (SA-β-galactosidase) and had enlarged cell shape, both of which are senescence indexes. The oh⁸dG-treated cells were also cell growth inhibited and arrested at G₁ in the cell cycle. The accumulation of cdk (cyclin dependent kinase) inhibitors, such as p16, p21, and p27, also implies that cellular senescence was induced in oh⁸dG-treated cells. However, these changes were not accompanied by cell differentiation or telomerase activity. Taken together, we conclude that oh⁸dG treatment of KG-1 cells induces cellular senescence.     

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.     

S-Phase-specific activation of PKC alpha induces senescence in non-small cell lung cancer cells

Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

Effects of Iejimalide B, a marine macrolide, on growth and apoptosis in prostate cancer cell lines

Iejimalide B, a marine macrolide, causes growth inhibition in a variety of cancer cell lines at nanomolar concentrations. We have investigated the effects of Iejimalide B on cell cycle kinetics and apoptosis in the p53+/AR+ LNCaP and p53-/AR- PC-3 prostate cancer cell lines. Iejimalide B, has a dose and time dependent effect on cell number (as measured by crystal violet assay) in both cell lines. In LNCaP cells Iejimalide B induces a dose dependent G0/G1 arrest and apoptosis at 48 h (as measured by Apo-BrdU staining). In contrast, Iejimalide B initially induces G0/G1 arrest followed by S phase arrest but does not induce apoptosis in PC-3 cells. qPCR and Western analysis suggests that Iejimalide B modulates the steady state level of many gene products associated with cell cycle (including cyclins D, E, and B and p21(waf1/cip1)) and cell death (including survivin, p21B and BNIP3L) in LNCaP cells. In PC-3 cells Iejimalide B induces the expression of p21(waf1/cip1), down regulates the expression of cyclin A, and does not modulate the expression of the genes associated with cell death. Comparison of the effects of Iejimalide B on the two cell lines suggests that Iejimalide B induces cell cycle arrest by two different mechanisms and that the induction of apoptosis in LNCaP cells is p53-dependent.     

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 revealed that, compared with control cells, the WI-38 cells in which p19ARFgene 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.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation

During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts.     

Growth-Regulatory Activity of the Growth Arrest-Specific Gene, GAS1, in NIH3T3 Fibroblasts

The growth arrest-specific gene, Gas-1, is preferentially expressed in quiescent NIH3T3 cells and inhibits DNA synthesis, suggesting that Gas-1 may be a tumor suppressor gene. When GAS1 cDNA, under the control of the strong constitutive CMV promoter, was transfected into NIH3T3 cells, no stable transfectant cell lines were produced, confirming that high levels of expression of GAS1 mRNA inhibit proliferation. GAS1, under the control of a dexamethasone-inducible promoter, was also transfected into NIH3T3 cells, resulting in normal numbers of transfectant clones. When expression of GAS1 mRNA was induced with dexamethasone, the growth rate was greatly inhibited. Morphological changes characteristic of growth arrest were also observed. To determine if antisense inhibition of expression of Gas-1 will transform normal fibroblasts, GAS1 cDNA, cloned in the antisense orientation, was transfected into NIH3T3 cells and expression of endogenous Gas-1 mRNA was inhibited. The GAS1-antisense cells had altered morphology and grew to a much higher saturation density than control cell lines with a loss of contact inhibition. However, there was no change in requirements for serum or any development of anchorage-independence. Antisense inhibition of expression of GAS1 is therefore insufficient to transform the cells, suggesting that additional genetic events are required for a fully malignant phenotype.