Cooperation between p53 and p130(Rb2) in induction of cellular senescence

To determine pathways cooperating with p53 in cellular senescence when the retinoblastoma protein (pRb)/p16INK4a pathway is defunct, we stably transfected the p16INK4a-negative C6 rat glioma cell line with a temperature-sensitive mutant p53. Activation of p53(Val-135) induces a switch in pocket protein expression from pRb and p107 to p130(Rb2) and stalls the cells in late G1, early S-phase at high levels of cyclin E. Maintenance of the arrest depends on the functions of p130(Rb2) repressing cyclin A. Inactivation of p53 in senescent cultures restores the pocket proteins to initial levels and initiates progression into S-phase, but the cells fail to resume proliferation, likely due to DNA damage becoming apparent in the arrest and activating apoptosis subsequent to the release from p53-dependent growth suppression. The data indicate that p53 can cooperate selectively with p130(Rb2) to induce cellular senescence, a pathway that may be relevant when the pRb/p16INK4a pathway is defunct.     

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.     

Dual role of MEK/ERK signaling in senescence and transformation of intestinal epithelial cells

The mitogen-activated protein kinase cascade operates downstream of Ras to convey cell-surface signals to the nucleus via nuclear translocation of ERK1 and ERK2. We and others have recently demonstrated that activation of ERK1/2 by growth factors is required for proliferation of intestinal epithelial crypt cells. However, it remained to be established whether ERK1/2 activation alone was sufficient to trigger intestinal epithelial cell (IEC) proliferation. To this aim, retrovirus encoding the hemagglutinin-tagged MAPK/ERK kinase (MEK)1 wild type (wtMEK), the upstream activator of ERK1/2, or a constitutively active mutant of MEK1 (MEK1-S218D/S222D; caMEK) were used to infect nonimmortalized human normal intestinal epithelial crypt cell cultures [human intestinal epithelial cells (HIEC)] and rodent immortalized intestinal crypt cells (IEC-6). Stable expression of caMEK but not wtMEK in HIEC led to the irreversible arrest of cellular proliferation (premature senescence). Concomitant with the onset of cell-cycle arrest was the induction of the cyclin-dependent kinase inhibitors p21(Cip), p53, and p16(INK4A). By contrast, overexpression of caMEK in IEC-6 cells induced growth factor relaxation for DNA synthesis, promoted morphological transformation and growth in soft agar, and did not affect expression of p21(Cip), p53, and p16(INK4A). We provided evidences that ERK1b, an alternatively spliced isoform of ERK1, is activated and may contribute to the deregulation of contact inhibition cell growth and transformation of these cells. Constitutive activation of MEK in IECs can produce either premature senescence or forced mitogenesis depending on the integrity of a senescence program controlled by the cell cycle inhibitors p53, p16(INK4A), and p21(CIP).     

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.     

Fez1/Lzts1 a new mitotic regulator implicated in cancer development

The retinoblastoma (Rb) tumor suppressor gene product, pRb, has an established role in the implementation of cellular senescence, the state of irreversible G1 cell cycle arrest provoked by diverse oncogenic stresses. In murine cells, senescence cell cycle arrest can be reversed by subsequent inactivation of pRb, indicating that pRb is required not only for the onset of cellular senescence, but also for the maintenance of senescence program in murine cells. However, in human cells, once pRb is fully activated by p16^INK4a, senescence cell cycle arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb, suggesting that p16^INK4a/Rb-pathway activates an alternative mechanism to irreversibly block the cell cycle in human senescent cells. Here, we discuss the molecular mechanism underlying the irreversibility of senescence cell cycle arrest and its potential towards tumor suppression.     

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.     

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

Failure of cell cleavage induces senescence in tetraploid primary cells

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen–transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-β-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.     

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

CDK inhibitors (p16/p19/p21) induce senescence and autophagy in cancer-associated fibroblasts, “fueling” tumor growth via paracrine interactions,without an increase in neo-angiogenesis

Here, we investigated the compartment-specific role of cell cycle arrest and senescence in breast cancer tumor growth. For this purpose, we generated a number of hTERT-immortalized senescent fibroblast cell lines overexpressing CDK inhibitors, such as p16(INK4A), p19(ARF) or p21(WAF1/CIP1). Interestingly, all these senescent fibroblast cell lines showed evidence of increased susceptibility toward the induction of autophagy (either at baseline or after starvation), as well as significant mitochondrial dysfunction. Most importantly, these senescent fibroblasts also dramatically promoted tumor growth (up to ~2-fold), without any comparable increases in tumor angiogenesis. Conversely, we generated human breast cancer cells (MDA-MB-231 cells) overexpressing CDK inhibitors, namely p16(INK4A) or p21(WAF1/CIP1). Senescent MDA-MB-231 cells also showed increased expression of markers of cell cycle arrest and autophagy, including β-galactosidase, as predicted. Senescent MDA-MB-231 cells had retarded tumor growth, with up to a near 2-fold reduction in tumor volume. Thus, the effects of CDK inhibitors are compartment-specific and are related to their metabolic effects, which results in the induction of autophagy and mitochondrial dysfunction. Finally, induction of cell cycle arrest with specific inhibitors (PD0332991) or cellular stressors [hydrogen peroxide (H?O?) or starvation] indicated that the onset of autophagy and senescence are inextricably linked biological processes. The compartment-specific induction of senescence (and hence autophagy) may be a new therapeutic target that could be exploited for the successful treatment of human breast cancer patients.     

Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein

The tumor suppressor gene p16INK4A is a cyclin-dependent kinase inhibitor (CDKI) and an important cell cycle regulator. We have previously constructed a recombinant adenovirus which expresses p16 (Adp16) and shown that infection in a variety of human tumor cell lines with this recombinant virus results in high levels of p16INK4A protein expression resulting in cell cycle arrest and loss of cyclin-cdk activity. Furthermore, adenoviral-mediated overexpression of wild-type p16INK4A is more toxic in cancer cells which express mutant forms of p16INK4A compared to cancer cell lines containing endogenous wild-type p16. TUNEL assay and DAPI staining following infection of MDA-MB 231 breast cancer cells with Adp16 indicate that p16INK4A-mediated cytotoxicity was associated with apoptosis. This is supported by studies demonstrating a decrease in cpp32 and cyclinB1 protein levels and induction of poly (ADP-ribose) polymerase (PARP) cleavage following infection of MDA-MB-231 cells with Adp16. These results suggest that gene therapy using Adp16 may be a promising treatment option for human cancers containing alterations in p16 expression.     

p16INK4a-induced senescence is disabled by melanoma-associated mutations

The p16(INK4a)-Rb tumour suppressor pathway is required for the initiation and maintenance of cellular senescence, a state of permanent growth arrest that acts as a natural barrier against cancer progression. Senescence can be overcome if the pathway is not fully engaged, and this may occur when p16(INK4a) is inactivated. p16(INK4a) is frequently altered in human cancer and germline mutations affecting p16(INK4a) have been linked to melanoma susceptibility. To characterize the functions of melanoma-associated p16(INK4a) mutations, in terms of promoting proliferative arrest and initiating senescence, we utilized an inducible expression system in a melanoma cell model. We show that wild-type p16(INK4a) promotes rapid cell cycle arrest that leads to a senescence programme characterized by the appearance of chromatin foci, activation of acidic beta-galactosidase activity, p53 independence and Rb dependence. Accumulation of wild-type p16(INK4a) also promoted cell enlargement and extensive vacuolization independent of Rb status. In contrast, the highly penetrant p16(INK4a) variants, R24P and A36P failed to arrest cell proliferation and did not initiate senescence. We also show that overexpression of CDK4, or its homologue CDK6, but not the downstream kinase, CDK2, inhibited the ability of wild-type p16(INK4a) to promote cell cycle arrest and senescence. Our data provide the first evidence that p16(INK4a) can initiate a CDK4/6-dependent autonomous senescence programme that is disabled by inherited melanoma-associated mutations.     

Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.     

Modulation of the expression of p16INK4a and p14ARF by hnRNP A1 and A2 RNA binding proteins: implications for cellular senescence

Cellular senescence is a terminal growth phase characteristic of normal human diploid fibroblasts. Altered gene expression during cellular senescence is numerous compared to that of younger proliferative cells in culture. We have previously reported that the levels and activities of hnRNP A1 and A2 RNA binding proteins are decreased in senescent human fibroblasts. Both proteins are multifunctional and may influence the expression of mRNA isoforms during development. In this study, we tested whether overexpression of either protein could modulate the mRNA isoforms of the INK4a locus, specifically p14(ARF) and p16(INK4a). Both INK4a mRNA isoforms have been shown to be growth suppressors and deletions of this locus allow cells to escape cellular senescence. We have found that increasing the ratio of either hnRNP A1 or A2 over that of splicing factor SF2/ASF results in the preferential generation of the p14(ARF) isoform. Overexpression of A1 or A2 RNA binding proteins also appear to increase the steady state mRNA levels of both isoforms, suggesting that in addition to alternative splicing, A1 and A2 may effect p14(ARF) and p16(INK4a) mRNA stability. A constitutive decrease in the ratio of hnRNP A1 or A2 to SF2/ASF in senescent fibroblasts is typically accompanied by an increase in the level of p16(INK4a) isoform. Our studies suggest that hnRNP A1 and A2 may exert an important role during replicative senescence by altering expression of cell cycle regulatory proteins through mRNA metabolism.     

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex

Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.