p16-pRB Pathways Research Articles

Antioxidants prevent particulate matter-induced senescence of lung fibroblasts

Particulate matter (PM) contributes to human diseases, particularly lung disease; however, the molecular mechanism of its action is yet to be determined. Herein, we found that prolonged PM exposure induced the cellular senescence of normal lung fibroblasts via a DNA damage-mediated response. This PM-induced senescence (PM-IS) was only observed in lung fibroblasts but not in A549 lung adenocarcinoma cells. Mechanistic analysis revealed that reactive oxygen species (ROS) activate the DNA damage response signaling axis, increasing p53 phosphorylation, ultimately leading to cellular senescence via an increase in p21 expression without affecting the p16-pRB pathway. A549 cells, instead, were resistant to PM-IS due to the PM-induced ROS production suppression. Water-soluble antioxidants, such as vitamin C and N-Acetyl Cysteine, were found to alleviate PM-IS by suppressing ROS production, implying that antioxidants are a promising therapeutic intervention for PM-mediated lung pathogenesis.

Does Type II Diabetes Induce Early Senescence and Degeneration in Human Intervertebral Discs? A Tissue Biomarker Evaluation

Diabetes mellitus is one of the leading causes of morbidity resulting in multi-organ dysfunction. Animal studies have shown that hyperglycemia results in stress-induced senescence through the p16-pRb pathway, thereby accelerating early disc degeneration. There is a paucity of literature on the effect of hyperglycemia in human intervertebral disc cells. We aimed to analyze the effect of diabetes mellitus in human intervertebral disc cells. This is a prospective study done in patients with degenerative disc disease. Patients were categorized into a control group (no diabetes: 26 patients) and a study group (type 2 diabetes for > 3 years: 24 patients). All patients underwent either discectomy or transforaminal lumbar interbody fusion and the removed disc was transported to pathology department. Tissue was prepared and histopathological grading was done followed by immunohistochemistry studies using antibodies for MMP-1, p21, p16, and pRb. Samples from diabetic patients had severe (grade 2) degenerative changes compared with the control group (grade 1). Changes were more intense in the nucleus pulposus with increased cellularity and clustering of chondrocytes, and disorganization and loss of nuclear matrix. Immunohistochemical staining for MMP1, p16, and pRb was more intense (Q score = 4) whereas the staining for p21 was less intense (Q score = 1) in the diabetic group compared with the control group. Our study demonstrates that type 2 diabetes mellitus accelerates stress-induced senescence in human intervertebral discs resulting in early disc degeneration. Also, the severity of disc degeneration is severe compared with the normal subjects. Hyperglycemia can affect the intervertebral discs similar to other organs and hence adequate control of blood glucose in diabetics can prevent the disc degeneration, which is the initiator of degeneration cascade in spine.

LncRNA RP11-670E13.6 Regulates Cell Cycle Progression in UVB Damaged Human Dermal Fibroblasts.

Long noncoding RNAs (lncRNAs) have gained extensive attention in recent years, however, their effects on ultraviolet (UV) radiation-induced skin photodamage remain to be elucidated. In this study, we performed high-throughput RNA sequencing and comprehensive bioinformatics analyses to characterize the transcriptome profiles including lncRNAs and mRNAs in UVB-irradiated primary human dermal fibroblasts (HDFs) and to explore the roles of lncRNAs in photoaging. Quantitative reverse transcription-polymerase chain reaction amplification was performed to verify the differentially expressed genes. We subsequently found that knocking down of RP11-670E13.6, an up-regulated lncRNA in UVB-irradiated HDFs, promoted a robust senescence phenotype, including increased numbers of the senescence-associated β-galactosidase-positive cells, decreased cell proliferation, accumulation of cells in G0/G1 phase and a characteristic gene expression signature of senescent cells. In addition, Western blot analysis showed that knocking down of RP11-670E13.6 activated the p16-pRB senescence pathway independent of the p53-p21 pathway. Therefore, we propose that RP11-670E13.6 may delay cellular senescence in UVB damaged HDFs through the p16-pRB pathway.

Ochratoxin A induced premature senescence in human renal proximal tubular cells.

Ochratoxin A (OTA) has many nephrotoxic effects and is a promising compound for the study of nephrotoxicity. Human renal proximal tubular cells (HKC) are an important model for the study of renal reabsorption, renal physiology and pathology. Since the induction of OTA in renal senescence is largely unknown, whether OTA can induce renal senescence, especially at a sublethal dose, and the mechanism of OTA toxicity remain unclear. In our study, a sublethal dose of OTA led to an enhanced senescent phenotype, β-galactosidase staining and senescence associated secretory phenotype (SASP). Cell cycle arrest and cell shape alternations also confirmed senescence. In addition, telomere analysis by RT-qPCR allowed us to classify OTA-induced senescence as a premature senescence. Western blot assays showed that the p53-p21 and the p16-pRB pathways and the ezrin-associated cell spreading changes were activated during the OTA-induced senescence of HKC. In conclusion, our results demonstrate that OTA promotes the senescence of HKC through the p53-p21 and p16-pRB pathways. The understanding of the mechanisms of OTA-induced senescence is critical in determining the role of OTA in cytotoxicity and its potential carcinogenicity.

MiR-29c-3p promotes senescence of human mesenchymal stem cells by targeting CNOT6 through p53–p21 and p16–pRB pathways

Mesenchymal stem cells (MSCs) are important seed cells for tissue engineering and are promising targets for cell-based therapies. However, the replicative senescence of MSCs during in vitro culture limits their research and clinical applications. The molecular mechanisms underlying the replicative senescence of MSCs are not fully understood. Evidence suggests that miRNAs play important roles in replicative senescence. A microarray analysis found that the miR-29c-3p level was significantly increased during the MSC senescence process. In our study, we investigated the roles of miR-29c-3p in senescence of MSCs. We cultured MSCs for long periods of time, up and down-regulated the miR-29c-3p expression in MSCs, and examined the senescent phenotype changes. The over-expression of miR-29c-3p led to enhanced senescence-associated-β-galactosidase (SA-β-gal) staining, senescence associated secretory phenotype (SASP), senescence associated heterochromatic foci (SAHF), reduced proliferation ability, retarded osteogenic differentiation and corresponding changes in senescence markers, whereas the miR-29c-3p down-regulation had the opposite results. Dual-luciferase reporter assays demonstrated that CNOT6 is the target gene of miR-29c-3p. Knockdown of CNOT6 confirmed its inhibitory effects on the senescence of MSCs. In addition, Western blot results showed that both the p53–p21 and the p16–pRB pathways were activated during the miR-29c-3p-induced senescence of MSCs. In conclusion, our results demonstrate that miR-29c-3p promotes the senescence of MSCs by targeting CNOT6 through p53–p21 and p16–pRB pathways and highlight the contribution of post-transcriptional regulation to stem cell senescence.

Rat Notochordal Cells Undergo Premature Stress-Induced Senescence by High Glucose.

Study DesignIn vitro cell culture.PurposeThe purpose of the study was to investigate the effect of high glucose on premature stress-induced senescence of rat notochordal cells.Overview of LiteratureGlucose-mediated increase of oxidative stress is a major causative factor for the development of diseases associated with diabetes mellitus such as senescence. However, no information is available for the effect of high glucose on premature stress-induced senescence of rat notochordal cells.MethodsNotochordal cells were isolated from 4-week-old rats, cultured and placed in either 10% fetal bovine serum (FBS, normal control) or 10% FBS plus two high glucose concentrations (0.1 M and 0.2 M, experimental conditions) for 1 and 3 days. We identified and quantified the mitochondrial damage (mitochondrial transmembrane potential), reactive oxygen species (ROS) and antioxidants, such as manganese superoxide dismutase (MnSOD) and catalase, for each condition. We also identified and quantified senescence and telomerase activity. Finally, we determined the expression of proteins related to replicative senescence (p53-p21-pRB) and stress-induced senescence (p16-pRB) pathways.ResultsTwo high glucose concentrations enhanced the disruption of mitochondrial transmembrane potential and excessive generation of ROS in notochordal cells for 1 and 3 days, respectively. The expressions of MnSOD and catalase were increased in notochordal cells treated with both high glucose concentrations at 1 and 3 days. The telomerase activity declined at 1 and 3 days. Two high glucose concentrations increased the occurrence of stress-induced senescence of notochordal cells by p16-pRB pathways at 1 and 3 days.ConclusionsDespite compensatory expression of antioxidants, high glucose-induced oxidative stress accelerates stress-induced senescence in rat notochordal cells. This may result in dysfunction of notochordal cells, leading to accelerated premature disc degeneration. The prevention of excessive generation of oxidative stress by strict blood glucose control is important to prevent or to delay premature disc degeneration in young patients with diabetes mellitus.

Protective Cytotoxic Clonal T-Cells in Myeloma Have the Characteristics of Telomere-Independent Senescence Rather Than an Exhausted or Anergic Phenotype: Implications for Immunotherapy

Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function.Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p<0.01) and maintain high cytokine secretion. CFSE proliferation assays of flow sorted T-cell clones demonstrated a significant lack of proliferation after stimulation with MACS iBeads (n=12; p<0.0001). They failed to respond in vitro to cytokines such as IL- 2, IL-12, IL-15 and OX40. In contrast, the T-cell clones were proliferative in a group of long term myeloma survivors in whom such T-cells are universally present suggesting that the impaired proliferation may be reversible. Geneset enrichment analysis of mRNA expression from Affymetrix U133 plus 2.0 arrays demonstrated pleiotropic dysfunction in multiple intracellular signalling pathways involved with proliferation and T cell inactivation. Utilizing phospho-flow techniques we demonstrated reduced phosphorylated ERK levels in clonal vs non-clonal T-cells (n=8; p<0.002) as well as elevated levels of phosphorylated Smad2/3 (n=10; p<0.02) and Bcl-xL (n=7; p<0.04) .These findings suggest that the ERK pathway, associated with proliferation, was suppressed, the TGFβ pathway was activated and that the dysfunctional T-cell clones have a survival advantage due to apoptotic resistance. In contrast, normal SHP-2 and phosphorylated ZAP-70 levels suggested there was no defect in upstream TCR signaling.Clonal T-cells lacked CD28, PD-1, CTLA-4, LAG-3, CD160 and Tim-3 expression and therefore are neither exhausted nor anergic. However, telomere assessment by qPCR demonstrated telomere T/S ratios were normal length for age and not significantly less than autologous CD57+TCR-Vβ- cells. T-cell clones also had normal p38 expression suggesting that the senescence was not due to TNF-α inhibiting the p38/MAPK pathway.The cytotoxic T-cell clones in patients with myeloma have the characteristics of telomere-independent senescence. The normal telomere length suggests that the hypo-responsiveness is induced independent of telomeres, and is therefore potentially reversible, though the pleiotropic nature of this dysfunction might require the targeting of multiple intracellular signalling pathways. The lack of PD-1 and CTLA-4 expression suggests that immune checkpoint blockade in myeloma would be unable to reverse the T cell dysfunction. DisclosuresNo relevant conflicts of interest to declare.

A human cancer xenograft model utilizing normal pancreatic duct epithelial cells conditionally transformed with defined oncogenes.

Pancreatic ductal adenocarcinomas (PDACs) are considered to arise through neoplastic transformation of human pancreatic duct epithelial cells (HPDECs). In order to evaluate the biological significance of genetic and epigenetic alterations in PDACs, we isolated primary HPDECs and established an in vitro carcinogenesis model. Firstly, lentivirus-mediated transduction of KRAS(G12V), MYC and human papillomavirus 16 (HPV16) E6/E7 under the control of a tetracyclin-inducible promoter efficiently immortalized and transformed primary HPDECs, which gave rise to adenocarcinomas subcutaneously in an immune-deficient mouse xenograft model, depending on expression of the four genes. The tumors regressed promptly upon shutting-off the oncogenes, and the remaining tissues showed histological features corresponding to normal ductal structures with simple columnar epithelium. Reexpression of the oncogenes resulted in development of multiple PDACs through pancreatic intraepithelial neoplasia-like structures. We also succeeded in efficient immortalization of primary HPDECs with transduction of mutant CDK4, cyclin D1 and TERT. The cells maintained a normal diploid status and formed duct-like structures in a three-dimensional culture. In combination with p53 silencing, KRAS(G12V) alone was sufficient to fully transform the immortalized HPDECs, and MYC markedly accelerated the development of tumors. Our PDAC model supports critical roles of KRAS mutations, inactivation of the p53 and p16-pRB pathways, active telomerase and MYC expression in pancreatic carcinogenesis and thus recapitulates many features of human PDAC development. The present system with reversible control of oncogene expression enabled de novo development of PDAC from quasinormal human tissues preformed subcutaneously in mice and might be applicable to carcinogenesis models in many organ sites.

Rat Induced Pluripotent Stem Cells Protect H9C2 Cells from Cellular Senescence via a Paracrine Mechanism

Objectives: Cellular senescence may play an important role in the pathology of heart aging. We aimed to explore whether induced pluripotent stem cells (iPSCs) could inhibit cardiac cellular senescence via a paracrine mechanism. Methods: We collected iPSC culture supernatant, with or without oxidative stress, as conditioned medium (CM) for the rat cardiomyocyte-derived cell line H9C2. Then we treated H9C2 cells, cultured with or without CM, with hypoxia/reoxygenation to induce cellular senescence and measured senescence-associated β-galactosidase (SA-β-gal) activity, G1 cell proportion and expression of the cell cycle regulators p16^INK4a, p21^Waf1/Cip1 and p53 at mRNA and protein levels in H9C2 cells. In addition, we used Luminex-based analysis to measure concentrations of trophic factors in iPSC-derived CM. Results: We found that iPSC-derived CM reduced SA-β-gal activity, attenuated G1 cell cycle arrest and reduced the expression of p16^INK4a, p21^Waf1/Cip1 and p53 in H9C2 cells. Furthermore, the CM contained more trophic factors, e.g. tissue inhibitor of metalloproteinase-1 and vascular endothelial growth factor, than H9C2-derived CM. Conclusions: Paracrine factors released from iPSCs prevent stress-induced senescence of H9C2 cells by inhibiting p53-p21 and p16-pRb pathways. This is the first report demonstrating that antisenescence effects of stem cell therapy may be a novel therapeutic strategy for age-related cardiovascular disease.

Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes

Purpose: Recently, it has been suggested that chondrocyte senescence plays an important role in the pathogenesis and development of Osteoarthritis (OA). SIRT6 is a member of sirtuin family of NAD+-dependent protein deacetylase. Recently, SIRT6 has been implicated in DNA repair, telomere maintenance, attenuation of inflammation and glucose homeostasis. Notably studies in mice have shown that SIRT6 deficiency produces premature aging phenotypes, whereas SIRT6 overexpression causes a moderate increase in the lifespan of male mice, strongly suggesting an important role of SIRT6 in aging and aging-associated diseases. However, its role in chondrocytes has so far not been explored. The purpose of this study is to examine the role of SIRT6 in human chondrocytes by inhibiting SIRT6 with a RNA interference (RNAi) technique. The hypothesis of the present study is that SIRT6 has a protective function toward human chondrocytes Methods: OA cartilage tissues were obtained from varus knee OA during total knee arthroplasty. Lateral and medial femoral condyles were used as cartilage with mild and severe OA, respectively. Articular cartilage tissues without OA were obtained from the patients underwent surgery for femoral neck fracture. These were used for histological experiments. Next, SIRT6 was depleted by RNAi and examined the influence of SIRT6 depletion on gene expression changes, proliferation, and senescence in normal human chondrocytes. Normal Human Articular Chondrocytes-knee (NHAC-kn) was used as a normal human chondrocytes. RNA or proteins were extracted 48 hours after the lipofection, and real-time PCR and western blotting analysis were respectively performed. The proliferation activity was examined using a Cell Counting Kit-8. Optical density was measured at 0, 24, 48, 72, 96 hours after the lipofection. Senescence associated β-Galactosidase (SA-β-Gal) staining was examined using a SA-β-Gal staining kit. Furthermore, to detect DNA damage and telomere dysfunction, the γH2AX foci and telomere dysfunction-induced foci (TIFs) where γH2AX foci co-localized with telomere repeat binding factor-1 (TRF-1) were examined using an immunofluorescence confocal microscopy. Results: Immunohistochemical analysis showed that SIRT6-positive cells were observed in normal cartilage, and mild and severe OA cartilage. In real-time PCR analysis, the depletion of SIRT6 significantly increased expression of matrix metalloproteinase (MMP) -1 and 13 mRNA. The proliferation assay showed that the absorbance was significantly decreased by the depletion of SIRT6 at 72 and 96 hours after lipofection, indicating reduced proliferation by the depletion of SIRT6. SA-β-Gal assay showed that the percentage of SA-β-Gal positive cells was significantly increased by the depletion of SIRT6, indicating that the depletion of SIRT6 induced premature senescence. Immunofluorescence microscopic analysis showed that γH2AX foci and TIFs was significantly increased in the SIRT6-depleted chondrocytes compared with control. Furthermore, to examine the downstream signaling pathways that mediate the induction of senescence by the depletion of SIRT6, the protein level of mediators for DNA damage induced-senescence, p16 and p21, was examined by western blotting. p16 protein level was increased while p21 protein level was reduced in the in SIRT6-depleted chondrocytes compared with control. Conclusions: The present study demonstrated that the depletion of SIRT6 in human chondrocytes caused increased expression of MMP-1 and 13, reduced proliferation and increased senescent cells. In addition, the depletion of SIRT6 caused accumulation of γH2AX and TIFs. These observations suggested that the depletion of SIRT6 induced premature senescence associated with a secretory phenotypic change and that the induction of the premature senescence may be caused by increased DNA damages and telomere dysfunction. Moreover, we observed p16 was increased by SIRT6 depletion. The p16-pRB pathway has been reported to be a major pathways that mediate premature senescence and proliferation arrest. Therefore taken together, these observations suggest the depletion of SIRT6 in human chondrocytes caused DNA damage and telomere dysfunction, followed by a premature senescence possibly via the p16-pRB pathway. SIRT6 may play an important role to protect chondrocytes from premature senescence, DNA damage, and telomere dysfunction.

In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes

Background contextOur previous in vivo study demonstrated that human nucleus pulposus chondrocytes (NPCs) in aging discs exhibited characteristic senescent features such as an increased senescence-associated β-galactosidase (SA-β-gal) expression, shortened telomere, and decreased telomerase activity. The replicative p53-p21-pRB pathway, rather than the stress-induced p16-pRB pathway, played a more important role in the senescence of NPCs in an in vivo condition, although there is a situation in which both the pathways can be activated simultaneously. However, the in vitro lifespan and senescence mechanisms of human NPCs remain unclear. PurposeTo evaluate the underlying mechanisms of in vitro lifespan and in vitro senescence of the human NPCs and to verify whether the in vitro senescence mechanisms of the human NPC can represent the in vivo aging mechanisms of the cell. Study design/settingAn in vitro study. MethodsWe serially cultivated human NPCs from patients of different ages (35, 42, 55, 66, and 76 years) until the cells reached the end of their in vitro lifespan. During each subcultivation, we calculated NPCs cumulative population doubling level (PDL) and examined senescence markers (SA-β-gal, telomere length, telomerase activity, and p53, p21, pRB, and p16 expressions). ResultsThe cumulative PDLs of the NPCs from patients aged 35, 42 55, 66, and 76 years were 32, 29, 11, 9, and 11, respectively. The younger patients (35 and 42 years) had a higher mean of cumulative PDLs than the elderly patients did (55, 66, and 76 years; 30.5 vs. 10.3; p=.001). In addition, there was a significant, negative correlation between the cumulative PDLs and patient's age (r=−0.89; p=.04). With advancing culture passages, the NPCs irrespective of patient's age exhibited characteristic senescent features, such as an increase in SA-β-gal expression, shortening of telomeres, decrease in telomerase activity, and activation of both replicative p53-p21-pRB and stress-induced p16-pRB pathways. However, all the senescent features occurred at the earlier passages in elderly compared with younger patients. ConclusionsThe present study demonstrated that the human NPCs had a finite in vitro lifespan, which declined with host aging. The in vitro lifespan was determined by both replicative and stress-induced senescence mechanisms. The similarity in the in vitro senescent features with those apparent in the previous in vivo study suggests a possibility of the in vitro senescence mechanisms of the human NPC as a model of the in vivo aging mechanisms of the cell for future studies.

Antitumor effects of naturally occurring oligomeric resveratrol derivatives

This study was designed to evaluate and characterize the molecular basis of antitumor activity of naturally occurring resveratrol (RES; 3,5,4'-trihydroxy-trans-stilbene) derivatives. The compounds were isolated from plants in previous studies and characterized spectroscopically. The antitumor activities of 31 RES derivatives, including dimers, trimers, and tetramers of RES, were evaluated using cell-based assays and validated on a murine model. Several trimeric and a tetrameric stilbenoids induced tumor cell apoptosis or growth arrest of several tumor cell lines with IC50 values (2.8-19.7 μM), significantly lower than that of RES (IC50>70 μM). Using pauciflorol B (PauB) as an example, we showed that the compound induced apoptosis p53 dependently, inducing p53 accumulation and p53-modulated gene expression in cells with wild-type p53, but not in those with nonfunctional p53. Reexpression of p53 in p53-null cells rescued cell death response. In parallel, the MAPK/p38 was activated and critical for PauB-induced killing. Interestingly, activation of p38 in p53 deficient cells was sufficient to drive cells into senescence via the p16-pRb pathway. Finally, PauB dose-dependently inhibited tumor growth on nude mice. Naturally occurring trimeric and tetrameric stilbenoids are potent antitumor agents. Those compounds exert antitumor effect through p53-dependent induction of apoptosis or senescence.

Induction of Cellular Senescence by Doxorubicin Is Associated with Upregulated miR-375 and Induction of Autophagy in K562 Cells

BackgroundCellular senescence is a specialized form of growth arrest that is generally irreversible. Upregulated p16, p53, and p21 expression and silencing of E2F target genes have been characterized to promote the establishment of senescence. It can be further aided by the transcriptional repression of proliferation-associated genes by the action of HP1γ, HMGA, and DNMT proteins to produce a repressive chromatin environment. Therefore, senescence has been suggested to functions as a natural brake for tumor development and plays a critical role in tumor suppression and aging.Methodology/Principal FindingsAn in vitro senescence model has been established by using K562 cells treated with 50 nM doxorubicin (DOX). Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells ought to be independent of p53 and p16-pRb pathways. Indeed, no change in the expression of the typical senescence-associated premalignant cell markers in the DOX-induced senescent K562 cells was found. MicroRNA profiling revealed upregulated miR-375 in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor was able to reverse the proliferation ability suppressed by DOX (p<0.05) and overexpression of miR-375 suppressed the normal proliferation of K562 cells. Upregulated miR-375 expression was associated with downregulated expression of 14-3-3zeta and SP1 genes. Autophagy was also investigated since DOX treatment was able to induce cells entering senescence and eventually lead to cell death. Among the 24 human autophagy-related genes examined, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted.Conclusions/SignificanceThis study has demonstrated that in the absence of p53 and p16, the induction of senescence by DOX was associated with upregulation of miR-375 and autophagy initiation. The anti-proliferative function of miR-375 is possibly exerted, at least in part, by targeting 14-3-3zeta and SP1 genes.

Differences in the expression of five senescence markers in oral cancer, oral leukoplakia and control samples in humans

Oncogene-induced senescence (OIS) may be a response to oncogenic activation, acting as a natural barrier against carcinogenesis at a premalignant stage. Thus, numerous cells in premalignant lesions enter senescence, but none or few in malignant tumours. This event could be due to the loss of senescence pathway effectors, including p16 (INK4a)-pRb or ARF-p53. The aim of this study was to characterize and compare the expression of certain senescent markers between oral precancer and cancer tissue samples. The expression of cyclin D1, Rb, maspin, p53 and mouse double minute 2 (MDM2) was analyzed in 20 paraffin-embedded tissue samples of normal oral mucosa (NOM), 14 samples of oral leukoplakia without dysplasia (OLD-), 11 samples of leukoplakia with dysplasia (OLD+) and 15 samples of oral squamous cell carcinoma (OSCC) by immunohistochemistry in tissue arrays. The expression of p16-pRb pathway markers, cyclin D1, maspin and Rb, was more frequent in OLD+ samples than in OSCC samples, although a statistical significance was only observed for maspin (P=0.036). Cyclin D1 expression was also significantly more frequent in OLD- samples vs. NOM samples. For the ARF-p53 pathway, the expression of p53 and MDM2 was significantly more frequent in the OLD- samples compared to in the NOM ones. These findings may indicate a role for cellular senescence in oral carcinogenesis, considering maspin as a reliable senescence marker and prognostic factor in oral premalignant lesions.

An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence

BackgroundCellular senescence is an irreversible cell cycle arrest that normal cells undergo in response to progressive shortening of telomeres, changes in telomeric structure, oncogene activation or oxidative stress and acts as an important tumour suppressor mechanism.ResultsTo identify the downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways crucial for mediating entry into senescence, we have carried out a loss-of-function RNA interference screen in conditionally immortalised human fibroblasts that can be induced to rapidly undergo senescence, whereas in primary cultures senescence is stochastic and occurs asynchronously. These cells are immortal but undergo a rapid irreversible arrest upon activation of the p53-p21 and p16-pRB pathways that can be readily bypassed upon their inactivation. The primary screen identified 112 known genes including p53 and another 29 shRNAmirs targetting as yet unidentified loci. Comparison of these known targets with genes known to be up-regulated upon senescence in these cells, by micro-array expression profiling, identified 4 common genes TMEM9B, ATXN10, LAYN and LTBP2/3. Direct silencing of these common genes, using lentiviral shRNAmirs, bypassed senescence in the conditionally immortalised cells.ConclusionThe senescence bypass screen identified TMEM9B, ATXN10, LAYN and LTBP2/3 as novel downstream effectors of the p53-p21 and p16-pRB tumour suppressor pathways. Although none of them has previously been linked to cellular senescence, TMEM9B has been suggested to be an upstream activator of NF-κB signalling which has been found to have a causal role in promoting senescence. Future studies will focus on determining on how many of the other primary hits also have a casual role in senescence and what is the mechanism of action.

Induction of Senescence by Doxorubicin Is Associated with Upregulated Mir-375 and Induction of Autophagy In CML Cell Line K562

AbstractAbstract 1843Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p < 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs

Background context The population of senescent disc cells has been shown to increase in degenerated or herniated discs. However, the mechanism and signaling pathway involved in the senescence of nucleus pulposus (NP) chondrocytes are unknown. Purpose To demonstrate the mechanisms involved in the senescence of NP chondrocytes. Study design/setting Senescence-related markers were assessed in the surgically obtained human NP specimens. Patient sample NP specimens remaining in the central region of the intervertebral disc were obtained from 25 patients (mean: 49 years, range: 20–75 years) undergoing discectomy. Based on the preoperative magnetic resonance images, there were 3 patients with Grade II degeneration, 17 patients with Grade III degeneration, and 5 patients with Grade IV degeneration. Outcome measures We examined cell senescence markers (senescence-associated β-galactosidase [SA-β-gal], telomere length, telomerase activity, p53, p21, pRB, and p16) and the hydrogen peroxide (H 2O 2) content as a marker for an oxidative stress in the human NP specimens. Methods SA-β-gal expression, telomere length, telomerase activity, and H 2O 2 content as well as their relationships with age and degeneration grades were analyzed. For the mechanism involved in the senescence of NP chondrocytes, expressions of p53, p21, pRB, and p16 in these cells were assessed with immunohistochemistry and Western blotting. Results The percentages of SA-β-gal-positive NP chondrocytes increased with age (r=.82, p<.001), whereas the telomere length and telomerase activity declined (r=−.41, p=.045; r=−.52, p=.008, respectively) However, there was no significant correlation between age and H 2O 2 contents (p=.18). The NP specimens with Grade III or Grade IV degeneration showed significantly higher percentages of SA-β-gal-positive NP chondrocytes than those with Grade II degeneration (p=.01 and p=.025, respectively). Immunohistochemistry showed that the senescent NP chondrocytes in all the specimens expressed p53, p21, and pRB, but a few NP chondrocytes in only two specimens expressed p16. Western blotting showed that the expressions of p53, p21, and pRB displayed a corresponding pattern, that is, a strong p53 expression led to strong p21 and pRB expressions and vice versa. Conclusions Our in vivo study demonstrated that senescent NP chondrocytes increased or accumulated in the NP with increasing age and advancing disc degeneration. The NP chondrocytes in the aging discs exhibited characteristic senescent features such as an increased SA-β-gal expression, shortened telomeres, and decreased telomerase activity. We further demonstrated that the telomere-based p53-p21-pRB pathway, rather than the stress-based p16-pRB pathway, plays a more important role in the senescence of NP chondrocytes in an in vivo condition. Our results suggest that prevention or reversal of the senescence of NP chondrocytes can be a novel therapeutic target for human disc degeneration.

Inactivation of tumor suppressor genes and the progression of adult T-cell leukemia-lymphoma

Almost three decades have passed since adult T-cell leukemia-lymphoma (ATLL) was proposed as a new disease entity. During this period, its causative agent, human T-cell leukemia virus type-1 (HTLV-1), was found and a crucial role of the viral product Tax in the development of ATLL was disclosed. However, the long latent period after infection with HTLV-1 indicates the need for additional factors for full-blown ATLL, most of which are supposed to be provided by somatic mutations of cellular genes. Recent progress in cell-cycle research has revealed that the uncontrolled and superior proliferative activity of malignant cells is mainly caused by the breakdown of cell-cycle regulation and that most malignancies carry aberrations in p16-pRB and/or p53 pathways. ATLL is not an exception, despite the consistent association of HTLV-1 in primary leukemia cells, and accumulating evidence indicates that the breakdown of these pathways is indeed involved in the leukemogenesis of ATLL, especially in its later steps, which serve as the key events for promotion of indolent ATLL to aggressive ATLL.

Profiles of the 2 INK4a gene products, p16 and p14 ARF, in human reference urothelium and bladder carcinomas, according to pRb and p53 protein status

The INK4a/ARF locus encodes 2 cell cycle regulatory proteins: p16 and p14 ARF. P16 inhibits the activities of cdks, which maintain the retinoblastoma protein (pRb) in its active hypophosphorylated state. P14 ARF blocks MDM2-induced p53 degradation and transactivational silencing. In this study, we investigated the expression of p16 and p14 ARF in reference human urothelium and in 51 urothelial carcinomas (UCs) of all stages and grades, by reverse transcription-polymerase chain reaction (RT-PCR). Patterns of p14 ARF and p16 expression were compared with each other and then with patterns of p53 and pRb protein expression, respectively, as determined by immunohistochemistry. P14 ARF and p16 mRNAs were present at low levels or were undetectable in reference urothelia and in most superficial tumors, whereas they were present at high levels in a subset of tumors of advanced stage and high grade. The expression profiles of these 2 mRNAs were correlated in all but 4 cases, indicating that the 2 INK4a products may have nonredundant functions. Forty-six of the 51 tumors (90%) presented changes to or a lack of activation of the p14 ARF-p53 pathway and were p53 positive (n = 10), p14 ARF negative (n = 23), or both p53 positive and p14 ARF negative (n = 13), suggesting that these 2 components of the pathway may be altered or nonactivated. Markedly high levels of p16 mRNA (n = 5) were associated with the absence of pRb expression, with the exception of 1 case in which the p16 gene contained a deletion mutation. A lack of p16 mRNA or low levels of this mRNA were associated with pRb detection in all but 1 case. In invasive UCs, the p16-pRb pathway, the p14 ARF-p53 pathway, or in many cases both pathways were altered or not activated, demonstrating the involvement of these pathways in invasive bladder tumorigenesis.

Alterations of p16-pRb Pathway and Chromosome Locus 9p21–22 in Sporadic Invasive Breast Carcinomas

The p16-pRb pathway represents a vital cell-cycle checkpoint. In the present study we investigated the alterations of this G1-phase protein pathway using immunohistochemical and molecular methods in a series of 55 breast carcinomas and correlated the findings with clinicopathological features of the patients. Furthermore, we examined its relationship with the status of the chromosomal region 9p21-22 performing a deletion map analysis because there are indications that, in addition to CDKN2 and MTS2/p15(INK4B) tumor suppressor genes (TSGs), this area harbors other TSG(s). Aberrant expression (Ab) of p16 and pRb was observed in 26 (47%) and 16 (29%) of the carcinomas, respectively. A statistical trend pointing out an inverse relationship between p16 and pRb expression was found (p = 0.079). Analysis of the region that encodes for p16 by deletion mapping, a PCR-based methylation assay and PCR-SSCP, revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16(INK4A) inactivation in breast carcinomas. The results of deletion mapping also suggest that another TSG(s) may reside at the 9p21-22 area particularly at the D9S162 loci and that co-deletion of this putative gene with CDKN2/p16(INK4A) may play a role in breast carcinogenesis. In addition, microsatellite instability (MI), a marker of replication error phenotype (RER+), was observed with a frequency of 16% in the area examined and was inversely related with loss of heterozygosity (LOH). Interestingly, most cases with MI at the region encoding for p16 were aggregated in a subgroup of breast carcinomas with no other obvious genetic and/or epigenetic CDKN2/p16(INK4A) alterations. We speculate that there is an additional mechanism of CDKN2/p16(INK4A) inactivation. The relationship of p16 protein level pRb, status, the p16-pRb combined immunoprofiles, and the microsatellite alterations detected at the 9p21-22 locus with the patients' clinicopathological parameters revealed two significant correlations: one between normal pRb expression and lymph node involvement (p = 0.0263), and the other between microsatellite alterations (LOH and or MI) and tumor size (p = 9.2 x 10(-3)). In view of the heterogenous nature of breast cancer, we suggest that in a significant proportion of breast carcinomas, deregulation of the p16-pRb pathway in association with another, as-yet unidentified, TSG(s) of the 9p21-22 region may play a role in initiating or progressing the oncogenic procedure, while in other subgroups, alternative molecules may play this role.