Elevated Expression Of p16INK4a Research Articles

Identification and functional validation of an enhancer variant in the 9p21.3 locus associated with glaucoma risk and elevated expression of p16INK4a.

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome-wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein-coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging.

Reduced stem cell aging in exercised human skeletal muscle is enhanced by ginsenoside Rg1.

Background: Stem cell aging, characterized by elevated p16INK4a expression, decreases cell repopulating and self-renewal abilities, which results in elevated inflammation and slow recovery against stress.Methods: Biopsied muscles were analyzed at baseline and 24 h after squat exercise in 12 trained men (22 ± 2 y). Placebo (PLA) and immunostimulant Rg1 (5 mg) were supplemented 1 h before a squat exercise, using a double-blind counterbalanced crossover design.Results: Perceived exertion at the end of resistance exercise session was significantly lowered after Rg1 supplementation. Exercise doubled endothelial progenitor cells (EPC) (p < 0.001) and decreased p16INK4a mRNA to 50% of baseline (d = 0.865, p < 0.05) in muscle tissues, despite p16INK4a+ cell and beta-galactosidase+ (ß-Gal+) cell counts being unaltered. Rg1 further lowered p16INK4a mRNA to 35% of baseline with greater effect size than the PLA level (d = 1.302, p < 0.01) and decreased myeloperoxidase (MPO) mRNA to 39% of baseline (p < 0.05). A strong correlation between MPO and p16INK4a expression in muscle tissues was observed (r = 0.84, p < 0.001).Conclusion: EPC in skeletal muscle doubled 1 d after an acute bout of resistance exercise. The exercised effects in lowering EPC aging and tissue inflammation were enhanced by immunostimulant Rg1, suggesting the involvement of immune stimulation on EPC rejuvenation.

Alpl prevents bone ageing sensitivity by specifically regulating senescence and differentiation in mesenchymal stem cells

Mutations in the liver/bone/kidney alkaline phosphatase (Alpl) gene cause hypophosphatasia (HPP) and early-onset bone dysplasia, suggesting that this gene is a key factor in human bone development. However, how and where Alpl acts in bone ageing is largely unknown. Here, we determined that ablation of Alpl induces prototypical premature bone ageing characteristics, including bone mass loss and marrow fat gain coupled with elevated expression of p16INK4A (p16) and p53 due to senescence and impaired differentiation in mesenchymal stem cells (MSCs). Mechanistically, Alpl deficiency in MSCs enhances ATP release and reduces ATP hydrolysis. Then, the excessive extracellular ATP is, in turn, internalized by MSCs and causes an elevation in the intracellular ATP level, which consequently inactivates the AMPKα pathway and contributes to the cell fate switch of MSCs. Reactivating AMPKα by metformin treatment successfully prevents premature bone ageing in Alpl+/- mice by improving the function of endogenous MSCs. These results identify a previously unknown role of Alpl in the regulation of ATP-mediated AMPKα alterations that maintain MSC stemness and prevent bone ageing and show that metformin offers a potential therapeutic option.

Abstract 2827: PD0332991-induced stromal senescence promotes melanoma growth in vivo

Abstract Cellular senescence, a process in which cells permanently exit the cell cycle, has pleiotropic biological effects. Several lines of evidence suggest that senescent cells within the tumor microenvironment can elicit a pro-inflammatory secretome that promotes cancer initiation and progression. This proinflammatory phenotype differs amongst senescent cells triggered by distinct stimuli; therefore, multiple forms of senescent cells likely exist within the tumor stroma during cancer evolution and treatment. Here, we sought to determine how stromal senescence initiated by clinically relevant treatments would influence melanoma growth. To generate senescent stromal populations, we treated presenescent MEFs (p4) with ultraviolet light (UV), Mitomycin C (MMC), or a CDK4/6 inhibitor (CDK4/6i). In contrast to p4 fibroblasts, cells treated with UV, MMC or CDK4/6i exhibited cell cycle arrest, increased SA-β-gal positivity and elevated p16INK4a expression. Pronounced 53BP1 and γH2AX foci were only observed in the UV and MMC treated cells, suggesting the CDK4/6i treatment did not induce DNA damage. Profiling of gene expression using a TaqMan mouse inflammation expression panel revealed distinctions amongst the 4 fibroblast lines. Of the senescent cell populations, CDK4/6i fibroblasts showed the highest number of elevated pro-inflammatory transcripts. To define the in vitro paracrine growth effects induced by stromal senescence, melanoma cell lines harboring either Braf600E, Ras, or unknown (wild-type) driver were co-cultured with each fibroblast population. In general, melanoma cells grew similarly on senescent and non-senescent fibroblasts; however, tumor cell growth was genotype-dependent. Notably, CDK4/6i fibroblasts never stimulated additional tumor cell growth in vitro. In contrast, syngeneic transplantations of fibroblasts and tumor cells into immune competent mice revealed that all senescent fibroblasts stimulated tumor growth in vivo. However, whether senescent and non-senescent fibroblasts promoted tumor growth at the same rate was genotype-dependent. Since this is the first study to examine the paracrine effects of senescent stromal cells using an immune competent mouse, we performed IHC for CD45 to assess whether immune cell recruitment differs in the presence of senescent stromal fibroblasts. No increase in CD45 positive infiltrates was observed in the melanoma lines co-injected with CDK4/6i fibroblasts. Together, this work reveals that melanoma growth in response to senescent stromal fibroblasts is genotype dependent, and that this stromal promotion of cancer growth can be accurately assessed only in the context of an immune competent, syngeneic host. Moreover, these data suggest that stromal senescence induced by emerging CDK4/6 inhibitor therapies and/or regimens combining chemotherapy/radiation with immune checkpoint blockade may also promote tumor growth through the paracrine effects of senescent bystanders. Citation Format: Xiangnan Guan, Kyle Lapak, Rebecca Hennessey, Christin Burd. PD0332991-induced stromal senescence promotes melanoma growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2827.

Purinergic P2Y14 receptor modulates stress-induced hematopoietic stem/progenitor cell senescence

Purinergic receptors of the P2Y family are G protein-coupled surface receptors that respond to extracellular nucleotides and can mediate responses to local cell damage. P2Y-dependent signaling contributes to thrombotic and/or inflammatory consequences of tissue injury by altering platelet and endothelial activation and immune cell phagocytosis. Here, we have demonstrated that P2Y14 modifies cell senescence and cell death in response to tissue stress, thereby enabling preservation of hematopoietic stem/progenitor cell function. In mice, P2Y14 deficiency had no demonstrable effect under homeostatic conditions; however, radiation stress, aging, sequential exposure to chemotherapy, and serial bone marrow transplantation increased senescence in animals lacking P2Y14. Enhanced senescence coincided with increased ROS, elevated p16(INK4a) expression, and hypophosphorylated Rb and was inhibited by treatment with a ROS scavenger or inhibition of p38/MAPK and JNK. Treatment of WT cells with pertussis toxin recapitulated the P2Y14 phenotype, suggesting that P2Y14 mediates antisenescence effects through Gi/o protein-dependent pathways. Primitive hematopoietic cells lacking P2Y14 were compromised in their ability to restore hematopoiesis in irradiated mice. Together, these data indicate that P2Y14 on stem/progenitor cells of the hematopoietic system inhibits cell senescence by monitoring and responding to the extracellular manifestations of tissue stress and suggest that P2Y14-mediated responses prevent the premature decline of regenerative capacity after injury.

Pro-senescence therapy for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequent human malignancies with poor prognosis. Due to the current limitations of therapeutic options, there is a pressing need to elucidate the molecular mechanisms underlying human hepatocarcinogenesis and to identify novel targets for systemic therapy. It has recently been shown that Deleted in Liver Cancer 1 (DLC1), encoding a RhoGAP (Rho GTPase activating protein), is a tumor suppressor whose allele is lost in ~ 50% of liver, breast, lung and 70% of colon cancers [1, 2]. Identification of the relevant signaling pathways initiated by DLC1 loss may therefore allow for a more specific and personalized therapy of HCC. Our earlier work showed that DLC1 loss results in constitutive nuclear localization of Megakaryoblastic Leukemia 1 and 2 (MKL1 and 2) proteins, which are coactivators of the transcription factor Serum Response Factor (SRF) governing fundamental biological processes such as cell migration, cell growth, differentiation and cytoskeletal organization [3, 4]. A key feature of MKL1/2 regulation is that the proteins reside in the cytoplasm in an inactive conformation and translocate into the nucleus upon serum stimulation and actin polymerization [5, 6]. MKL1 nuclear localization following DLC1 loss is brought about by activation of the RhoA/actin signaling pathway and impairment of MKL1 phosphorylation, resulting in constitutive activation of tumor-relevant MKL1/2 target genes and enhanced HCC cell proliferation [4]. These findings led us to test whether depletion of MKL1/2 could block the proliferation of DLC1-deficient HCC cells. Indeed, DLC1-deficient HCC cells cease to grow in response to MKL1/2 depletion and display characteristic senescence-associated features including flat morphology, G1 arrest and induction of senescence-associated beta-galactosidase activity [7]. The MKL-knockdown mediated senescence response is caused by activation of the oncogene Ras and results in elevated p16INK4a expression, hypophosphorylation of the retinoblastoma (Rb) protein and upregulation of components of the senescence-messaging secretome. The same repertoire of oncogene-induced senescence factors is induced upon DLC1 reconstitution, suggesting that DLC1 exerts its tumor suppressive effects via engagement of the same key effector pathways [7] (Figure ​(Figure1).1). To assess the in vivo relevance for our observations, we employed polyethylenimine (PEI) complexation as an efficient tool for in vivo siRNA delivery into athymic nude mice bearing HCC tumor xenografts. Depletion of MKL1/2, as well as MKL1 alone completely abolishes tumor growth. Moreover, the regression of HCC xenografts is associated with oncogene-induced senescence [7]. This study raises a number of important questions. First, how does MKL1/2 depletion lead to Ras activation? Does MKL1/2 engage a RasGAP (RasGTPase activating protein) that causes senescence by activation of endogenous Ras and Ras effector pathways? Likewise, activation of one of the nine RasGEF (RasGTPase guanine nucleotide exchange factors) catalyzing the exchange of Ras-GDP for Ras-GTP could lead to hyperactivation of Ras. At present, it is not known which of the myriad of MKL1/2 target genes affects the senescence response (Figure ​(Figure1).1). This will be a challenging task to tackle in the future. Second, why is MKL1 knockdown alone sufficient to curb HCC xenograft growth? It should be noted that inhibition of Rho-actin-dependent signalling to SRF in HeLa, NIH3T3 and MDA-MB-231 cells requires ablation of both MKL1 and MKL2 [3, 8]. Our work suggests that MKL1 depletion is sufficient to suppress HCC-relevant MKL1/2 target genes. Additional experiments are needed to identify the MKL target genes driving hepatocarcinogenesis. Third, which mechanisms are employed by MKL1/2 to evade senescence? Figure 1 Model for senescence induction in HCC with DLC1 loss Interestingly, senescence pathways can be re-engaged upon depletion of MKL1/2 or reintroduction of DLC1 (Figure ​(Figure1).1). Given the efficacy of MKL1/2 downregulation by PEI complexes, a therapeutic strategy to induce senescence by antagonizing MKL1/2 can be utilized to combat HCC. Such a strategy gains even more impetus since tumor suppressors such as DLC1 are generally not amenable to direct therapeutic targeting and blocking of Rho by geranylgeranyltransferase inhibition appears to be a suboptimal option due to the widespread cellular distribution of geranylgeranylation of proteins. Identifying MKL1/2 inhibitors will therefore be a promising goal for the future.

Activation of p53 with Nutlin-3a radiosensitizes lung cancer cells via enhancing radiation-induced premature senescence

Radiotherapy is routinely used for the treatment of lung cancer. However, the mechanisms underlying ionizing radiation (IR)-induced senescence and its role in lung cancer treatment are poorly understood. Here, we show that IR suppresses the proliferation of human non-small cell lung cancer (NSCLC) cells via an apoptosis-independent mechanism. Further investigations reveal that the anticancer effect of irradiation correlates well with IR-induced premature senescence, as evidenced by increased senescence-associated β-glactosidase (SA-β-gal) staining, decreased BrdU incorporation and elevated expression of p16INK4a (p16) in irradiated NSCLC cells. Mechanistic studies indicate that the induction of senescence is associated with activation of the p53–p21 pathway, and that inhibition of p53 transcriptional activity by PFT-α attenuates IR-induced tumor cell killing and senescence. Gain-of-function assays demonstrate that restoration of p53 expression sensitizes H1299 cells to irradiation, whereas knockdown of p53 expression by siRNA inhibits IR-induced senescence in H460 cells. Furthermore, treatment with Nutlin-3a, a small molecule inhibitor of MDM2, enhances IR-induced tumor cell killing and senescence by stabilizing the activation of the p53–p21 signaling pathway. Taken together, these findings demonstrate for the first time that pharmacological activation of p53 by Nutlin-3a can sensitize lung cancer cells to radiation therapy via promoting IR-induced premature senescence.

RB-Pathway Disruption Is Associated with Improved Response to Neoadjuvant Chemotherapy in Breast Cancer

We sought to determine whether dysregulation of the retinoblastoma (RB) tumor suppressor pathway was associated with improved response to neoadjuvant chemotherapy in breast cancer. An RB-loss signature was used to analyze the association between pathway status and pathologic complete response in gene expression datasets encompassing three different neoadjuvant regimens. Parallel immunohistochemical analysis of the RB pathway was conducted on pretreatment biopsies to determine the association with pathologic response to neoadjuvant chemotherapy. An RB-loss gene expression signature was associated with increased pathologic complete response in datasets from breast cancer patients treated with 5-fluorouracil/adriamycin/cytoxan (FAC; P < 0.001), T/FAC (P < 0.001), and Taxane/Adriaymcin (P < 0.001) neoadjuvant therapy encompassing approximately 1,000 patients. The association with improved response to neoadjuvant chemotherapy was true in both estrogen receptor (ER)-positive and ER-negative breast cancer. Elevated expression of p16ink4a is associated with the RB-loss signature (R = 0.493-0.5982), and correspondingly p16ink4a mRNA levels were strongly associated with pathologic complete response in the same datasets analyzed. In an independent cohort, immunohistochemical analyses of RB and p16ink4a revealed an association of RB loss (P = 0.0018) or elevated p16ink4a (P = 0.0253) with pathologic complete response. In addition, by Miller-Payne and clinicopathologic scoring analyses, RB-deficient tumors experienced an overall improved response to neoadjuvant chemotherapy. Disruption of the RB pathway as measured by several independent methods was associated with improved response to neoadjuvant chemotherapy. The RB-pathway status was relevant for pathologic response in both ER-positive and ER-negative breast cancer with similar results observed with multiple chemotherapy regimens. Combined, these data indicate that RB status is associated with the response to neoadjuvant chemotherapy in breast cancer and could be used to inform treatment.

Senescence‐escape in melanoma

Cellular senescence is an important defense against uncontrolled cellular replication and is a critical tumor suppressor mechanism. Although initially described as an in vitro phenomenon, in recent years, several lines of evidence have indicated that senescence also occurs in vivo. Disparate cellular stresses can provoke senescence including telomere shortening, activation of certain oncogenes, oxidative stress and stalled replication forks. These stresses are best known to engage the p16INK4a-RB and ARF-p53 pathways to effect growth arrest and prevent neoplasia. Among these, ‘oncogene-induced senescence’ (OIS) is particularly known for the prevention of melanoma, resulting from the expression of mutant B-RAF or N-RAS in this disease.

HIF1α is required for survival maintenance of chronic myeloid leukemia stem cells

AbstractHypoxia-inducible factor-1α (HIF1α), a master transcriptional regulator of the cellular and systemic hypoxia response, is essential for the maintenance of self-renewal capacity of normal HSCs. It is still unknown whether HIF1α has a role in survival regulation of leukemia stem cells (LSCs) in chronic myeloid leukemia (CML). Using a mouse model of CML, here we report that HIF1α plays a crucial role in survival maintenance of LSCs. Deletion of HIF1α impairs the propagation of CML through impairing cell-cycle progression and inducing apoptosis of LSCs. Deletion of HIF1α results in elevated expression of p16Ink4a and p19Arf in LSCs, and knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α−/− LSCs. Compared with normal HSCs, LSCs appear to be more dependent on the HIF1α pathway. Together, these results demonstrate that HIF1α represents a critical pathway in LSCs and inhibition of the HIF1α pathway provides a therapeutic strategy for eradicating LSCs in CML.

Association of RB/p16-Pathway Perturbations with DCIS Recurrence: Dependence on Tumor versus Tissue Microenvironment

The prevalence of ductal carcinoma in situ (DCIS) diagnoses has significantly increased as a result of active radiographic screening. Surgical resection and hormone and radiation therapies are effective treatments, but not all DCIS will progress to invasive breast cancer. Therefore, markers are needed to define tumors at low risk of recurrence and progression that can be treated by surgery alone rather than by adjuvant therapies. Initial analyses indicate that retinoblastoma (RB)-pathway perturbations occur at high frequency in DCIS and mirror the molecular alterations observed in invasive breast cancer. Particularly, the elevated expression of p16ink4a in DCIS was associated with loss of RB function and estrogen receptor-negative biology. Furthermore, high expression of p16ink4a in conjunction with Ki-67 was associated with increased risk of DCIS recurrence and progression to invasive disease in multivariate analyses. These data are consistent with a functional role for RB in modulating the invasive behavior of mammary epithelial cells. The tissue microenvironment is particularly relevant to the behavior of DCIS, and, surprisingly, elevated expression of p16ink4a in nonproliferative stroma was observed in a substantial fraction of cases. In this tissue compartment, p16ink4a expression was strongly associated with disease recurrence, independent of standard histopathologic features. Together, the data herein describe dual aspects of RB-pathway biology that are associated with disease recurrence through the epithelial or stromal compartment of DCIS.

MiRNA-205 Suppresses Melanoma Cell Proliferation and Induces Senescence via Regulation of E2F1 Protein

MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-205 is significantly suppressed in melanoma specimens when compared with nevi and is correlated inversely with melanoma progression. miRNA target databases predicted E2F1 and E2F5 as putative targets. The expression levels of E2F1 and E2F5 were correlated inversely with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3'-UTR sequences complementary to either E2F1 or E2F5. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels. The proliferative capacity of melanoma cells was suppressed by miR-205 and mediated by E2F-regulated AKT phosphorylation. miR-205 overexpression resulted in induction of apoptosis, as evidenced by increased cleaved caspase-3, poly-(ADP-ribose) polymerase, and cytochrome c release. Stable overexpression of miR-205 suppressed melanoma cell proliferation, colony formation, and tumor cell growth in vivo and induced a senescence phenotype accompanied by elevated expression of p16INK4A and other markers for senescence. E2F1 overexpression in miR-205-expressing cells partially reversed the effects on melanoma cell growth and senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma.

Abstract 142: Novel tumor suppressor role of microRNA-205 in melanoma via regulation of E2F1

Abstract MicroRNAs (miRNAs) regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Altered miRNA expression has been demonstrated in several human cancers. Limited information is available concerning the expression and role of miRNAs in melanoma. Here, we report that expression of miR-205 is significantly suppressed in melanoma tumor samples when compared with nevi, and is correlated inversely with melanoma progression. miR-205 expression was also significantly downregulated in a panel of melanoma cell lines when compared to a normal melanocyte line. miRNA target databases predicted a conserved target site for miRNA-205 in the 3’ untranslated region (UTR) of E2F1 and E2F5. The expression levels of E2F1 and E2F5 were inversely correlated with that of miR-205 in melanoma cell lines. miR-205 significantly suppressed the luciferase activity of reporter plasmids containing the 3’UTR sequences complementary to either E2F1 or E2F5, which was abolished by mutations in these 3’UTR regions. Overexpression of miR-205 in melanoma cells reduced E2F1 and E2F5 protein levels without alteration of mRNA expression. The proliferative ability of melanoma cells was suppressed by miR-205, mediated by E2F-regulated AKT phosphorylation. Transient overexpression of miR-205 in melanoma cells resulted in induction of apoptosis, as indicated by increases in cleaved caspase-3, poly-(ADP-ribose) polymerase (PARP) and release of cytochrome-c. Stable overexpression of miR-205 suppressed cell proliferation and colony formation, and resulted in inhibition of tumor cell growth in vivo and induction of a senescence phenotype accompanied by elevated p16INK4A expression and other markers for senescence. These results demonstrate a novel role for miR-205 as a tumor suppressor in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 142. doi:10.1158/1538-7445.AM2011-142

Cell cycle arrest and cytochrome c-mediated apoptotic induction by MCS-5A is associated with up-regulation of p16 INK4a in HL-60 cells

MCS-5A, an analog of sangivamycin, selectively inhibits the cyclin-dependent kinases CDK1 and 4 in HL-60 cells in vitro (IC 50: 9.6 and 8.8 μΜ, respectively), while weakly inhibiting other housekeeping protein kinases. MCS-5A effectively induces HL-60 cell cycle arrest at the G 1 and G 2/M phases through direct inhibition of CDK1 and 4 activity. In addition, elevated expression of p16 INK4a and a reduction in the level of hyperphosphorylated pRb showed that 3 μΜ MCS-5A also induces p16 INK4a-mediated cell cycle arrest at the G 1 phase. Furthermore, apoptotic induction in MCS-5A-treated HL-60 cells is associated with the release of cytochrome c from mitochondria, which, in turn, results in the activation of procaspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). In addition, the involvement of p16 INK4a in this apoptotic induction was demonstrated using A549 cells with a homozygous deletion of p16 INK4a. Based on these results, we conclude that MCS-5A is a candidate therapeutic agent for the treatment of human promyelocytic leukemia via the up-regulation of p16 INK4a.

Concurrence of replicative senescence and elevated expression of p16 INK4A with subculture-induced but not calcium-induced differentiation in normal human oral keratinocytes

Primary normal human oral keratinocytes (NHOKs) undergo differentiation in the presence of calcium concentrations higher than 0.15 mM in vitro, which is useful in investigating the mechanisms involved in the differentiation of epithelial cells. Serial subculture of NHOKs to the postmitotic stage also induces terminal differentiation. However, the detailed mechanisms of both differentiation processes remain substantially unknown. To investigate the molecular differences in these processes, NHOKs were induced to differentiate by exposure to 1.2 mM of calcium and by serial subculture to the postmitotic stage. To study whether the cells were induced to differentiate and to undergo replicative senescence, the amount of cellular involucrin and the expression of senescence-associated β-galactosidase (SA- β-gal) were measured respectively. The expression of replicative senescence-associated genes and the activity of telomerase from the differentiated cells were also determined. Both calcium treatment and serial subculture to the postmitotic stage notably elevated the cellular involucrin. The percentage of SA- β-gal-positive cells was significantly elevated by the continued subculture, but such changes were not observed in keratinocytes exposed to calcium. The concentration of cellular p16 INK4A protein was progressively increased by the continued subculture but was not changed by calcium treatment. On the other hand, the concentrations of cellular p53 were similar in both differentiation processes. However, telomerase activity was lost in NHOKs that had undergone differentiation by both calcium treatment and serial subculture. The results indicate that calcium-induced differentiation of NHOKs has similar characteristics to their serial subculture-induced differentiation, but that the differentiation processes are not identical, because calcium-induced differentiation does not concur with either replicative senescence or the gradually increased concentration of p16 INK4A.

Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice, but not in hepatocytes from p16INK4a-null mice

In primary rat hepatocytes, prolonged activation of the p42/44 mitogen-activated protein kinase (MAPK) pathway is associated with a decrease in DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor (CKI) proteins p21Cip-1/WAF1 and p16INK4a. To evaluate the relative importance of these CKIs in mediating this response, we determined the impact of prolonged MAPK activation on DNA synthesis in primary cultures of hepatocytes derived from mice embryonically deleted (null) for either p21Cip-1/WAF1 or p16INK4a. When MAPK was activated in wild-type mouse hepatocytes for 24 h, via infection with a construct to express an inducible oestrogen receptor-Raf-1 fusion protein (DeltaRaf:ER), the expression of p21Cip-1/WAF1 and p16INK4a CKI proteins increased, cyclin-dependent kinase 2 (cdk2) and cdk4 activities decreased, and DNA synthesis decreased. Inhibition of RhoA GTPase function increased the basal expression of p21Cip-1/WAF1 and p27Kip-1 but not p16INK4a, and enhanced the ability of MAPK signalling to decrease DNA synthesis. Ablation of the expression of CCAATT enhancer-binding protein alpha (C/EBPalpha), but not of the expression of C/EBPbeta, decreased the ability of MAPK signalling to induce p21Cip-1/WAF1. When MAPK was activated in p16INK4a-null hepatocytes for 24 h, the expression of p21Cip-1/WAF1 increased, cdk2 and cdk4 activities decreased and DNA synthesis decreased. In contrast with these findings, prolonged activation of the MAPK pathway in hepatocytes from p21Cip-1/WAF1-null mice enhanced cdk2 and cdk4 activities and caused a large increase in DNA synthesis, despite elevated expression of p16INK4a. Inhibition of RhoA GTPase activity in p21Cip-1/WAF1-null cells partly blunted both the basal levels of DNA synthesis and the ability of prolonged MAPK signalling to increase DNA synthesis. Expression of anti-sense p21Cip-1/WAF1 in either wild-type or p16INK4a-null hepatocytes decreased the ability of prolonged MAPK signalling to increase the expression of p21Cip-1/WAF1, and permitted MAPK signalling to increase both cdk2 and cdk4 activities and DNA synthesis. These results argue that the ability of prolonged MAPK signalling to inhibit DNA synthesis in hepatocytes requires the expression of p21Cip-1/WAF1, and that the increased expression of p16INK4a has a smaller role in the ability of this stimulus to mediate growth arrest. Our results also suggest that RhoA function can modulate DNA synthesis in primary hepatocytes via the expression of p21Cip-1/WAF1 and p27Kip-1.