Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated beta-galactosidase (SA-beta-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-beta-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.     

Hallmarks for senescence in carcinogenesis: novel signaling players

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.     

Dual roles of telomere dysfunction in initiation and suppression of tumorigenesis

Human carcinomas arise through the acquisition of genetic changes that endow precursor cancer cells with a critical threshold of cancer-relevant genetic lesions. This complex genomic alterations confer upon precursor cancer cells the ability to grow indefinitely and to metastasize to distant sites. One important mechanism underlying a cell's tumorigenic potential is the status of its telomere. Telomeres are G-rich simple repeat sequences that serve to prevent chromosomal ends from being recognized as DNA double-strand breaks (DSBs). Dysfunctional telomeres resemble DSBs, leading to the formation of dicentric chromosomes that fuel high degrees of genomic instability. In the setting of an intact p53 pathway, this instability promotes cellular senescence, a potent tumor suppressor mechanism. However, rare cells that stochastically lose p53 function emerge from this sea of genomic instability and progress towards cancer. In this review, we describe the use of mouse models to probe the impact of dysfunctional telomeres on tumor initiation and suppression.     

Rplp1 bypasses replicative senescence and contributes to transformation

To determine whether genes expressed by embryonic stem cells have a proliferative effect in primary cells, primary mouse embryonic fibroblasts were infected with an ES cell cDNA library. This led to identification of the ribosomal protein, Rplp1, a member of the P group of ribosomal proteins, whose putative role for bypassing replicative senescence in MEFs was investigated. Our results show that Rplp1 produces a two-fold increase in the expression of an E2F1 promoter and upregulation of cyclin E in MEFs. Therefore, this study is the first to show that overexpression of a single ribosomal protein, Rplp1, is a cause and not a consequence of cell proliferation. In addition, co-expression of Rplp1 with mutant ras^Val12 contributed to transformation in NIH3T3 cells, as was evidenced by colony production in soft-agar assays. Moreover, the Rplp1 protein was upregulated in MEFs and NIH3T3 cells upon expression of a p53 dominant negative mutant gene designated p53R175H. Hence, mutation of p53 may facilitate immortalization in vitro by upregulating Rplp1. Lastly, Rplp1 mRNA was found to be upregulated in 16 of 26 human colon cancer biopsy specimens, a finding that may be of relevance to cancer research.     

Genetic instability and the quasispecies model

Genetic instability is a defining characteristic of cancers. Microsatellite instability (MIN) leads to by elevated point mutation rates, whereas chromosomal instability (CIN) refers to increased rates of losing or gaining whole chromosomes or parts of chromosomes during cell division. CIN and MIN are, in general, mutually exclusive. The quasispecies model is a very successful theoretical framework for the study of evolution at high mutation rates. It predicts the existence of an experimentally verified error catastrophe. This catastrophe occurs when the mutation rates exceed a threshold value, the error threshold, above which replicative infidelity is incompatible with cell survival. We analyse the semiconservative quasispecies model of both MIN and CIN tumors. We consider the role of post-methylation DNA repair in tumor cells and demonstrate that DNA repair is fundamental to the nature of the error catastrophe in both types of tumors. We find that CIN introduces a plateau in the maximum viable mutation rate for a repair-free model, which does not exist in the case of MIN. This provides a plausible explanation for the mutual exclusivity of CIN and MIN.     

Increase in mitochondrial mass in human fibroblasts under oxidative stress and during replicative cell senescence

Abnormal proliferation of mitochondria generally occurs in muscle of aged individuals and patients with mitochondrial myopathy. An increase in the mitochondrial DNA (mtDNA) copy number has also been observed in aging human tissues. However, the molecular mechanism underlying the increase in mitochondrial mass and mtDNA is still unclear. In a previous study, we demonstrated that sublethal levels of oxidative stress caused an increase in mitochondrial mass in human lung cells. In this communication, we report our recent findings that the mitochondrial mass in human lung fibroblasts (MRC-5) in a later proliferation stage is significantly increased compared to that in the early stages of proliferation. The extent of the increase in mitochondrial mass in the senescent cells was similar to that in cells in the early stages of proliferation that had been treated with low concentrations ( 180 µM) of hydrogen peroxide (H₂O₂). Moreover, we found that the rate of reactive oxygen species (ROS) production was higher in cells in the later proliferation stage compared to cells in the early proliferation stages. A similar phenomenon was also observed in cells in the early proliferation stages under low levels of oxidative stress. On the other hand, the mRNA levels of many nuclear DNA-encoded proteins involved in mitochondrial biogenesis, particularly nuclear respiratory factor-1, were found to increase in cells in later proliferation stages and in cells in early proliferation stages that had been treated with 180 µM H₂O₂. Interestingly, the increase in mitochondrial mass in the cells under oxidative stress could be repressed by treatment with cycloheximide orm-chlorocarbonyl cyanide phenylhydrazone but not by chloramphenicol. Furthermore, the mitochondrial mass of mtDNA-less ° cells was also significantly increased by exposure to low concentrations (e.g. 180 µM) of H₂O₂. These results suggest that the increase in mitochondrial mass in replicative senescent cells may result from an increase in ROS production, and that it is dependent on both de novo synthesis of nuclear DNA-encoded proteins and their import into mitochondria, dictated by the membrane potential of mitochondria.     

High glucose-induced replicative senescence: point of no return and effect of telomerase

Primary human cells enter senescence after a characteristic number of population doublings (PDs). In the current study, human skin fibroblasts were propagated in culture under 5.5mM glucose (normoglycemia); addition of 16.5mM D-glucose to a concentration of 22 mM (hyperglycemia); and addition of 16.5mM L-glucose (osmotic control). Hyperglycemia induced premature replicative senescence after 44.42+/-1.5 PDs compared to 57.9+/-3.83 PDs under normoglycemia (p<0.0001). L-Glucose had no effect, suggesting that the effect of hyperglycemia was not attributed to hyperosmolarity. Activated caspase-3 measurement showed a significantly higher percentage of apoptotic cells in high glucose medium. Telomerase overexpression circumvented the effects of hyperglycemia on replicative capacity and apoptosis. The "point of no return," beyond which hyperglycemia resulted in irreversible progression to premature replicative senescence, occurred after exposure to hyperglycemia for as few as 20 PDs. These results may provide a biochemical basis for the relationship between hyperglycemia and those complications of diabetes, which are reminiscent of accelerated senescence.     

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes

The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.     

Pathways connecting telomeres and p53 in senescence, apoptosis, and cancer

The ends of eukaryotic chromosomes are protected by specialized structures termed telomeres that serve in part to prevent the chromosome end from activating a DNA damage response. However, this important function for telomeres in chromosome end protection can be lost as telomeres shorten with cell division in culture or in self-renewing tissues with advancing age. Impaired telomere function leads to induction of a DNA damage response and activation of the tumor suppressor protein p53. p53 serves a critical role in enforcing both senescence and apoptotic responses to dysfunctional telomeres. Loss of p53 creates a permissive environment in which critically short telomeres are inappropriately joined to generate chromosomal end-to-end fusions. These fused chromosomes result in cycles of chromosome fusion-bridge-breakage, which can fuel cancer initiation, especially in epithelial tissues, by facilitating changes in gene copy number.     

Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.     

Molecular chaperones regulate p53 and suppress senescence programs

Many types of cancer cells constitutively express major molecular chaperones at high levels. Recent findings demonstrate that specific depletion of individual chaperones, including various members of the Hsp70 family, small heat shock proteins, or VCP/p97, leads to activation of p53 pathway and subsequently triggers cellular senescence. Here, we discuss a possibility that in cancer cells high levels of chaperones serve to keep the p53 signaling under control, thus allowing cancer cells to evade the default senescence and form tumors.     

Model of the developing tumorigenic phenotype in mammalian cells and the roles of sustained stress and replicative senescence

The molecular mechanisms that drive mammalian cells to the development of cancer are the subject of intense biochemical, genetic and medical studies. But for the present, there is no comprehensive model that might serve as a general framework for the interpretation of experimental data. This paper is an attempt to create a conceptual model of the mechanism of the developing tumorigenic phenotype in mammalian cells, defined as having high genomic instability and proliferative activity. The basic statement in the model is that mutations acquired by tumor cells are not caused directly by external DNA damaging agents, but instead are produced by the cell itself as an output of a Mutator Response similar to the bacterial "SOS response" and characterized by the initiation of error-prone cell cycle progression and an elevated rate of mutation. This response may be induced in arrested mammalian cells by intracellular and extracellular proliferative signals combined with blocked apoptosis. The mutant cells originated by this response are subjected to natural selection via apoptosis and turnover. This selection process favors the survival of cells with high proliferative activity and the suppression of apoptosis resulting in the long run in the appearance of immortalized cells with high proliferative activity. Either a sustained stressful environment accompanied by continuing apoptotic cell death, or replicative senescence, provides conditions suitable for activation of the Mutator Response, namely the emergence of arrested cells with blocked apoptosis and the induction of proliferative signal. It also accelerates the selection process by providing continuing cell turnover. The proposed mechanism is described at the level of involved metabolic pathways and proteins and substantiated by the related experimental data available in the literature.     

Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21WAF1, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.     

Identification of 30 protein species involved in replicative senescence and stress-induced premature senescence

Exposure of human proliferative cells to subcytotoxic stress triggers stress-induced premature senescence (SIPS) which is characterized by many biomarkers of replicative senescence. Proteomic comparison of replicative senescence and stress-induced premature senescence indicates that, at the level of protein expression, stress-induced premature senescence and replicative senescence are different phenotypes sharing however similarities. In this study, we identified 30 proteins showing changes of expression level specific or common to replicative senescence and/or stress-induced premature senescence. These changes affect different cell functions, including energy metabolism, defense systems, maintenance of the redox potential, cell morphology and transduction pathways.     

On the proportion of cancer stem cells in a tumour

It is now generally accepted that cancers contain a sub-population, the cancer stem cells (CSCs), which initiate and drive a tumour’s growth. At least until recently it has been widely assumed that only a small proportion of the cells in a tumour are CSCs. Here we use a mathematical model, supported by experimental evidence, to show that such an assumption is unwarranted. We show that CSCs may comprise any possible proportion of the tumour, and that the higher the proportion the more aggressive the tumour is likely to be.     

Protein maintenance in aging and replicative senescence: a role for the peptide methionine sulfoxide reductases

Cellular aging is characterized by the build-up of oxidatively modified protein that results, at least in part, from impaired redox homeostasis associated with the aging process. Protein degradation and repair are critical for eliminating oxidized proteins from the cell. Oxidized protein degradation is mainly achieved by the proteasomal system and it is now well established that proteasomal function is generally impaired with age. Specific enzymatic systems have been identified which catalyze the regeneration of cysteine and methionine following oxidation within proteins. Protein-bound methionine sulfoxide diastereoisomers S and R are repaired by the combined action of the enzymes MsrA and MsrB that are subsequently regenerated by thioredoxin/thioredoxin reductase. Importantly, the peptide methionine sulfoxide reductase system has been implicated in increased longevity and resistance to oxidative stress in different cell types and model organisms. In a previous study, we reported that peptide methionine sulfoxide reductase activity as well as gene and protein expression of MsrA are decreased in various organs as a function of age. More recently, we have shown that gene expression of both MsrA and MsrB2 (Cbs-1) is decreased during replicative senescence of WI-38 fibroblasts, and this decline is associated with an alteration in catalytic activity and the accumulation of oxidized protein. In this review, we will address the importance of protein maintenance in the aging process as well as in replicative senescence, with a special focus on regulation of the peptide methionine sulfoxide reductase systems.     

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining beta-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.     

On the role of endothelial progenitor cells in tumor neovascularization

The exact role that bone marrow (BM)-derived endothelial progenitor cells (EPCs) play in tumor neovascularization is heavily debated. We develop a quantitative three-compartment model with predictive power regarding the dynamics of tumorigenesis. There are two distinct processes by which tumor neovasculature can be built: angiogenesis is the formation of new blood vessels from preexisting vessels; vasculogenesis is the formation of new vessels by recruiting circulating EPCs. We show that vasculogenesis-driven and angiogenesis-driven tumors grow in different ways. (i) If angiogenesis is the prevailing process, then the tumor mass (and volume) will grow as a cubic power of time, and BM-derived EPCs will stay at a constant level. (ii) If vasculogenesis is the dominant process, then the tumor mass will be characterized by a linear growth in time, and the number of circulating EPCs (after possibly increasing to a maximum) will decrease to low levels. With this information, one can identify the signature of each of the processes in the observations of tumor growth and the dynamics of the relevant characteristics, such as the level of BM-derived EPCs. We show how our results can help explain some apparently contradictory experimental data. We also propose ways to couple this study with directed experiments to identify the exact role of vasculogenesis in tumor progression.