Expression profiles of p53-, p16(INK4a)-, and telomere-regulating genes in replicative senescent primary human, mouse, and chicken fibroblast cells.

Replicative senescence is known to be an intrinsic mechanism in determining the finite life span of in vitro cultured cells. Since this process is recognized as an evolutionarily conserved mechanism from yeast to mammalian cells, we compared the senescence-associated genetic alterations in the p53, p16(INK4a), and telomere regulatory pathways using replicative senescent human, mouse, and chicken fibroblast cells. Normal human diploid fibroblast (HDF; WI38) and chicken embryonic fibroblast (CEF) cells were shown to have a more extended in vitro proliferative potential than their mouse embryonic fibroblast (MEF) counterpart. In contrast to the HDF and CEF cells, MEF cells were shown to express telomerase mRNA and maintain telomerase activity throughout their in vitro life span. Functional p53 activity was shown to increase in the replicative senescent HDF and CEF cells, but not in replicative senescent MEF cells. On the other hand, there was a gradual elevation of p16(INK4a) expression with increased cell passages which reached a maximum in replicative senescent MEF cells. Taken together, the present study demonstrates that the p53, p16(INK4a), and telomere regulatory functions may be differentially regulated during replicative senescence in human, mouse, and chicken fibroblast cells.     

Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.     

CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.     

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.     

Significant role for p16INK4a in p53-independent telomere-directed senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

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.     

Significant Role for p16 in p53-Independent Telomere-Directed Senescence

Telomere attrition in primary human fibroblasts induces replicative senescence accompanied by activation of the p53 and p16(INK4a)/RB tumor suppressor pathways. Although the contribution of p53 and its target, p21, to telomere-driven senescence have been well established, the role of p16(INK4a) is controversial. Attempts to dissect the significance of p16(INK4a) in response to telomere shortening have been hampered by the concomitant induction of p16(INK4a) by cell culture conditions. To circumvent this problem, we studied the role of p16(INK4a) in the cellular response to acute telomere damage induced by a dominant negative allele of TRF2, TRF2(Delta B Delta M). This approach avoids the confounding aspects of culture stress because parallel cultures with and without telomere damage can be compared. Telomere damage generated with TRF2(Delta B Delta M) resulted in induction of p16(INK4a) in the majority of cells as detected by immunohistochemistry. Inhibition of p16(INK4a) with shRNA or overexpression of BMI1 had a significant effect on the telomere damage response in p53-deficient cells. While p53 deficiency alone only partially abrogated the telomere damage-induced cell cycle arrest, combined inhibition of p16(INK4a) and p53 led to nearly complete bypass of telomere-directed senescence. We conclude that p16(INK4a) contributes to the p53-independent response to telomere damage.     

Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype

Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16(INK4a), a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16(INK4a) and another cyclin-dependent kinase inhibitor, p21(CIP1/WAF1), induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16(INK4a). Cells induced to senesce by ectopic p16(INK4a) expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16(INK4a) by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16(INK4a) in suppressing the SASP. These findings suggest that p16(INK4a)-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16(INK4a) activation or senescence per se, but rather is a damage response that is separable from the growth arrest.     

Mechanisms that limit the in vitro proliferative potential of human CD8+ T lymphocytes

Human T lymphocytes can be numerically expanded in vitro only to a limited extent. The cyclin-dependent kinase inhibitor p16(INK4a) is essential in the control of cellular proliferation, and its expression, in epithelial cells, is associated with irreversible growth arrest. Using long-term cultured CD8+ T lymphocytes, we have investigated the role of the p16/pRb pathway in the regulation of T cell proliferation and senescence. In this study, we describe at least two mechanisms that cause replicative growth arrest in cultured lymphocytes. The first one depends on the expression of p16(INK4a) and is directly responsible for the exit of a significant proportion of CD8+ T cells from the proliferative population. This induced p16 expression pattern is observed during each round of mitogen stimulation and is not related to activation-induced cell death. Importantly, knocking down p16(INK4a) expression allows increased proliferation of T cells. The second one is a phenomenon that resembles human fibroblast senescence, but is independent of p16(INK4a) and of telomere attrition. Interestingly, virtually all pRb proteins in the senescent population are found in the active form. Our data indicate that newly synthesized p16(INK4a) limits the proliferation of T lymphocytes that respond to mitogen, but is not required for the loss of mitogen responsiveness called senescence.     

Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock

We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21(WAF1) and p16(INK4A), but not p14(ARF), in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21(WAF) genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16(INK4A) (but not p14(ARF)) were also resistant to SDB-induced senescence, suggesting that the p16(INK4A)/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53-/- mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.     

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.     

Retinoic acid extends the in vitro life span of normal human oral keratinocytes by decreasing p16(INK4A) expression and maintaining telomerase activity

Retinoic acid (RA) plays an important role in the regulation of cell growth and differentiation. To investigate whether RA extends in vitro the life span of human epithelial cells, we examined the effect of all-trans RA on both the cumulative population-doubling level (PDL) and the replicative senescence of cultured oral keratinocytes. When proliferating oral keratinocytes were cultured in medium containing 1 nM of all-trans RA, the in vitro life span of the cells was increased 1.5- to 1.8-fold compared to the vehicle control and the replicative senescence of the cells was significantly inhibited. Since the replicative senescence of human epithelial cells is associated with a steady increase of p16(INK4A) and a loss of telomerase activity, we expected that RA could delay the replicative senescence of oral keratinocytes by decreasing p16(INK4A) expression and/or inhibiting the loss of telomerase activity. To test this possibility, we examined the expression of replicative senescence-associated genes and the telomerase activities of different PDL numbers of oral keratinocytes exposed to 1 nM of all-trans RA. The protein level of cellular p16(INK4A) in the RA-treated oral keratinocytes was gradually but significantly enhanced by an increased PDL number; however, the level was significantly lower than that of the vehicle control at all of the same PDL numbers. In contrast, the telomerase activity was maintained in oral keratinocytes with increasing PDL numbers induced by RA treatment. Summarizing, these results indicate that RA induces the in vitro life-span extension of oral keratinocytes, which is linked to a decreased cellular level of p16(INK4A) and the maintenance of telomerase activity.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.     

Senescence: a telomeric limit to immortality or a cellular response to physiologic stresses?

Cells entering a state of senescence undergo a irreversible cell cycle arrest, associated by a set of functional and morphological changes. Senescence occurs following telomeres shortening (replicative senescence) or exposure to other acute or chronic physiologic stress signals (a phenomenon termed stasis: stress or aberrant signaling-induced senescence). In this review, I discuss the pathways of cellular senescence, the mechanisms involved and the role that these pathways have in regulating the initiation and progression of cancer. Telomere-initiated senescence or loss of telomere function trigger focal recruitement of protein sensors of the DNA double-strand breaks leading to the activation of the DNA damage checkpoint responses and the tumour suppressor gene product, p53, which in turn induces the cell-cycle inhibitor, p21(WAF1). Loss of p53 and pRb function allows continued cell division despite increasing telomere dysfunction and eventually entry into telomere crisis. Immortalisation is an essential prerequisite for the formation of a tumour cell. Therefore, a developing tumour cell must circumvent at least two proliferative barriers--cellular senescence and crisis--to achieve neoplastic transformation. These barriers are regulated by telomere shortening and by the p16(INK4a)/Rb and p53 tumour suppressor pathways. Elucidation of the genes and emerging knowledge about the regulatory mechanisms that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.     

p16INK4a as a Second Effector of the Telomere Damage Pathway

Telomere damage resulting from telomere shortening can potentially suppress tumorigenesis by permanently arresting or eliminating incipient cancer cells. Dysfunctional telomeres activate the canonical DNA damage signaling pathway, resulting in a p53-mediated G1/S arrest and senescence or apoptosis. Experimental induction of telomere damage through inhibition of the telomeric protein TRF2 recapitulates aspects of telomere attrition, including a p53-mediated cell cycle arrest. Using this system, we have shown that telomere damage can also elicit a G1/S arrest through the RB-regulator p16INK4a, especially in cells lacking p53 function. Here we discuss the significance of p16INK4a as a second effector of the telomere damage response.     

Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1

The polycomb protein Bmi-1 represses the INK4a locus, which encodes the tumor suppressors p16 and p14(ARF). Here we report that Bmi-1 is downregulated when WI-38 human fibroblasts undergo replicative senescence, but not quiescence, and extends replicative life span when overexpressed. Life span extension by Bmi-1 required the pRb, but not p53, tumor suppressor protein. Deletion analysis showed that the RING finger and helix-turn-helix domains of Bmi-1 were required for life span extension and suppression of p16. Furthermore, a RING finger deletion mutant exhibited dominant negative activity, inducing p16 and premature senescence. Interestingly, presenescent cultures of some, but not all, human fibroblasts contained growth-arrested cells expressing high levels of p16 and apparently arrested by a p53- and telomere-independent mechanism. Bmi-1 selectively extended the life span of these cultures. Low O(2) concentrations had no effect on p16 levels or life span extension by Bmi-1 but reduced expression of the p53 target, p21. We propose that some human fibroblast strains are more sensitive to stress-induced senescence and have both p16-dependent and p53/telomere-dependent pathways of senescence. Our data suggest that Bmi-1 extends the replicative life span of human fibroblasts by suppressing the p16-dependent senescence pathway.