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PubMed2004

Extracellular invertase is an essential component of cytokinin-mediated delay of senescence.

Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.

Agrobacterium tumefaciens↗

PubMed1993

Investigation of the role of G1/S cell cycle mediators in cellular senescence.

Cellular senescence is a state of irreversible cell cycle arrest in which normal cells at the end of their lifespan fail to enter into DNA synthesis upon serum or growth factor stimulation. We examined whether proteins required for G1/S cell cycle progression were irreversibly down-regulated in senescent human fibroblasts. Both the 44- and 42-kDa forms of the MAP-kinase protein were expressed at similar levels in young and senescent cells. In contrast to young cells where both forms were phosphorylated on tyrosine in response to serum, the p42MAP-kinase was not tyrosine phosphorylated upon serum stimulation, whereas p44MAP-kinase was phosphorylated on tyrosine in serum-starved or serum-stimulated senescent cells. Examination of p53 protein in growing, quiescent, and senescent cells revealed no significant differences in levels between the different growth states. In contrast, cdk2 and cyclin A mRNAs were completely down-regulated in stimulated senescent fibroblasts, while the G1 cyclins, C, D1, and E mRNAs, were still expressed in stimulated senescent cells although at reduced levels compared to young cells. The expression of early G1 markers, but not late G1 markers, indicates that senescent cells may be blocked at a point in late G1. We investigated whether transfection of cyclin A, alone or in combination with cdc2, was sufficient for extension of lifespan or escape from senescence. Clones expressing the transfected human cyclin A or cdc2 genes senesced at a population doubling similar to controls, thereby showing that cyclin A or cdc2 expression alone was insufficient for escape from senescence.

Base Sequence↗

PubMed1995

Diminished responsiveness of senescent normal human fibroblasts to TNF-dependent proliferation and interleukin production is not due to its effect on the receptors or on the activation of a nuclear factor NF-kappa B.

The limited life span in culture of normal human diploid fibroblasts (HDF) has provided a model of cellular senescence. The short-term growth of these cells in culture is regulated by a number of different cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and fibroblast growth factor (FGF). However, the effect of senescence on the responsiveness of HDF to these cytokines is not known. In the present report, we examined the effects of TNF on foreskin-derived HDF at different passage levels. We compared the response of HDF cells at population doubling (PD) 23 (young) with that of cells at PD 70 (senescent). Young cells proliferated in response to TNF in a dose-dependent manner. Under these conditions TNF had no effect on senescent HDF. The decrease in TNF responsiveness was found to be dependent on PD. The lack of response of senescent HDF was not unique to TNF, since FGF and IL-1 were also ineffective. In contrast to senescent HDF, TNF-dependent proliferation of young HDF could be further potentiated by IL-1 and FGF, suggesting an independent signaling mechanism. On exposure to TNF, senescent HDF produced IL-6 and IL-8, but to a much lower degree than that produced by young HDF. The diminished responsiveness of senescent HDF to TNF does not appear to be due to the difference in either receptor number or affinity, since senescent cells had two- to threefold higher number of TNF receptors than young HDF but the same affinity. TNF induced the activation of a nuclear transcriptional factor, NF-kappa B, equally in both young and senescent cells, which indicates the lack of a defect in the early events of TNF signal transduction in senescent fibroblasts. Overall, our results indicate that there is an age-dependent decline in TNF-induced proliferation and in the production of interleukins by fibroblasts; this unresponsiveness appears not to be due to TNF receptors or NF-kappa B activation. These results may have importance in understanding the diminished immune response, inflammation, and wound healing associated with aging.

Base Sequence↗

PubMed2002

Identification of a candidate tumor-suppressor gene specifically activated during Ras-induced senescence.

Normal cells display protective responses against oncogenes. Notably, oncogenic Ras triggers an irreversible proliferation arrest that is reminiscent of replicative senescence and that is considered a relevant tumor-suppressor mechanism. Here, we have used microarrayed filters to identify genes specifically upregulated in Ras-senescent human fibroblasts. Among the initial set of genes selected from the microarrays, we found the cell-cycle inhibitor p21(Cip1/Waf1), thus validating the potency of the screening to identify markers and mediators of Ras-senescence. A group of six genes, formed by those more highly upregulated during Ras-senescence, was analyzed in further detail to evaluate their specificity. In particular, we examined their expression in cells overexpressing Ras but rendered resistant to Ras-senescence by the viral oncoprotein E1a; also, we have studied their expression during replicative senescence, organismal aging, H(2)O(2)-induced senescence, and DNA damage. In this manner, we have identified a novel gene, RIS1 (for Ras-induced senescence 1), which is not upregulated in association to any of the above-mentioned processes, but exclusively during Ras-senescence. Furthermore, RIS1 is also upregulated by the transcriptional factor Ets2, which is a known mediator of Ras-induced senescence. Interestingly, RIS1 is located at chromosomal position 3p21.3 and, more specifically, it is included in a short segment of just 1 Mb previously defined by other investigators for its tumor-suppressor activity. In summary, we report the identification of a novel gene, RIS1, as a highly specific marker of Ras-induced senescence and a candidate tumor-suppressor gene.

Adenovirus E1A Proteins↗

PubMed2004

Vascular cell senescence and vascular aging.

Vascular cells have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest called "cellular senescence". A number of genetic animal models carrying targeted disruption of the genes that confer the protection against senescence in vitro have been reported to exhibit the phenotypes of premature aging. Similar mutations have been found in the patients with premature aging syndromes. Many of the changes in senescent vascular cell behavior are consistent with the changes seen in age-related vascular diseases. We have demonstrated the presence of senescent vascular cells in human atherosclerotic lesions but not in non-atherosclerotic lesions. Moreover, these cells express increased levels of pro-inflammatory molecules and decreased levels of endothelial nitric oxide synthase, suggesting that cellular senescence in vivo contributes to the pathogenesis of human atherosclerosis. One widely discussed hypothesis of senescence is the telomere hypothesis. An increasing body of evidence has established the critical role of the telomere in vascular cell senescence. Another line of evidence suggests that telomere-independent mechanisms are also involved in vascular cell senescence. Activation of Ras, an important signaling molecule involved in atherogenic stimuli, induces vascular cell senescence and thereby promotes vascular inflammation in vitro and in vivo. It is possible that mitogenic-signaling pathways induce telomere-dependent and telomere-independent senescence, which results in vascular dysfunction. Further understanding of the mechanism underlying cellular senescence will provide insights into the potential of antisenescence therapy for vascular aging.

Animals↗

PubMedDate not supplied

Replicative senescence of human fibroblasts: the role of Ras-dependent signaling and oxidative stress.

Replicative senescence of human fibroblasts is a widely used cellular model for human aging. While it is clear that telomere erosion contributes to the development of replicative senescence, it is assumed that additional factors contribute to the senescent phenotype. The free radical theory of aging suggests that oxidative damage is a major cause of aging; furthermore, the expression of activated oncogenes, such as oncogenic Ras, can induce premature senescence in primary cells. The functional relation between the various inducers of senescence is not known. The present study was guided by the hypothesis that constitutive activation of normal, unmutated Ras may contribute to senescence-induced growth arrest in senescent human fibroblasts. When various branches of Ras-dependent signaling were investigated, constitutive activation of the Ras/Raf/MEK/ERK pathway was not observed. To evaluate the role of oxidative stress for the senescent phenotype, we also investigated stress-related protein kinases. While we found no evidence for alterations in the activity of p38, we could detect an increased activity of Jun kinase in senescent fibroblasts. We also found higher levels of reactive oxygen species (ROS) in senescent fibroblasts compared to their younger counterparts. The accumulation of ROS in senescent cells may be related to the constitutive activation of Jun kinase.

Blotting, Western↗

PubMed2002

Replicative senescence in organ transplantation-mechanisms and significance.

In the past two decades, transplantation has become a preferred modality of treatment of end-stage failure of vital organs. Currently, with the significant improvement in short-term graft survival rates, the main effort is concentrated on prolonging the functional life span of transplanted organs. One of the theories which were put forward to explain the progressive deterioration of transplant function was that of replicative senescence. Senescence of an organ or tissue results from age and/or environmental stress-dependant modification of cellular function. With time, the accumulation of cellular alterations may lead to deleterious effects in various organs and tissues and adversely affect transplants. In this article we are reviewing the candidate mechanisms of senescence such as telomere shortening, genetic regulation and environmental-'toxic' factors and are examining the implications of the theory of replicative senescence for organ allograft. We are also presenting our experiments with renal ischemia/reperfusion in rat serving as a model of kidney transplantation, where baseline kidney telomere length and novel marker of cellular senescence--senescence associated beta-Galactosidase (SA-Gal) expression in tissue served as markers. For the first time in vivo, we were able to show that with aging of the animals the amount of senescent cells in kidney tissue was increasing, while the average renal tissue telomere length was decreasing. The degree of tissue senescence, as determined by amount of SA-Gal positively stained cells, was inversely correlated with the recovery of the kidney function after ischemia/reperfusion injury. These results confirm the theory of replicative senescence in organ ischemia for the first time in vivo, and quantitatively validate the direct correlation between the amount of senescent cells in the organ and its susceptibility to ischemic injury. We conclude that recent advances in study of the cellular basis of senescence, in vitro and especially in vivo, may hold clues to the understanding of events which could be implicated in the damage or protection of organ allografts.

Animals↗

PubMed2004

Hallmarks of senescence in carcinogenesis and cancer therapy.

Cellular senescence is a signal transduction program leading to irreversible cell cycle arrest. This growth arrest can be triggered by many different mechanisms including recognition by cellular sensors of DNA double-strand breaks leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. Senescence is initiated by the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals (STASIS: stress or aberrant signaling-induced senescence). The process of carcinogenesis involves a series of changes that allow tumor cells to bypass the senescence program. Nevertheless, tumor cells retain the capacity to undergo senescence. Treatment of tumor cells with many conventional anticancer therapies activates DNA damage signaling pathways, which induce apoptosis in some cells and senescence in others. Overexpression of tumor suppressors or inhibition of oncogenes can also induce rapid senescence in tumor cells. Senescent cells, while not dividing, remain metabolically active and produce many secreted factors, some of which stimulate and others inhibit the growth of tumors. The emerging knowledge about the pathways that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.

Animals↗

PubMed2006

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Aging↗

PubMed2001

Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.

There is growing evidence that senescent cells accumulate in vivo and are associated with the aging process in parallel with the progressive erosion of telomeres. Because recent data show that telomere shortening is involved in the pathogenesis of liver cirrhosis, we looked for replicative senescence cells in normal livers, chronic hepatitis C, and hepatocellular carcinoma (HCC). Replicative senescent cells were detected on liver tissue cryosections using expression of a specific marker, senescence-associated beta-galactosidase, a cytoplasmic enzyme detected at pH 6. A total of 57 frozen liver samples (15 normal liver, 32 chronic hepatitis C, and 10 HCCs) were studied. Replicative senescence was graded as absent in 56% of cases (32 of 57) and present in 44% (25 of 57). Replicative senescence was considered present in 3 of 15 normal livers (20%), 16 of 32 chronic hepatitis cases (50%), and 6 of 10 HCCs (60%). In the group of nontumoral livers, the presence of senescent cells in liver was associated with older age (P =.03). In the group with chronic hepatitis C, fibrosis stage, but not activity grade, was significantly correlated with the accumulation of replicative senescent cells (P <.001). Finally, beta-Gal staining in nontumoral tissue was strongly correlated with the presence of HCC in the surrounding liver (P <.001). These results suggest that chronic hepatitis C represents a relevant model of accelerated replicative senescence and that accumulation of replicative senescent cells predispose to HCC development. Detection of replicative senescent cells may then serve as a predictive marker of a hepatocellular carcinoma in the surrounding tissue. HUM PATHOL 32:327-332.

Carcinoma, Hepatocellular↗

PubMed1996

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Carrier Proteins↗

PubMed2004

Morphological adjustment of senescent cells by modulating caveolin-1 status.

Morphological change is one of the cardinal features of the senescent phenotype; for example, senescent human diploid cells have a flat large shape. However, the mechanisms underlying such senescence-related morphological alterations have not been well studied. To investigate this situation, we characterized the senescence-dependent changes of cellular structural determinants in terms of their levels and activities. These determinants included integrins, focal adhesion complexes, and small Rho GTPases, and special emphasis was placed on their relationships with caveolin-1 status. We observed that the expression integrin beta(1) and focal adhesion kinase (FAK) were increased and that the phosphorylations of FAK and paxillin, hallmarks of focal adhesion formation, were also increased in senescent human diploid fibroblast cells. Moreover, the Rho GTPases Rac1 and Cdc42 were found to be highly activated in senescent cells. In addition, focal adhesion complexes and Rho GTPases were up-regulated in the caveolin-rich membrane domain in the senescent cells. Activated Rac1 and Cdc42 directly interacted with caveolin-1 in senescent cells. Interestingly, caveolin-1 knock-out senescent cells, achieved by using small interfering RNA and antisense oligonucleotide, showed disrupted focal adhesion formation and actin stress fibers via the inactivation of FAK, which resulted in morphological adjustment to the young cell-like small spindle shape. Based on the results obtained, we propose that caveolin-1 plays an important role in senescence-associated morphological changes by regulating focal adhesion kinase activity and actin stress fiber formation in the senescent cells.

Caveolin 1↗

PubMed2002

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.

3T3 Cells↗

PubMed1992

A conserved region in human and Chinese hamster X chromosomes can induce cellular senescence of nickel-transformed Chinese hamster cell lines.

Cellular senescence is the genetically programmed cessation of cellular proliferation. We have recently mapped a putative senescence gene(s) on the X chromosome of Chinese hamster embryo (CHE) cells. In the present study, we have utilized microcell-mediated chromosome transfer (microcell fusion) to test whether: (i) the human X chromosome exhibits similar genetic potential to induce senescence and (ii) the deletion or inactivation of the X-linked senescence gene(s) in CHE cells is associated with nickel-induced immortalization. A normal CHE or human X chromosome was first introduced into mouse-cell hybrids, then transferred by microcell fusion into a nickel-transformed, immortal male CHE cell line (Ni-2/TGR) with an X deletion (Xq1). Microcell fusion of the normal CHE X chromosome into tumorigenic Ni-2/TGR cells yielded senescence of all X recipient clones. The normal human X chromosome induced dominant senescence of tumorigenic Ni-2/TGR cells in only 17% of the resulting microcell hybrids (14/81). Karyotypic analyses of 13 non-senescing human X chromosome-derived microcell hybrid clones revealed that none of these clones retained the complete X. A normal CHE X chromosome induced senescence of 75% of hybrids obtained with another immortal and tumorigenic nickel-transformed male CHE cell line (Ni-6/TGR), which exhibited no visible deletion of the X chromosome, while the normal human X chromosome, only induced senescence in 19% of these hybrids. Transfer of the normal CHE or human X chromosome into spontaneously transformed and tumorigenic cell lines, CHO/TGR or V79/TGR, had little or no effect on their growth. These data suggest that both human and CHE cells possess similar X-linked genetic activities that regulate the process of cellular senescence, and that in Chinese hamster cells nickel-induced immortalization but not that of CHO or V79 cells is associated with inactivation of an X-linked senescence gene.

Animals↗

PubMed2004

Chondrocyte senescence, joint loading and osteoarthritis.

cellular level is not completely understood, but both aging and loading-induced stresses have been shown to undermine cell functions related to the maintenance and restoration of the cartilage matrix. Based on precedents set by studies of other age-related degenerative diseases, we have focused our laboratory work on senescence as the cause of age-dependent decline in chondrocytes and on the impact of excessive mechanical stresses in promoting senescence. We hypothesized that senescent chondrocytes accumulate with age in articular cartilage and we propose that excessive mechanical stress plays a role in this process by promoting oxidative damage in chondrocytes that ultimately causes them to senesce. To test this hypothesis, we measured cell senescence markers (beta-galactosidase expression, mitotic activity, and telomere length) in human articular cartilage chondrocytes, and determined the effects of chronic exposure to oxidative stress on chondrocyte growth and senescence. In addition, we measured the effects of abnormally high levels of mechanical shear stress on the release of oxidants in cartilage explants. We found that senescent chondrocytes accumulated with age in articular cartilage. In vitro studies showed that chronic oxidative stress caused by repeated exposure to peroxide, or by growth under superphysiologic oxygen tension caused chondrocyte populations to senesce prematurely, before extensive telomere erosion occurred. Mechanical shear stress applied to cartilage explants considerably increased the production of oxidants. These observations support the hypothesis that senescence accounts for age-related decline in chondrocyte function and indicate that mechanically induced oxidative damage plays a role in this process. This suggests that new efforts to prevent the development and progression of osteoarthritis should include strategies that slow the progression of chondrocyte senescence or replace senescent cells.

Adolescent↗

PubMed2001

Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence.

In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB(-/-) tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27(KIP1) that is accompanied by an increase in p27(KIP1) specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27(KIP1) and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27(KIP1) and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21(CIP1) or p27(KIP1) induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27(KIP1) expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27(KIP1) is critical for pRb-induced senescence.

Carrier Proteins↗

PubMed2000

IP(3), IP(3) receptor, and cellular senescence.

Herein we demonstrate that replicative cellular senescence in vitro results in sharply reduced inositol 1,4,5-trisphosphate (IP(3)) receptor levels, reduced mitogen-evoked IP(3) formation and Ca(2+) release, and Ca(2+) store depletion. Human diploid fibroblasts (HDFs) underwent either 30 mean population doublings [mean population doublings (MPDs) thymidine labeling index (TI) >92% ("young") or between 53 and 58 MPDs (TI < 28%; "senescent")]. We found that the cytosolic Ca(2+) release triggered by either ionomycin or by several IP(3)-generating mitogens, namely bradykinin, thrombin, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), was attenuated markedly in senescent HDFs. Notably, the triggered cytosolic Ca(2+) transients were of a smaller magnitude in senescent HDFs. However, the response latency seen with both PDGF and EGF was greater for senescent cells. Finally, a smaller proportion of senescent HDFs showed oscillations. In parallel, IP(3) formation in response to bradykinin or EGF was also attenuated in senescent HDFs. Furthermore, senescent HDFs displayed a sharply diminished Ca(2+) release response to intracellularly applied IP(3). Finally, to compare IP(3) receptor protein levels directly in young and senescent HDFs, their microsomal membranes were probed in Western blots with a highly specific anti-IP(3) receptor antiserum, Ab(40). A approximately 260-kDa band corresponding to the IP(3) receptor protein was noted; its intensity was reduced by approximately 50% in senescent cells. Thus, we suggest that reduced IP(3) receptor expression, lowered IP(3) formation, and Ca(2+) release, as well as Ca(2+) store depletion, all contribute to the deficient Ca(2+) signaling seen in HDFs undergoing replicative senescence.

Bradykinin↗

PubMed2004

Tumor suppressor maspin is up-regulated during keratinocyte senescence, exerting a paracrine antiangiogenic activity.

Cell senescence is a physiological program of terminal growth arrest, which is believed to play an important role in cancer prevention. Senescent cells secrete multiple growth-regulatory proteins, some of which can affect tumor growth, survival, invasion, or angiogenesis. Changes in expression of different senescence-associated genes were analyzed in cultured human skin keratinocytes (KCs) that underwent replicative senescence or confluence-induced accelerated senescence. Senescent KC cultures showed a strong increase in mRNA and protein expression of maspin, a member of serine protease inhibitor family and an epithelial cell tumor suppressor with anti-invasive and antiangiogenic activities. Immunohistochemical analysis of 14 normal human skin samples (age range from 3 months to 84 years) showed that maspin is expressed by KCs in vivo and that the extent and intensity of maspin expression in the skin is significantly (P = 0.01) correlated with chronological age. Antiangiogenic activity of maspin secreted by senescent KCs was investigated in vitro by testing the effect of conditioned media from different KC cultures on endothelial cell migration in the presence or absence of several angiogenic factors. Media conditioned by senescent cultures (undergoing replicative or accelerated senescence), but not by proliferating KCs, strongly inhibited the stimulation of endothelial cell migration by all of the tested angiogenic factors. Neutralizing antibody against maspin abrogated this effect of conditioned media. These findings indicate that senescent KCs exert a paracrine antiangiogenic activity, and maspin is the principal contributor to this potentially tumor-suppressive effect of cellular senescence.

Cellular Senescence↗