Different telomere damage signaling pathways in human and mouse cells

Programmed telomere shortening in human somatic cells is thought to act as a tumor suppressor pathway, limiting the replicative potential of developing tumor cells. Critically short human telomeres induce senescence either by activating p53 or by inducing the p16/RB pathway, and suppression of both pathways is required to suppress senescence of aged human cells. Here we report that removal of TRF2 from human telomeres and the ensuing de-protection of chromosome ends induced immediate premature senescence. Although the telomeric tracts remained intact, the TRF2(DeltaBDeltaM)-induced premature senescence was indistinguishable from replicative senescence and could be mediated by either the p53 or the p16/RB pathway. Telomere de-protection also induced a growth arrest and senescent morphology in mouse cells. However, in this setting the loss of p53 function was sufficient to completely abrogate the arrest, indicating that the p16/RB response to telomere dysfunction is not active in mouse cells. These findings reveal a fundamental difference in telomere damage signaling in human and mouse cells that bears on the use of mouse models for the telomere tumor suppressor pathway.     

Suppression of human tumor cell proliferation by Smurf2-induced senescence

The limitation of proliferative potential in human somatic cells imposed by replicative senescence has been proposed as a mechanism of tumor suppression. The E3 ubiquitin ligase Smurf2 is up-regulated during replicative senescence in response to telomere shortening, and induces senescence when expressed adventitiously in early passage or telomerase-immortalized human fibroblasts. To investigate the generality of Smurf2's control of cell proliferation, we have studied the effects of Smurf2 up-regulation on cell proliferation in early passage human mammary epithelial cells which normally do not show elevated expression of Smurf2 during senescence, and in 16 human cancer cell lines derived from both sarcomas and carcinomas. Here we report that Smurf2 up-regulation induced senescence in a wide variety of human cell types, including highly neoplastic cell lines. Consistent with our previous findings, the ability of Smurf2 to arrest cell proliferation did not require its ubiquitin ligase activity. Furthermore, expression of the cyclin-dependent kinase inhibitor p21 was increased in tumor cells undergoing Smurf2-induced senescence, and such increase occurred independently of the transactivation function of p53. Our results, which reveal a previously unsuspected tumor suppression function for Smurf2-induced senescence, suggest that modulation of Smurf2 action may be a useful strategy for inhibition of cancer cell growth.     

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-κB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-κB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65^−/− murine fibroblasts immortalize at considerably faster rates than RelA/p65^+/+ cells. The ability of RelA/p65^−/− fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65^−/− fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-κB. Altogether, our findings present a fresh perspective on the role of NF-κB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.     

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.     

The M‐type receptor PLA2R regulates senescence through the p53 pathway

Senescence is a stable proliferative arrest induced by various stresses such as telomere erosion, oncogenic or oxidative stress. Compelling evidence suggests that it acts as a barrier against tumour development. Describing new mechanisms that favour an escape from senescence can thus reveal new insights into tumorigenesis. To identify new genes controlling the senescence programme, we performed a loss‐of‐function genetic screen in primary human fibroblasts. We report that knockdown of the M‐type receptor PLA2R (phospholipase A2 receptor) prevents the onset of replicative senescence and diminishes stress‐induced senescence. Interestingly, expression of PLA2R increases during replicative senescence, and its ectopic expression results in premature senescence. We show that PLA2R regulates senescence in a reactive oxygen species–DNA damage–p53‐dependent manner. Taken together, our study identifies PLA2R as a potential new tumour suppressor gene crucial in the induction of cellular senescence through the activation of the p53 pathway.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.     

The specific role of pRb in p16INK4A-mediated arrest of normal and malignant human breast cells

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G₁ was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.Key words: breast cancer, senescence, retinoblastoma, p130, p107     

Direct evidence from siRNA-directed "knock down" that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation

The selective pressure for disruption of the cyclin-dependent kinase inhibitor p16(INK4a) in human cancer has been postulated to reflect its role in mediating growth arrest, both in response to telomere erosion (replicative senescence) and to oncogene-induced and other "stress" signals. Given the known species-specific differences in regulation of senescence, we have tested this hypothesis in human, as opposed to rodent, cells by designing a small interfering RNA (siRNA) to knock down p16(INK4a) expression. Transfection of this siRNA into late-passage normal human diploid fibroblasts allowed at least temporary escape from entry into replicative senescence. Furthermore, in our in vitro model of early-stage, RAS-induced thyroid tumorigenesis, sequential transfections with this siRNA allowed outgrowth of small clusters of proliferating epithelial cells, consistent with escape from the spontaneous "senescence", which normally curtails their proliferative response to mutant RAS. These data provide the first direct evidence that p16(INK4a) is necessary for the initiation of both telomere-dependent and telomere-independent senescence in human cells.     

Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence

p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.     

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

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

Cellular senescence induced by cathepsin X downregulation

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker β-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.     

Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts.

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.     

miR-22 represses cancer progression by inducing cellular senescence

Cellular senescence acts as a barrier to cancer progression, and microRNAs (miRNAs) are thought to be potential senescence regulators. However, whether senescence-associated miRNAs (SA-miRNAs) contribute to tumor suppression remains unknown. Here, we report that miR-22, a novel SA-miRNA, has an impact on tumorigenesis. miR-22 is up-regulated in human senescent fibroblasts and epithelial cells but down-regulated in various cancer cell lines. miR-22 overexpression induces growth suppression and acquisition of a senescent phenotype in human normal and cancer cells. miR-22 knockdown in presenescent fibroblasts decreased cell size, and cells became more compact. miR-22-induced senescence also decreases cell motility and inhibits cell invasion in vitro. Synthetic miR-22 delivery suppresses tumor growth and metastasis in vivo by inducing cellular senescence in a mouse model of breast carcinoma. We confirmed that CDK6, SIRT1, and Sp1, genes involved in the senescence program, are direct targets of miR-22. Our study provides the first evidence that miR-22 restores the cellular senescence program in cancer cells and acts as a tumor suppressor.     

Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16(INK4a) cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16(INK4a)-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT(+) keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16(INK4a) expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16(INK4a)-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems.     

Signaling pathway requirements for induction of senescence by telomere homolog oligonucleotides

Cellular senescence is a major defense against cancer. In human fibroblasts, suppressing both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence induced by ectopic expression of a dominant negative form of the telomere repeat binding factor 2, TRF2(DN). We recently reported that exposure to oligonucleotides homologous to the telomere 3' overhang (T-oligos) activates both the p53 and pRb pathways and leads to senescence in primary human fibroblasts. To further characterize T-oligo-induced senescence, we compared established isogenic fibroblast cell lines lacking functional p53 and/or pRb pathways to the normal parental line. Here, we report that, as in physiologic senescence, inactivation of both the p53 and pRb pathways is necessary to suppress T-oligo-induced senescence. Moreover, T-oligo rapidly induces senescence in a malignant fibroblast-derived cell line, demonstrating the potential of using T-oligo as a novel anticancer therapeutic. Our data support the hypothesis that exposure of the TTAGGG tandem repeat telomere 3' overhang sequence is the event that initiates signaling through DNA damage response pathways after experimental telomere disruption, serial passage, or acute genomic damage of normal cells.     

dNTP metabolism links mechanical cues and YAP/TAZ to cell growth and oncogene‐induced senescence

YAP/TAZ, downstream transducers of the Hippo pathway, are powerful regulators of cancer growth. How these factors control proliferation remains poorly defined. Here, we found that YAP/TAZ directly regulate expression of key enzymes involved in deoxynucleotide biosynthesis and maintain dNTP precursor pools in human cancer cells. Regulation of deoxynucleotide metabolism is required for YAP‐induced cell growth and underlies the resistance of YAP‐addicted cells to chemotherapeutics targeting dNTP synthesis. During RAS‐induced senescence, YAP/TAZ bypass RAS‐mediated inhibition of nucleotide metabolism and control senescence. Endogenous YAP/TAZ targets and signatures are inhibited by RAS/MEK1 during senescence, and depletion of YAP/TAZ is sufficient to cause senescence‐associated phenotypes, suggesting a role for YAP/TAZ in suppression of senescence. Finally, mechanical cues, such as ECM stiffness and cell geometry, regulate senescence in a YAP‐dependent manner. This study indicates that YAP/TAZ couples cell proliferation with a metabolism suited for DNA replication and facilitates escape from oncogene‐induced senescence. We speculate that this activity might be relevant during the initial phases of tumour progression or during experimental stem cell reprogramming induced by YAP.     

Quantitative analysis of cellular senescence phenotypes using an imaging cytometer

Until now, various stimuli as well as serial passaging have been known to induce cellular senescence in normal human diploid fibroblasts. However, in many cases, we have encountered difficulty in quantitatively analyzing the cellular senescence phenotypes of senescent cells in a physiological condition. High-content screening (HCS)-based image analysis is becoming an important and powerful research tool. In the present study, an automated and quantitative cellular image-analysis system was employed to quantify the cellular senescence phenotypes induced in normal human diploid fibroblasts, TIG-1 cells, and found to be a powerful tool in the cellular senescence study.     

Nek6 overexpression antagonizes p53-induced senescence in human cancer cells

Nek6 is an NIMA-related kinase that plays a critical role in mitotic cell cycle progression. Recent studies have shown that Nek6 is upregulated in various human cancers, but the function of Nek6 in tumorigenesis is largely unknown. Here, we examined the role of Nek6 in cellular senescence. Our data revealed that Nek6 expression is decreased both in both the replicative senescence of human normal fibroblasts and premature senescence induced by p53 expression in EJ human bladder cancer cells and H1299 human lung cancer cells. Interestingly, the enforced expression of Nek6 in EJ and H1299 cells completely suppresses p53-induced senescence, whereas the expression of kinase-dead Nek6 did not affect p53-induced senescence. Mechanistic studies revealed that cell cycle arrest in the G1 and G2/M phases, as well as the reduction of cyclin B and cdc2 protein level upon p53 expression were significantly reduced by Nek6 overexpression. In addition, p53-induced increases in intracellular levels of ROS were also inhibited in cells overexpressing Nek6. These results suggest that the downregulation of Nek6 expression is required for the onset of p53-induced cellular senescence and imply a possible role of Nek6 in tumorigenesis.     

DNA damage checkpoint kinase Chk2 triggers replicative senescence

Telomere shortening in normal human cells causes replicative senescence, a p53-dependent growth arrest state, which is thought to represent an innate defence against tumour progression. However, although it has been postulated that critical telomere loss generates a 'DNA damage' signal, the signalling pathway(s) that alerts cells to short dysfunctional telomeres remains only partially defined. We show that senescence in human fibroblasts is associated with focal accumulation of gamma-H2AX and phosphorylation of Chk2, known mediators of the ataxia-telangiectasia mutated regulated signalling pathway activated by DNA double-strand breaks. Both these responses increased in cells grown beyond senescence through inactivation of p53 and pRb, indicating that they are driven by continued cell division and not a consequence of senescence. gamma-H2AX (though not Chk2) was shown to associate directly with telomeric DNA. Furthermore, inactivation of Chk2 in human fibroblasts led to a fall in p21(waf1) expression and an extension of proliferative lifespan, consistent with failure to activate p53. Thus, Chk2 forms an essential component of a common pathway signalling cell cycle arrest in response to both telomere erosion and DNA damage.