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.     

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.     

A two-stage,p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.     

Replicative senescence as a barrier to human cancer

There is evidence that one critically short telomere may be recognized as DNA damage and, as a consequence, induce a p53/p21WAF- and p16INK4A-dependent G1 cell cycle checkpoint to cause senescence. Additionally, senescence via a p53- and p16(INK4A)-dependent mechanism can be induced by the over- or under-stimulation of certain signalling pathways that are involved in cancer. Central to this alternative senescence mechanism is the p14ARF protein, which connects oncogene activation, but not DNA damage, to p53 activation and senescence. We find that immortal keratinocytes almost invariably have dysfunctional p53 and p16 and have high levels of telomerase, but very often express a wild-type p14(ARF). Furthermore, when normal keratinocytes senesce they show a striking elevation of p16 protein, but not of p14(ARF) or its downstream targets p53 and p21(WAF). These results suggest that p16, rather than p14(ARF), is the more important gene in human keratinocyte senescence, but do not exclude a co-operative role for p14(ARF), perhaps in the induction of senescence by activated oncogenes in neoplasia. Regardless of mechanism, these results suggest that replicative senescence acts as a barrier to human cancer development.     

Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3sigma, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3sigma-dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16(INK4a) tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.     

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.     

Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts

Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3-20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-beta-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.     

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.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

Senescence delay of human diploid fibroblast induced by anti-sense p16INK4a expression

p16(INK4a), a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and cyclin-dependent kinase 6, is also implicated in the mechanisms underlying replicative senescence, because its RNA and protein accumulate as cells approach their finite number of population doublings in tissue culture. To further explore the involvement of p16(INK4a) in replicative senescence, we constructed a retroviral vector containing antisense p16(INK4a), pDOR-ASp16, and introduced it into early passages of human diploid fibroblasts. The introduction of this construct significantly suppressed the expression of wild-type p16(INK4a). It also imposed a finite increase in proliferative life span and significant delay of several other cell senescent features, such as cell flattening, cell cycle arrest, and senescence-associated beta-galactosidase positivity. Moreover, telomere shortening and decline in DNA repair capacity, which normally accompany cell senescence, are also postponed by the ASp16 transfection. The life span of fibroblasts was significantly extended, but the onset of replicative senescence could not be totally prevented. Telomerase could not be activated even though telomere shortening was slowed. These observations suggest that the telomere pathway of senescence cannot be bypassed by ASp16 expression. These data not only strongly support a role for p16(INK4a) in replicative senescence but also raise the possibility of using the antisense p16(INK4a) therapeutically.     

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.     

Id-1 delays senescence but does not immortalize keratinocytes

Defining the molecular basis responsible for regulating the proliferative potential of keratinocytes has important implications for normal homeostasis and neoplasia of the skin. Under current culture conditions, neonatal foreskin-derived human keratinocytes possess a relatively short replicative lifespan. Recently it was reported that forced overexpression of the helix-loop-helix protein Id-1 was capable of immortalizing keratinocytes, secondary to activation of telomerase activity and suppression of p16/Rb-mediated growth arrest pathways. To investigate the relationship between Id-1, telomerase activity, telomere length, p16, Rb cell cycle regulators, and senescence, whole populations of keratinocytes were infected with a retrovirus to induce overexpression of Id-1. In these unselected cultures, enhanced Id-1 levels clearly extended the lifespan of keratinocytes, but Id-1 did not prevent the onset of replicative senescence. Under these experimental conditions, Id-1 expression did not trigger induction of telomerase activity, and there was progressive shortening of the telomeres that was accompanied by elevated p16 levels and prevalence of active Rb. The ability of Id-1 to postpone, but not prevent, senescence may be related to partial inhibition of p16 expression, as the Id-1-overexpressing cultures displayed a decreased capacity for 12-O-tetradecanoylphorbol-13-acetate-mediated p16 induction. Thus, while no immortalization was observed, Id-1 could delay the onset of replicative senescence in unselected human keratinocyte populations.     

Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization

A large proportion of the CD8(+) T cell pool in persons chronically infected with HIV consists of cells that show features of replicative senescence, an end stage characterized by irreversible cell cycle arrest, multiple genetic and functional changes, and shortened telomeres. The objective of our research was to determine whether constitutive expression of the gene for the human telomerase (hTERT) can prevent senescence-induced impairments in human virus-specific CD8(+) T cells, particularly in the context of HIV-1 disease. Our results indicate that hTERT-expressing HIV-specific CD8(+) lymphocytes show both an enhanced and sustained capacity to inhibit HIV-1 replication in in vitro coculture experiments, as well as prolonged ability to produce IFN-gamma and TNF-alpha in response to stimulation with HIV-1-derived peptides, as compared with vector-transduced controls. Loss of CD28 expression, the signature change of replicative senescence in cell culture, was retarded in those CD8(+) T cell cultures that had high levels of CD28 at the time of hTERT transduction. These findings suggest that telomere shortening may be the primary driving force behind several aspects of CD8(+) T cell dysfunction associated with replicative senescence. We also demonstrate reduced accumulation of the p16(INK4a) and p21(WAF1) cell cycle inhibitors in hTERT-transduced lymphocytes, providing a possible mechanism by which stable hTERT expression is able to circumvent the senescence barrier in CD8(+) T cells. Given the key role of CD8(+) T cell function in controlling a variety of acute and latent viral infections, approaches to retard the functional decrements associated with replicative senescence may lead to novel types of immunotherapy.     

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.