An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.     

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.     

Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts

Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3-20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-beta-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.     

Attenuation of NF-kappaB signaling response to UVB light during cellular senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-kappaB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-kappaB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-kappaB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-kappaB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-kappaB complexes. Supershift experiments showed that the specific NF-kappaB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IkappaBalpha showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-kappaB-responsive sites of the TNFalpha promoter were used to analyze the functional activity of the NF-kappaB complexes. Results showed that UVB exposure induced an increase in NF-kappaB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-kappaB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.     

Age-dependent alterations of c-fos and growth regulation in human fibroblasts expressing the HPV16 E6 protein.

Normal human cells in culture become senescent after a limited number of population doublings. Senescent cells display characteristic changes in gene expression, among which is a repression of the ability to induce the c-fos gene. We have proposed a two-stage model for cellular senescence in which the mortality stage 1 (M1) mechanism can be overcome by agents that bind both the product of the retinoblastoma susceptibility gene (pRB)-like pocket proteins and p53. In this study we determined whether the repression of c-fos at M1 was downstream of the p53 or pRB-like "arms" of the M1 mechanism. We examined c-fos expression during the entire lifespan of normal human fibroblasts carrying E6 (which binds p53), E7 (which binds pRB), or both E6 and E7 of human papilloma virus type 16. The results indicate a dramatic change in cellular physiology at M1. Before M1, c-fos inducibility is controlled by an E6-independent mechanism that is blocked by E7. After M1, c-fos inducibility becomes dependent on E6 whereas E7 has no effect. In addition, a novel oscillation of c-fos expression with an approximately 2-h periodicity appears in E6-expressing fibroblasts post-M1. Accompanying this shift at M1 is a dramatic change in the ability to divide in low serum. Before M1, E6-expressing fibroblasts growth arrest in 0.3% serum, although they continue dividing under those conditions post-M1. These results demonstrate the unique physiology of fibroblasts during the extended lifespan between M1 and M2 and suggest that p53 might participate in the process that represses the c-fos gene at the onset of cellular senescence.     

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.     

A progressive reduction in autophagic capacity contributes to induction of replicative senescence in Hs68 cells

Autophagy has been implicated in delayed aging and extended longevity. Here, we aimed to study the possible effects of autophagy during the progression of replicative senescence, which is one of the major features of aging. Human foreskin fibroblasts, Hs68 cells, at an initial passage of 15 were serially cultured for several months until they reached cellular senescence. A decrease in cell proliferation was observed during the progression of senescence. Induction of replicative senescence in aged cells (at passage 40) was confirmed by senescence-associated β-galactosidase (SA-β-gal) activity that represents a sensitive and reliable marker for quantifying senescent cells. We detected a significantly increased percentage (%) of SA-β-gal-positive cells at passage 40 (63%) when compared with the younger SA-β-gal-positive cells at passage 15 (0.5%). Notably, the gradual decrease in basal autophagy coincided with replicative senescence induction. However, despite decreased basal autophagic activity in senescent cells, autophagy inducers could induce autophagy in senescent cells. RT-PCR analysis of 11 autophagy-related genes revealed that the decreased basal autophagy in senescent cells might be due to the downregulation of autophagy-regulatory proteins, but not autophagy machinery components. Moreover, the senescence phenotype was not induced in the cells in which rapamycin was added to the culture to continuously induce autophagy from passage 29 until passage 40. Together, our findings suggest that reduced basal autophagy levels due to downregulation of autophagy-regulatory proteins may be the mechanism underlying replicative senescence in Hs68 cells.     

Pleiotropic action of AP-1 in v-Src-transformed cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.     

Altered regulation of platelet-derived growth factor A-chain and c-fos gene expression in senescent progeria fibroblasts

The study of human genetic disorders known as premature aging syndromes may provide insight into the mechanisms of cellular senescence. These diseases are clinically characterized by the premature onset and accelerated progression of numerous features normally associated with human aging. Previous studies have indicated that fibroblasts derived from premature aging syndrome patients have in vitro growth properties similar to senescent fibroblasts from normal individuals. As an initial approach to determine whether gene expression is altered in premature aging syndrome fibroblasts, RNA was prepared from various cell strains and used for gel blot hybridization experiments. Although normal fibroblasts only express platelet-derived growth factor (PDGF) A-chain mRNA for a brief period following mitogenic stimulation, one strain of Hutchinson-Gilford (progeria) syndrome fibroblasts, AG3513, constitutively expresses PDGF A-chain mRNA and PDGF-AA homodimers. The PDGF A-chain gene does not appear to be amplified or rearranged in these fibroblasts. AG3513 progeria fibroblasts have properties characteristic of senescent cells, including an altered morphology and a diminished mitogenic response to growth promoters. The diminished response of AG3513 progeria fibroblasts to PDGF stimulation was examined in some detail. Studies using 125I-PDGF-BB, which binds with high affinity to both A- and B-type PDGF receptors, indicate that normal and AG3513 progeria fibroblasts have a similar number of PDGF receptors. Although receptor autophosphorylation occurs normally in PDGF-stimulated AG3513 progeria fibroblasts, c-fos mRNA induction does not. The senescent phenotype of AG3513 fibroblasts is probably unrelated to their constitutive PDGF A-chain gene expression; further studies are necessary in order to directly address this issue. Also, additional analysis of this progeria fibroblast strain may provide information on the control of mitogen-inducible gene expression in normal cells.     

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.     

Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O2) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H2O2 represents a critical signal mediating replicative senescence.     

Selective Loss of CDC2 and CDK2 Induction by Tumor Necrosis Factor-α in Senescent Human Diploid Fibroblasts

Normal human diploid fibroblasts undergo a finite number of doublings in culture. This process of senescence is accompanied by a loss in the ability to respond to proliferative stimuli and is therefore distinct from the quiescent state induced by nutrient deprivation. We have studied changes in gene expression induced in these cells following exposure to the cytokine, tumor necrosis factor-α (TNF). We observed that TNF induced CDC2 and CDK2 expression in early-passage quiescent WI-38 fibroblasts. However, as cells approached senescence, their ability to induce CDC2 and CDK2, as well as stimulate DNA synthesis in response to TNF, progressively declined, with minimal to absent induction in senescent cells. This occurred despite the TNF-dependent induction of such proliferation-independent genes as manganese superoxide dismutase and interleukin-6 in senescent and quiescent cells. Serum was similarly unable to induce CDC2 or CDK2 expression in senescent cells. These results demonstrate that senescent cells are selectively deficient in TNF-mediated induction of CDC2 and CDK2, genes crucial to DNA synthesis and mitosis.     

Cisplatin-induced premature senescence with concomitant reduction of gap junctions in human fibroblasts

To examine the role of gap junctions in cell senescence, the changes of gap junctions in cisplatin-induced premature senescence of primary cultured fibroblasts were studied and compared with the replicative senescent human fibroblasts.Dye transfer assay for gap junction function and immunofluorescent staining for connexin 43 protein distribution were done respectively. Furthermore, cytofluorimetry and DAPI fluorescence staining were performed for cell cycle and apoptosis analysis, p53 gene expression level was detected with indirect immunofluorescence. We found that cisplatin(10mM) treatment could block cell growth cycle at G1 and induced premature senescence. The premature senescence changes included high frequency of apoptosis, elevation of p53 expression, loss of membranous gap junctions and reduction of dye-transfer capacity. These changes were comparable to the changes of replicative senescence of human fibroblasts. It was also concluded that cisplatin could induce premature senescence concomitant with inhibition of gap junctions in the fibroblasts. Loss of functional gap junctions from the cell membrane may account for the reduced intercellular communication in the premature senescent fibroblasts. The cell system we used may provide a model useful for the study of the gap junction thus promoting agents against premature senescence.