Expression Of p16INK4A Gene Research Articles

Induction of p16Ink4a Gene Expression in Heme Protein-Induced AKI and by Heme: Pathophysiologic Implications.

Understanding the pathogenetic basis for acute kidney injury (AKI) involves the study of ischemic and nephrotoxic models of AKI, the latter including heme protein-induced AKI (HP-AKI). Recently, interest has grown regarding the role of senescence as a mechanism of kidney injury, including AKI. We examined whether senescence occurs in HP-AKI, and potential inducers of, and the role of a key driver of senescence, namely, p16Ink4a, in HP-AKI. The long-established murine glycerol model of HP-AKI was utilized, and indices of senescence were examined. To evaluate the interaction of heme and p16Ink4a expression, murine models of genetic deficiency of hemopexin (HPX) and heme oxygenase-1 (HO-1) were employed. To determine the involvement of p16Ink4a in HP-AKI, the population of p16Ink4a-expressing cells was reduced using the INK-ATTAC model. Using multiple indices, a senescence phenotype appears in the kidney within hours after the induction of HP-AKI. This phenotype includes significant upregulation of p16Ink4a. p16Ink4a is upregulated in the kidney following the individual administration of myoglobin, hemoglobin, and heme, as well as in renal epithelial cells exposed to heme in vitro. Genetic deficiencies of HPX and HO-1, which, independently, are expected to increased heme content in the kidney exaggerate induction of p16Ink4a in the kidney and exacerbate HP-AKI, the latter shown in the present studies involving HPX-/- mice, and in prior studies involving HO-1-/- mice. Finally, reduction in the population of p16Ink4a-expressing cells in the kidney improves renal function in HP-AKI even within 24 hours. The pathogenesis of HP-AKI involves senescence and the induction of p16Ink4a, the latter driven, in part, by hemoglobin, myoglobin, and heme.

Senescence ofbone marrow mesenchymal stem cells in Wistar malerats receiving normal chow/high-calorie diets with/without vitamin D.

Bone marrow mesenchymal stem cells (BM-MSCs) have a momentous function in the composition of the bone marrow microenvironment because of their many valuable properties and abilities, such as immunomodulation and hematopoiesis. The features and actions of MSCs are influenced by senescence, which may be affected by various factors such as nutritional/micronutrients status, e.g., vitamin D. This study aimed to examine the effects of a high-calorie diet (HCD) with/without vitamin D on BM-MSCs senescence. In the first phase, 48 middle-aged rats were fed a normal chow diet (NCD, n = 24) and an HCD (n = 24) for 26 weeks. Afterward, the rats in each group were randomly divided into three equal subgroups. Immediately, eight-rat from each diet group were sacrificed to assess the HCD effects on the first phase measurements. In the second phase, the remaining 4 groups of rats were fed either NCD or HCD with (6 IU/g) or without vitamin D (standard intake: 1 IU/g); in other words, in this phase, the animals were fed (a) NCD, (b) NCD plus vitamin D, (c) HCD, and (d) HCD plus vitamin D for 4 months. BM-MSCs were isolated and evaluated for P16INK4a, P38 MAPK, and Bmi-1 gene expression, reactive oxygen species (ROS) levels, SA-β-gal activity, and cell cycle profile at the end of both phases. After 26 weeks (first phase), the ROS level, SA-β-gal-positive cells, and cells in the G1 phase were significantly higher in HCD-fed rats than in NCD-fed ones (P < 0.05). HCD prescription did not significantly affect cells in the S and G2 phases (p > 0.05). Compared with the NCD-fed animals, P16INK4a and P38 MAPK gene expression were up-regulated in the HCD-fed animals; also, Bmi-1 gene expression was down-regulated (P < 0.05).BM-MSCs from vitamin D-treated rats (second phase) exhibited reduced mRNA levels of P16INK4a and P38 MAPK genes and increased Bmi-1 mRNA levels (all P<0.05). Vitamin D prescription also declined the percentage of SA-β-gal-positive cells, ROS levels, and the cells in the G1 phase and increased the cells in the S phase in both NCD and HCD-fed animals (P <0.05). The reduction of the cells in the G2 phase in rats fed with an NCD plus vitamin D was statistically non-significant (P=0.128) and significant in HCD plus vitamin D rats (P=0.002).HCD accelerates BM-MSCs senescence, and vitamin D reduces BM-MSCs senescence biomarkers.

Stat1 is an inducible transcriptional repressor of neural stem cells self-renewal program during neuroinflammation.

A central issue in regenerative medicine is understanding the mechanisms that regulate the self-renewal of endogenous stem cells in response to injury and disease. Interferons increase hematopoietic stem cells during infection by activating STAT1, but the mechanisms by which STAT1 regulates intrinsic programs in neural stem cells (NSCs) during neuroinflammation is less known. Here we explored the role of STAT1 on NSC self-renewal. We show that overexpressing Stat1 in NSCs derived from the subventricular zone (SVZ) decreases NSC self-renewal capacity while Stat1 deletion increases NSC self-renewal, neurogenesis, and oligodendrogenesis in isolated NSCs. Importantly, we find upregulation of STAT1 in NSCs in a mouse model of multiple sclerosis (MS) and an increase in pathological T cells expressing IFN-γ rather than interleukin 17 (IL-17) in the cerebrospinal fluid of affected mice. We find IFN-γ is superior to IL-17 in reducing proliferation and precipitating an abnormal NSC phenotype featuring increased STAT1 phosphorylation and Stat1 and p16ink4a gene expression. Notably, Stat1-/- NSCs were resistant to the effect of IFN-γ. Lastly, we identified a Stat1-dependent gene expression profile associated with an increase in the Sox9 transcription factor, a regulator of self-renewal. Stat1 binds and transcriptionally represses Sox9 in a transcriptional luciferase assay. We conclude that Stat1 serves as an inducible checkpoint for NSC self-renewal that is upregulated during chronic brain inflammation leading to decreased self-renewal. As such, Stat1 may be a potential target to modulate for next generation therapies to prevent progression and loss of repair function in NSCs/neural progenitors in MS.

MicroRNA-19a-3p Decreases with Age in Mice and Humans and Inhibits Osteoblast Senescence.

Aging is a major risk factor for most chronic diseases, including osteoporosis, and is characterized by an accumulation of senescent cells in various tissues. MicroRNAs (miRNAs) are critical regulators of bone aging and cellular senescence. Here, we report that miR-19a-3p decreases with age in bone samples from mice as well as in posterior iliac crest bone biopsies of younger versus older healthy women. miR-19a-3p also decreased in mouse bone marrow stromal cells following induction of senescence using etoposide, H2O2, or serial passaging. To explore the transcriptomic effects of miR-19a-3p, we performed RNA sequencing of mouse calvarial osteoblasts transfected with control or miR-19a-3p mimics and found that miR-19a-3p overexpression significantly altered the expression of various senescence, senescence-associated secretory phenotype-related, and proliferation genes. Specifically, miR-19a-3p overexpression in nonsenescent osteoblasts significantly suppressed p16 Ink4a and p21 Cip1 gene expression and increased their proliferative capacity. Finally, we established a novel senotherapeutic role for this miRNA by treating miR-19a-3p expressing cells with H2O2 to induce senescence. Interestingly, these cells exhibited lower p16 Ink4a and p21 Cip1 expression, increased proliferation-related gene expression, and reduced SA-β-Gal+ cells. Our results thus establish that miR-19a-3p is a senescence-associated miRNA that decreases with age in mouse and human bones and is a potential senotherapeutic target for age-related bone loss. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Remission of insomnia in older adults treated with cognitive behavioral therapy for insomnia (CBT-I) reduces p16INK4a gene expression in peripheral blood: secondary outcome analysis from a randomized clinical trial

Late life insomnia may increase risk for accelerated biological aging. Intervening to treat insomnia may provide protection from biological aging by reducing the prevalence of senescent cells in the immune system, as indicated by gene expression of a marker of cellular senescence, p16INK4a. In the present study, we determine whether treatment of insomnia in older adults with cognitive behavioral therapy for insomnia (CBT-I) would reduce p16INK4a gene expression in peripheral blood mononuclear cells (PBMC), compared to a sleep education therapy (SET), an active comparator condition. Secondly, we investigate the relationship between sustained insomnia remission and reduced expression of p16INK4a. Participants 60 + years old with insomnia were enrolled in a randomized controlled trial and assigned to CBT-I or SET. Analyses of 231 older adults (CBT-I = 119; SET = 112) examine baseline, post (2 months), and 24 months gene expression of p16INK4a. Compared to baseline, expression of p16INK4a increased in the SET group over 24 months (P = 0.03), but showed no change in the CBT-I group. Those who received CBT-I and experienced sustained remission of insomnia had a significant decline in p16INK4a expression by 24 months compared to baseline (P = 0.02). Individuals not sustaining remission of insomnia exhibited overall increase expression of p16INK4a by 24 months (P = 0.03). In older adults with insomnia, p16INK4a increases over 24 months, while CBT-I treatment of insomnia mitigates the increase in p16INK4a. Further, sustained remission of insomnia using CBT-I leads to a decrease in p16INK4a. These results suggest that behavioral interventions that are effective at treating insomnia might reduce the population of senescent cells in circulating blood.

Changes in hormones, Leukocyte Telomere Length (LTL), and p16INK4a expression in SM-exposed individuals in favor of the cellular senescence

Sulfur mustard (SM) is a chemical warfare agent with well-known severe toxic effects and may cause long-term debilitating injuries. We aimed to evaluate aging and longevity in Iranian SM-exposed survivors using some endocrine and molecular biomarkers for the first time. Dehydroepiandrosterone (DHEA), prolactin (PRL), cortisol, testosterone, and luteinizing hormone (LH) were measured in 289 male SM-veterans and 66 age-matched males using the ELISA method. Leukocyte Telomere Length (LTL) measurement and p16INK4a expression were measured in the peripheral blood leukocytes of 55 males who were exposed to SM. We found a significantly lower serum DHEAS level and higher serum PRL level in SM-exposed groups (without any related to the severity of lung injuries) compared to healthy controls, but no significant difference in serum levels of cortisol, testosterone, and LH. The molar ratio of DHEAS/cortisol was significantly higher in controls compared to the SM-exposed individuals especially those with severe lung damage. Some biological parameters of allostatic load score such as DHEAS and DHEAS/cortisol ratio significantly decreased long-term after the SM exposure. Additionally, we found that LTL was shorter in SM-exposed veterans rather than unexposed controls while p16INK4a gene expression significantly increased in these groups. It seems that DHEAS, DHEAS/cortisol ratio, LTL, and p16INK4a gene expression have changed significantly in favor of cellular senescence in SM-exposed patients. Therefore, it seems that SM exposure increases biological age compared to chronological age in SM-exposed survivors.

Delayed effects of radiation in adipose tissue reflect progenitor damage and not cellular senescence.

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAβ-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6months and 3years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-βgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

Molecular phenotypes of colorectal cancer.

e15506 Background: Despite the positive results of colorectal cancer treatment, there is a group of patients that progresses despite successful treatment for I–II stage of the disease. it may be that modern predictors in do not fully cover the entire spectrum of molecular genetic traits, especially the tumor microenvironment. Perhaps expression analysis is not only tumor tissue, but resection margin could also reach a consensus on the molecular gene phenotypes of colorectal cancer with the achievement of the goal of prognostic efficiency and therapeutic and prophylactic diagnostic tactics. Methods: The expression level of 64 genes in tumor tissue in samples obtained from 172 patients with stage I–III CRC was analyzed by real-time Polymerase chain reaction (PCR). The mean follow-up time was 28.15±14.7 months. Data analysis and predictive modeling arrays using the R 3.6.3 Computation Evaluation Framework (R Foundation for statistical computing, Vienna, Austria) using additional packages rms 5.1-4, survival 3.2-7 and pROC 1.16.2. Results: As a result of statistical analysis, data were obtained on the presence of predictors of the studied unfavorable source code among the selected sets of genes carcinogenesis in colorectal cancer. The next step revealed progression status with BIRC5, IL2, SCUBE2, MYC and P16INK4A gene expression as in tissues tumors, and in resection margin. 2 regression models were built to predict survival without progression in patients with the inclusion of the level of expression in the tumor tissue- nor, the inclusion of the level of gene expression in tumor and resection margin. Model with inclusion only tumor transcripts statistics did not differ from models without predictors. Conclusions: In this study, it was possible to identify a group of genes associated with certain favorable prognosis and responsible in one way or another for proliferation, angiogenesis, immune status of the tumor. It can be concluded that the study of the level of expression of numerical genes in tumor tissue can become a promising direction for the selection of patients in the group of poor prognosis in colorectal cancer.

Isatis tinctoria L. Leaf Extract Inhibits Replicative Senescence in Dermal Fibroblasts by Regulating mTOR-NF-κB-SASP Signaling.

Senescent fibroblasts progressively deteriorate the functional properties of skin tissue. Senescent cells secrete senescence-associated secretory phenotype (SASP) factor, which causes the aging of surrounding non-senescent cells and accelerates aging in the individuals. Recent findings suggested the senomorphic targeting of the SASP regulation as a new generation of effective therapeutics. We investigated whether Isatis tinctoria L. leaf extract (ITE) inhibited senescence biomarkers p53, p21CDKN1A, and p16INK4A gene expression, and SASP secretions by inhibiting cellular senescence in the replicative senescent human dermal fibroblast (RS-HDF). ITE has been demonstrated to inhibit the secretion of SASP factors in several senomorphic types by regulating the MAPK/NF-κB pathway via its inhibitory effect on mTOR. ITE suppressed the inflammatory response by inhibiting mTOR, MAPK, and IκBα phosphorylation, and blocking the nuclear translocation of NF-κB. In addition, we observed that autophagy pathway was related to inhibitory effect of ITE on cellular senescence. From these results, we concluded that ITE can prevent and restore senescence by blocking the activation and secretion of senescence-related factors generated from RS-HDFs through mTOR-NF-κB regulation.

Expression OF p16INK4a Gene in Premalignant and Malignant Lesions of Oral Cavity

Squamous cell carcinoma is the summits malignant neoplasm of the oral cavity. Tobacco and alcohol is identified as risk factors, but squamous cell carcinoma can occur in patients with no known risk factors. Oral cancer is the sixth most common malignancy and is one of the major causes of cancer morbidity and mortality worldwide. Cancer is caused due to a series of alteration in genetic and epigenetic factors that occur in multiple steps and is influenced by the genetic predisposition of the individual and by exogenous environmental factors. These factors result in a series of molecular alteration, including inactivation of tumor suppressor genes expression of p16 has been proposed as a marker for malignant transformation. The p16 staining was correlated between the control and study groups and p 16 was shown to be increasing expressed in premalignant and less expressed in malignant category and was found to be statistically significant by Fischer’s exact test. This study concluded that p16 was increasingly expressed in premalignant lesions and less expressed in malignant lesion. In the present study 9 of the control cases were p16 negative and one case showed sporadic staining. The study group I showed 1 case of sporadic staining, 6 cases of focal staining and 8 cases of diffuse staining. The study II showed 14 cases of sporadic staining, 6 cases of focal staining and 5 cases of diffuse staining. Hence variations cannot be accurately assessed, but it plays a crucial role in assessing pre-malignant lesions progressing to malignancy. To confirm this, a larger sample study is required. As advances in research have leads to greater understanding of potentially malignant lesions in the oral cavity.

Glycine alleviates fluoride‐induced oxidative stress, apoptosis and senescence in a porcine testicular Sertoli cell line

Glycine is a well-known free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. Excessive fluoride exposure is associated with multiple types of cellular damage in humans and animals. The objective of the present study was to investigate the protective effects of glycine on sodium fluoride (NaF) exposure and the possible underlying mechanisms in a porcine testicular Sertoli cell line model. Cellular viability and proliferation were examined following NaF exposure and glycine supplementation, and glycine dramatically ameliorated the decreases in NaF-induced porcine testicular Sertoli cell viability and proliferation. Further investigations revealed that glycine decreased NaF-induced intracellular reactive oxygen species production, DNA fragment accumulation and the apoptosis incidence in the porcine testicular Sertoli cell line; in addition, glycine improved mitochondrial function and ATP production. Notably, results of the SPiDER-β-Gal analysis suggested that glycine alleviated NaF-induced cellular senescence and downregulated P53, P21, HMGA2 and P16INK4a gene expression in the porcine testicular Sertoli cell line. Collectively, the beneficial effects of glycine alleviate NaF-induced oxidative stress, apoptosis and senescence, and together with our previous findings, support the hypothesis that glycine plays an important role in protecting against NaF exposure-induced impairments in the porcine testicular Sertoli cell line.

Myeloma Derived Extracellular Vesicles Containing Endoplasmic Reticulum Remodel the Bone Marrow Microenvironment Towards a Pro-Tumoral Senescent Phenotype

Multiple myeloma (MM) is a tumor characterised by the generation of large quantities of immunoglobulin which undergoes protein folding and secretion through the endoplasmic reticulum (ER). Many studies have shown that primary MM cells have high ER stress (Lee et al, 2003 PNAS, Obeng et al 2006 Blood and Mimura et al, 2012 Blood,). Moreover, it has also been shown that serum from MM contains significantly more extracellular vesicles (EVs) than serum from normal patients (Caivano et al, 2015 Tumour Biol). Here we explore if these two observations are connected and determine whether primary MM cells export ER inside EVs and the impact this has on the tumor microenvironment. Primary MM and primary MM bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. To determine if MM secrete ER inside EV, patient derived MM and MM cell lines were transduced with rLV.EF.mCherry-ER lentivirus which fluorescently tags the ER. High-resolution imaging combined with image cytometry shows that CD38+ vesicles containing ER are formed by budding from the MM cell surface. Analysis of MM derived EVs using a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that MM actively export ER in the large EVs, approximately 0.6micron-1.5micron in diameter. To track the recipient cell for large EV packaged ER in vivo, U266 MM cell line (transduced with rLV.EF.mCherry-ER9 lentivirus) was injected into the tail of NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence, as evidence of MM derived ER uptake. Murine CD45-/CD31-Ter119-/CD105+/CD140a+ BMSC had increased mCherry fluorescence but not F4/80+/GR1-/CD115int BM macrophages or CD45-/Ter119-/CD31+ endothelial cells or CD45+ leukocytes. Two proteins detected at high levels in the proteomics analysis of large EV were endoplasmic reticulum oxidoreductin 1 (ERO1) and protein disulfide-isomerase (P4B4) which functionally catalyze the formation, breakage and rearrangement of disulfide bonds resulting in the production of superoxide. Next, we analysed large EVs containing ER for superoxide. MM derived ER+ and ER- large EVs were isolated by sorting for mCherry fluorescence (MM transduced with rLV.EF.mCherry-ER9 lentivirus) and Amplex Red assay confirmed that ER+ large EVs had increased levels of H2O2. In vivo analysis of the BMSC from MM engrafted NSG mice confirmed high oxidative stress as measured by increased H2DCFDA fluorescence. To determine the impact of MM derived ER containing EVs on the function of the BMSC isolated ER containing EVs were incubated with BMSC repeatedly for up to 7 days and senescent markers were assessed. Beta-galactosidase staining, p16ink4a gene expression and a senescence associated secretary phenotype (SASP) were all upregulated in BMSC cultured with MM derived ER+ large EV and not ER- large EV. To determine if MM induced BMSC senescence in vivo we injected U266 and primary MM into NSG mice, humanised NSG mice were used as a control. Post MM engraftment, animals were sacrificed and BMSC were isolated by cell sorting for CD45-/CD31-Ter119-/CD105+/CD140a+ cells and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BMSC from U266 and primary MM engrafted NSG mice and not from humanised NSG mice. Knockdown of p16 in BMSC prevents ER+ large EV from inducing a SASP and conditioned media had no effect on MM proliferation compared to conditioned media from ER+ large EV treated BMSC. Finally, we used an NSG mouse model whereby we transplanted p16ink4a KD BMSC or control KD BMSC with MM cells subcutaneously into the flank. MM combined with p16ink4a KD BMSC has reduced tumor volume compared with animals with control KD BMSC. Data indicates that MM secrete ER in large EV and that MM derived EVs containing ER function as a signal which then changes the physiology of BMSC, towards a senescent phenotype which in turn promotes malignant plasma cell survival and proliferation. Disclosures Bowles: Janssen: Research Funding; Abbvie: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Autofluorescence is a Reliable in vitro Marker of Cellular Senescence in Human Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSC) are used in cell therapies, however cellular senescence increases heterogeneity of cell populations and leads to uncertainty in therapies’ outcomes. The determination of cellular senescence is time consuming and logistically intensive. Here, we propose the use of endogenous autofluorescence as real-time quantification of cellular senescence in human MSC, based on label-free flow cytometry analysis. We correlated cell autofluorescence to senescence using senescence-associated beta-galactosidase assay (SA-β-Gal) with chromogenic (X-GAL) and fluorescent (C12FDG) substrates, gene expression of senescence markers (such as p16INK4A, p18INK4C, CCND2 and CDCA7) and telomere length. Autofluorescence was further correlated to MSC differentiation assays (adipogenesis, chondrogenesis and osteogenesis), MSC stemness markers (CD90/CD106) and cytokine secretion (IL-6 and MCP-1). Increased cell autofluorescence significantly correlated with increased SA-β-Gal signal (both X-GAL and C12FDG substrates), cell volume and cell granularity, IL-6/MCP-1 secretion and with increased p16INK4A and CCND2 gene expression. Increased cell autofluorescence was negatively associated with the expression of the CD90/CD106 markers, osteogenic and chondrogenic differentiation potentials and p18INK4C and CDCA7 gene expression. Cell autofluorescence correlated neither with telomere length nor with adipogenic differentiation potential. We conclude that autofluorescence can be used as fast and non-invasive senescence assay for comparing MSC populations under controlled culture conditions.

Evaluation of UHRF1 and P16INK4A expression levels in newly diagnosed AML patients

Introduction: Gene mutation is an infrequent cause of tumor suppressor gene (TSG) defect in de novo AML patients. Instead, it seems that leukemic cells employ epigenetic tricks to attenuate the negative impacts of intact TSGs. Ordinarily, critical TSGs, such as p16INK4A, is hyper-methylated in AML blasts under the impact of master epigenetic regulators, such as UHRF1. In this study, we investigated the correlation between UHRF1 and p16INK4A gene expression levels in newly diagnosed AML patients.&#x0D; Methods: Bone marrow and peripheral blood samples were obtained from 50 newly diagnosed AML patients and 18 healthy normal control subjects. Gene expression levels of UHRF1 and P16INK4A were surveyed using SYBR Green Quantitative Real-time PCR. Statistical analyses were done using SPSS statistical software 21.0.&#x0D; Results: P16INK4A gene expression showed reduced levels in 80.64% of patients above 45 years of age, while only 32% of patients below 45 years had reduced expression levels. The Spearman correlation test also demonstrated a significant negative correlation between UHRF1 and p16INK4A gene expression levels in AML patients, which was not observed in the control group (r=0.343 and P= 0.015).&#x0D; Conclusion: Regarding the age-related patterns of UHRF1 and p16INK4A gene expression, and also the presence of negative correlation between them, we conclude that UHRF1 may potentially be involved in p16INK4A down-regulation in elderly AML patients, which may subsequently facilitate the progression of AML in older ages.&#x0D;

Influence of aging in the modulation of epigenetic biomarkers of carcinogenesis after exposure to air pollution.

Classified as carcinogenic to humans by the IARC in 2013, fine air particulate matter (PM2.5) can be inhaled and retained into the lung or reach the systemic circulation. This can cause or exacerbate numerous pathologies to which the elderly are often more sensitive. In order to estimate the influence of age on the development of early cellular epigenetic alterations involved in carcinogenesis, peripheral blood mononuclear cells sampled from 90 patients from three age classes (25-30, 50-55 and 75-80 years old) were ex vivo exposed to urban PM2.5. Particles exposure led to variations in telomerase activity and telomeres length in all age groups without any influence of age. Conversely, P16INK4A gene expression increased significantly with age after exposure to PM2.5. Age could enhance MGMT gene expression after exposure to particles, by decreasing the level of promoter methylation in the oldest people. Hence, our results demonstrated several tendencies in cells modification depending on age, even if all epigenetic assays were carried out after a limited exposure time allowing only one or two cell cycles. Since lung cancer symptoms appear only at an advanced stage, our results underline the needs for further investigation on the studied biomarkers for early diagnosis of carcinogenesis to improve survival.

A Retrospective 20-Year Analysis of Proliferative Verrucous Leukoplakia and Its Progression to Malignancy and Association with High-risk Human Papillomavirus.

Proliferative verrucous leukoplakia (PVL) is defined as an aggressive, relentless and recalcitrant form of leukoplakia that has a high propensity for malignant transformation. The aim of this study was to evaluate the malignant potential of PVL and determine its possible association with high-risk human papillomavirus (HPV). Twenty cases with a clinical and biopsy proven diagnosis of PVL were collected from the University of Florida Oral Medicine clinic database. Immunohistochemistry was performed to evaluate the expression of p16INK4A and p53 genes in the PVL lesions. The lesions were also tested for high-risk HPV by DNA in-situ hybridization. The average age of the patients at the time of first biopsy was 62.7years. Most patients had multiple sites of involvement, gingiva being the most common location. The lesions progressed to malignancy in approximately 50% of patients. The expression of p16INK4A gene was considered negative, with at least a 50-65% immunoreactivity observed in only three cases that progressed to malignancy. No expression of high-risk HPV was detected, whereas p53 staining was positive in less than 25% of the cells demonstrating gene expression. No definite association between PVL and high-risk HPV infection could be established. Due to the high transformation potential of PVL, early recognition with aggressive treatment, including multiple biopsies, and continued close clinical follow-up, remain the mainstay of favorable management of this condition.

ZBP-89 function in colonic stem cells and during butyrate-induced senescence.

ZBP-89 (Zfp148, ZNF148) is a Kruppel-type zinc-finger family transcription factor that binds to GC-rich DNA elements. Earlier studies in cell lines demonstrated that ZBP-89 cooperates with Wnt β-catenin signaling by inducing β-catenin gene expression. Since β-catenin levels are normally highest at the crypt base, we examined whether ZBP-89 is required for stem cell maintenance. Lineage-tracing using a Zfp148CreERT2 transgenic line demonstrated expression in both intestine and colonic stem cells. Deleting the Zfp148 locus in the colon using the Cdx2NLSCreERT2 transgene, reduced the size and number of polyps formed in the Apc-deleted mice. Since colon polyps form in the presence of butyrate, a short chain fatty acid that suppresses cell growth, we examined the direct effect of butyrate on colon organoid survival. Butyrate induced senescence of colon organoids carrying the Apc deletion, only when Zfp148 was deleted. Using quantitative PCR and chromatin immunoprecipitation, we determined that butyrate treatment of colon cell lines suppressed ZNF148 gene expression, inducing CDKN2a (p16Ink4a) gene expression. Collectively, Zfp148 mRNA is expressed in CBCs, and is required for stem cell maintenance and colonic transformation. Butyrate induces colonic cell senescence in part through suppression of ZBP-89 gene expression and its subsequent occupancy of the CDKN2A promoter.

The tumour suppressor CDKN2A/p16INK4a regulates adipogenesis and bone marrow-dependent development of perivascular adipose tissue.

The genomic CDKN2A/B locus, encoding p16INK4a among others, is linked to an increased risk for cardiovascular disease and type 2 diabetes. Obesity is a risk factor for both cardiovascular disease and type 2 diabetes. p16INK4a is a cell cycle regulator and tumour suppressor. Whether it plays a role in adipose tissue formation is unknown. p16INK4a knock-down in 3T3/L1 preadipocytes or p16INK4a deficiency in mouse embryonic fibroblasts enhanced adipogenesis, suggesting a role for p16INK4a in adipose tissue formation. p16INK4a-deficient mice developed more epicardial adipose tissue in response to the adipogenic peroxisome proliferator activated receptor gamma agonist rosiglitazone. Additionally, adipose tissue around the aorta from p16INK4a-deficient mice displayed enhanced rosiglitazone-induced gene expression of adipogenic markers and stem cell antigen, a marker of bone marrow-derived precursor cells. Mice transplanted with p16INK4a-deficient bone marrow had more epicardial adipose tissue compared to controls when fed a high-fat diet. In humans, p16INK4a gene expression was enriched in epicardial adipose tissue compared to other adipose tissue depots. Moreover, epicardial adipose tissue from obese humans displayed increased expression of stem cell antigen compared to lean controls, supporting a bone marrow origin of epicardial adipose tissue. These results show that p16INK4a modulates epicardial adipose tissue development, providing a potential mechanistic link between the genetic association of the CDKN2A/B locus and cardiovascular disease risk.

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells.

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.

Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2′-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5′ CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.