Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging

Mammalian cells can respond to damage or stress by entering a state of arrested growth and altered function termed cellular senescence. Several lines of evidence suggest that the senescence response suppresses tumorigenesis. Cellular senescence is also thought to contribute to aging, but the mechanism is not well understood. We show that senescent human fibroblasts stimulate premalignant and malignant, but not normal, epithelial cells to proliferate in culture and form tumors in mice. In culture, the growth stimulation was evident when senescent cells comprised only 10% of the fibroblast population and was equally robust whether senescence was induced by replicative exhaustion, oncogenic RAS, p14(ARF), or hydrogen peroxide. Moreover, it was due at least in part to soluble and insoluble factors secreted by senescent cells. In mice, senescent, much more than presenescent, fibroblasts caused premalignant and malignant epithelial cells to form tumors. Our findings suggest that, although cellular senescence suppresses tumorigenesis early in life, it may promote cancer in aged organisms, suggesting it is an example of evolutionary antagonistic pleiotropy.     

Role of endonuclease G in senescence-associated cell death of human endothelial cells

Mitotic cells in culture show a limited replicative potential and after extended subculturing undergo a terminal growth arrest termed cellular senescence. When cells reach the senescent phenotype, this is accompanied by a significant change in the cellular phenotype and massive changes in gene expression, including the upregulation of secreted factors. In human fibroblasts, senescent cells also acquire resistance to apoptosis. In contrary, in human endothelial cells, both replicative and stress-induced premature senescence is accompanied by increased cell death; however mechanisms of cell death are poorly explored. In this communication, we addressed the role of endonuclease G (EndoG), a mitochondrial mediator of caspase-independent cell death, in senescence-associated cell death of human endothelial cells. Using immunofluorescence microscopy, we found, that EndoG is localized in the mitochondria in young cells, but relocalizes to the nucleus upon senescence. When EndoG gene expression was downregulated by lentiviral shRNA vectors, we found a significant reduction in the replicative life span and a corresponding increase in cell death. We also observed a slight shift in the cell death phenotype from necrosis to apoptosis. Together these observations suggest an important role of EndoG in the senescence program of human endothelial cells.     

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.     

细胞周期抑制因子p16可诱导人成纤维细胞发生衰老样变化

目的 探讨细胞周期抑制因子p16在细胞衰老中的作用。方法 利用逆转录病毒载体将p16基因转染入人胚肺二倍体成纤维细胞2BS中,获得高表达,检测其对2BS细胞衰老的影响。结果 与对照组细胞相比,p16基因转染后,2BS细胞生长速度下降了约50％,细胞周期阻滞于G1期,细胞对生长因子刺激的反应性下降了79．4％,细胞形态呈衰老细胞样变化。结论 p16在人二倍体成纤维细胞的衰老过程中起促进作用。     

Identification of altered expression of ADP/ATP translocase during cellular senescence in vitro

In this study, we have used the mRNA differential display technique to investigate the changes in gene expression that occur in the process of cellular aging. A number of cDNAs whose corresponding mRNAs are either increasingly or decreasingly expressed in senescent cells were thereby isolated. Through DNA sequencing, one of these differentially displayed mRNAs was identified as mitochondrial ADP/ATP translocase. The altered expression of ADP/ATP translocase in different stages of senescent fibroblasts was futher confirmed by Northern blots and semiquantitative RT-PCR. Our results demonstrate that expression of ADP/ATP translocase is progressively decreased during the process of in vitro cellular senescence. Further analyses with MTT assays indicate that the decreased expression of ADP/ATP translocase in senescent cells is in parallel with the decline of mitochondrial functions, suggesting that altered expression of this important mitochondrial enzyme might play an active role in the process of cellular senescence.     

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.     

Constitutive expression of growth-related mRNAs in proliferating and nonproliferating lung epithelial cells in primary culture: evidence for growth-dependent translational control.

We describe the control of proliferation and growth-related gene expression in primary cultures of epithelial cells derived from rat lung. Type 2 epithelial cells line the gas-exchange surface of the alveoli where they produce and secrete surfactant. When isolated from adult animals, type 2 cells do not proliferate in culture, although they have a limited ability to do so in vivo. We show that type 2 cells isolated from neonatal rats proliferate in culture and that growth can be reversibly arrested by withdrawing serum from the medium. We studied the expression of five genes whose mRNA levels fluctuate with the state of proliferation in several cell systems: the c-myc and c-Ha-ras protooncogenes and the genes encoding actin, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17), and histone 3.2. All five mRNAs were constitutively expressed at identical levels in proliferating and nonproliferating (serum deprived) neonatal cells and in adult cells. Thus, at the level of mRNA abundance, the expression of these five genes was uncoupled from the growth state of the cells. By contrast, synthesis of the replication-dependent histones and the activity of ornithine decarboxylase were detectable only in proliferating neonatal cells and not in serum-deprived neonatal cells or in adult cells. The results suggest that, in type 2 cells, growth factors might regulate the translation, rather than the mRNA abundance, of at least some growth-related genes and that the ability to respond to this translational control may be developmentally regulated.     

Subtractive hybridization of mRNA from early passage and senescent endothelial cells

Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions.     

The biological clock that measures the mitotic life-span of mouse embryo fibroblasts continues to function in the presence of simian virus 40 large tumor antigen.

Normal mammalian fibroblasts cultured in vitro undergo a limited number of divisions before entering a senescent phase in which they can be maintained for long periods but cannot be induced to divide. In rodent fibroblasts senescence can be prevented by expression of simian virus 40 large tumor antigen (T antigen). Cells expressing T antigen can proliferate indefinitely; however, such cells are absolutely dependent upon continued expression of T antigen for maintenance of growth; inactivation of T antigen results in a rapid and irreversible entry into a postmitotic state. To determine when, after the initial expression of T antigen, fibroblasts become dependent upon it for continued growth, we serially cultivated embryonic fibroblasts prepared from H-2Kb-tsA58 transgenic mice. We show that these fibroblasts become dependent upon T antigen for maintenance of proliferation only when their normal mitotic life-span has elapsed and that the biological clock that limits the mitotic potential continues to function normally, even in cells expressing this immortalizing gene. Our results suggest that random accumulation of cellular damage is unlikely to be the factor that limits fibroblast division but support the hypothesis that senescence is regulated via a genetic program.     

抑制性消减杂交筛选细胞衰老相关基因

目的 研究人胚肺二倍体成纤维细胞2BS在衰老过程中的基因表达变化。方法 对年轻2BS细胞[低于30PD(population doubling)]和衰老2BS细胞(高于55PD)样品进行双向抑制性消减杂交，并筛选分析。结果 构建了2个抑制性消减文库，分别代表年轻细胞和衰老细胞高表达基因，点杂交筛选获得30个差异表达片断(差异比例大于2)包含多种细胞功能的变化。部分差异基因表达在新生儿脐带血和老年人外周血样中差异也有显著性。结论首次报道了RBM4、FBXO7、TOM1等基因在2BS细胞衰老时的表达变化，并表明体外培养细胞与个体的衰老变化过程有一定相似性。     

Cellular senescence: from growth arrest to immunogenic conversion

Cellular senescence was first reported in human fibroblasts as a state of stable in vitro growth arrest following extended culture. Since that initial observation, a variety of other phenotypic characteristics have been shown to co-associate with irreversible cell cycle exit in senescent fibroblasts. These include (1) a pro-inflammatory secretory response, (2) the up-regulation of immune ligands, (3) altered responses to apoptotic stimuli and (4) promiscuous gene expression (stochastic activation of genes possibly as a result of chromatin remodeling). Many features associated with senescent fibroblasts appear to promote conversion to an immunogenic phenotype that facilitates self-elimination by the immune system. Pro-inflammatory cytokines can attract and activate immune cells, the presentation of membrane bound immune ligands allows for specific recognition and promiscuous gene expression may function to generate an array of tissue restricted proteins that could subsequently be processed into peptides for presentation via MHC molecules. However, the phenotypes of senescent cells from different tissues and species are often assumed to be broadly similar to those seen in senescent human fibroblasts, but the data show a more complex picture in which the growth arrest mechanism, tissue of origin and species can all radically modulate this basic pattern. Furthermore, well-established triggers of cell senescence are often associated with a DNA damage response (DDR), but this may not be a universal feature of senescent cells. As such, we discuss the role of DNA damage in regulating an immunogenic response in senescent cells, in addition to discussing less established “atypical” senescent states that may occur independent of DNA damage.     

Effects of human Na(+)/dicarboxylate cotransporter 3 on the replicative senescence of human embryonic lung diploid fibroblasts

To investigate the role of human Na(+)/dicarboxylate cotransporter 3 (hNaDC3) in the replicative senescence of normal human embryonic lung diploid fibroblasts (WI-38), a retroviral vector containing hNaDC3 was constructed. hNaDC3 was introduced into normal WI-38 cells through infection with the retroviral virus. Monoclones were selected with G418. The integration and expression of exotic genes were confirmed by Northern blot and Western blot. When compared with the control cells, WI-38 cells transfected with hNaDC3 cDNA showed significant suppression of growth rate (by 40%), increase of positive rate of SA-beta-gal staining, decrease of mitochondrial membrane potential, shortening of telomere length, and increase of P16 and P21 expression. The morphology characteristics of senescent fibroblasts appeared earlier. Our results have, for the first time, demonstrated that high expression of hNaDC3 may be able to, at least partly, promote the cellular senescence of human diploid fibroblasts.     

Up-regulation of S100C in normal human fibroblasts in the process of aging in vitro

S100 proteins belonging to the EF-hand Ca(2+)-binding protein family regulate a variety of cellular processes via interaction with different target proteins. Several diseases, including cancer and Alzheimer's disease, are related to a disorder of multifunctional S100 proteins, which are expressed in cell- and tissue-specific manners. We previously demonstrated that S100C could move to and accumulate in the nuclei of normal human fibroblasts but not in the nuclei of immortalized and neoplastic cells. In addition, we found that its nuclear accumulation resulted in suppression of DNA synthesis in normal cells at a confluent stage. In the present study, we investigated whether S100C was associated with cellular senescence in vitro. We found that S100C expression increased in normal human fibroblasts in the process of aging in culture and was accompanied by accumulation of its protein in the nuclei of senescent fibroblasts. In addition, the nuclear accumulation of S100C increased expression of a cyclin-dependent kinase inhibitor p21(Sdi1), a strong inhibitor of cell growth. These findings suggest that an increase in the cells having nuclear accumulation of S100C is closely related to the process of cellular senescence of normal human fibroblasts.     

Is increased arachidonic acid release a cause or a consequence of replicative senescence?

Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation. During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells. To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis. Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells. Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells. Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis. Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis. So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging.     

Polyamine metabolism and cell-cycle-dependent gene expression in IMR-90 human diploid fibroblasts during senescence in culture

Aging of IMR-90 human diploid fibroblasts in culture is accompanied by specific changes of polyamine metabolism including: (a) a fivefold decrease of serum-induced activity of ornithine decarboxylase (ODC¹ EC 4.1.1.17); (b) a six to tenfold increase of polyamine catabolism; and (c) a reduction of putrescine uptake. These changes apparently led to a significant reduction of putrescine accumulation in senescent cells following serum stimulation. Since the induction of ODC is a mid-G₁ event, the change of polyamine metabolism may be related to changes of expression of other cell-cycle-dependent genes during cellular aging. In addition to ODC gene, we have examined the expression of two early G₁ genes, c-erbB and c-myc, and one late G₁/S gene thymidine kinase, at mRNA levels, in both young and old IMR-90 cells. We have also compared the enzyme activities of two late G₁/S genes, thymidine kinase and thymidylate synthetase, in young and old cells following serum stimulation. We did not observe significant changes of c-erbB, c-myc, and ODC mRNA levels during cellular senescence. However, we found that serum-induced mRNA level of thymidine kinase gene in old IMR-90 cells was significantly reduced compared to that in the young cells. Results also demonstrate that aging of IMR-90 cells was accompanied by significant decrease of both thymidine kinase and thymidylate synthetase activities. In view of the recognized importance of polyamines in growth regulation, it is possible that alteration of polyamine metabolism may contribute to the impairment of expression of some key G₁/S genes and such impairment may contribute to the ultimate loss of dividing potential in senescent cells.     

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.     

Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis

Human ageing is characterized by a progressive loss of physiological functions, increased tissue damage and defects in various tissue renewal systems. Age-related decreases of the cellular replicative capacity can be reproduced by in vitro assays of cellular ageing. When diploid human fibroblasts reach their finite lifespan, they enter an irreversible G1 growth arrest status referred to as replicative senescence. While deregulation of programmed cell death (apoptosis) is a key feature of age-related pathology in several tissues, this is not reflected in the standard in vitro senescence model of human fibroblasts, and the role of apoptosis during cellular ageing remains unclear. We have analyzed replicative senescence of human umbilical vein endothelial cells (HUVEC) in vitro and found that senescent HUVEC also arrest in the G1 phase of the cell cycle but, unlike fibroblasts, accumulate with a 4N DNA content, indicative of polyploidization. In contrast to human fibroblasts, senescent endothelial cells display a considerable increase in spontaneous apoptosis. The data imply that age-dependent apoptosis is a regular feature of human endothelial cells and suggest cell type specific differences in human ageing.     

Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro

Widespread application of beta-cell replacement strategies for diabetes is dependent upon the availability of an unlimited supply of cells exhibiting appropriate glucose-responsive insulin secretion. Therefore, a great deal of effort has been focused on understanding the factors that control beta-cell growth. Previously, we found that human beta-cell-enriched islet cultures can be stimulated to proliferate, but expansion was limited by growth arrest after 10-15 cell divisions. Here, we have investigated the mechanism behind the growth arrest. Our studies, including analyses of the expression of senescence-associated beta-galactosidase, p16(INK4a) levels, and telomere lengths, indicate that cellular senescence is responsible for limiting the number of cell divisions that human beta-cells can undergo. The senescent phenotype was not prevented by retroviral transduction of the hTERT gene, although telomerase activity was induced. These results have implications for the use of primary human islet cells in cell transplantation therapies for diabetes.     

Identification of Hsc70 as target for AGE modification in senescent human fibroblasts

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bona fide target for AGE modification in human fibroblasts.