p16INK4a Expression Levels Research Articles

Sodium valproate affects the expression of p16INK4a and p21WAFI/Cip1 cyclin‑dependent kinase inhibitors in HeLa cells.

p16INK4a and p21WAF1/Cip1 are cyclin-dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus-induced cervical cancer, p16 INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21 WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16 INK4a and p21 WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription-quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16 INK4a and a tendency for p16INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16 INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16 INK4a and p21 WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Topical application of a BCL-2 inhibitor ameliorates imiquimod-induced psoriasiform dermatitis by eliminating senescent cells

BackgroundPsoriasis is an inflammatory skin disease with unclear pathogenesis and unmet therapeutic needs. ObjectiveTo investigate the role of senescent CD4+ T cells in psoriatic lesion formation and explore the application of senolytics in treating psoriasis. MethodsWe explored the expression levels of p16INK4a and p21, classical markers of cellular senescence, in CD4+ T cells from human psoriatic lesions and imiquimod (IMQ)-induced psoriatic lesions. We prepared a senolytic gel using B-cell lymphoma 2 (BCL-2) inhibitor ABT-737 and evaluated its therapeutic efficacy in treating psoriasis. ResultsUsing multispectrum immunohistochemistry (mIHC) staining, we detected increased expression levels of p16INK4a and p21 in CD4+ T cells from psoriatic lesions. After topical application of ABT-737 gel, significant alleviation of IMQ-induced psoriatic lesions was observed, with milder pathological alterations. Mechanistically, ABT-737 gel significantly decreased the percentage of senescent cells, expression of T cell receptor (TCR) α and β chains, and expression of Tet methylcytosine dioxygenase 2 (Tet2) in IMQ-induced psoriatic lesions, as determined by mIHC, high-throughput sequencing of the TCR repertoire, and RT-qPCR, respectively. Furthermore, the severity of psoriatic lesions in CD4creTet2f/f mice was milder than that in Tet2f/f mice in the IMQ-induced psoriasis model. ConclusionWe revealed the roles of senescent CD4+ T cells in developing psoriasis and highlighted the therapeutic potential of topical ABT-737 gel in treating psoriasis through the elimination of senescent cells, modulation of the TCR αβ repertoire, and regulation of the TET2-Th17 cell pathway.

Senolytic effects on dental pulp stem cell's proliferation and differentiation during long-term expansion

Objective: To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells (DPSC) in long-term, large-scale expansion, and to explore strategies for maintaining the youthful state of DPSC in vitro. Methods: Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons, provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology, Sichuan University. Long-term, large-scale in vitro expansion of DPSC was conducted. The study compared young DPSC (passage 5) with aged DPSC (passage 25) using cellular senescence-associated β-galactosidase staining, colony formation assay, and Alizarin Red S staining for osteogenic differentiation induction. To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability. Medicine screening for the most effective senolytic was compared among 5 common senolytics [Navitoclax (ABT-263), curcumin, dasatinib, fisetin, and quercetin]. The clearance efficacy was compared using cellular senescence-associated β-galactosidase staining to reflect the changes in senescent cell ratio. The senolytic with the highest efficacy was chosen for further experiments. The passage at which the proportion of senescent cells significantly increased was identified, and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells. Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associated β-galactosidase staining, real-time fluorescence quantitative PCR (RT-qPCR), colony formation assay, wound healing assay, and Alizarin Red S staining for osteogenic differentiation induction. Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase (ALP) immunohistochemical staining. Results: The proportion of senescent cells increased as the expansion extended, leading to decreased proliferation and osteogenic differentiation ability of senescent DPSC compared to young DPSC (P<0.05). Senescent DPSC exhibited altered mRNA expression levels of senescence-related genes, including p21, p16INK4a, IL-6, and Ki67 (P<0.001). Among the five senolytics, ABT-263 had the biggest decreases in the proportion of senescent cells. After intermittent ABT-263 treatment during expansion, the proportion of senescent cells in the senolytic-treated group [(6.72±2.34)%] was significantly lower than that in the control group [(31.82±0.57)%] (P<0.001). RT-qPCR confirmed that compared with the control group, mRNA expressions of p21, p16INK4a, and IL-6 in the senolytic-treated group were significantly decreased (P<0.05), while mRNA expressions of Ki67 were significantly increased (P<0.01). Furthermore, the cell healing ability and osteogenic differentiation ability of the senolytic-treated group were higher than those of the control group (P<0.05). In vivo experimental results indicated that the relative new bone area [(2.36±0.48)%] after DPSC transplantation in the senolytic-treated group was greater than that in the control group [(1.00±0.46)%] (P<0.05), and the expression of ALP was higher than that in the control group (P<0.01). Conclusions: ABT-263 can effectively eliminate senescent cells in long-term large-scale DPSC expansion. Continuous treatment with ABT-263 during cultivation can maintain the proliferation and differentiation ability of DPSC both in vivo and in vitro.

Endogenous sulfur dioxide deficiency as a driver of cardiomyocyte senescence through abolishing sulphenylation of STAT3 at cysteine 259

ObjectiveCardiomyocyte senescence is an important contributor to cardiovascular diseases and can be induced by stressors including DNA damage, oxidative stress, mitochondrial dysfunction, epigenetic regulation, etc. However, the underlying mechanisms for the development of cardiomyocyte senescence remain largely unknown. Sulfur dioxide (SO2) is produced endogenously by aspartate aminotransferase 2 (AAT2) catalysis and plays an important regulatory role in the development of cardiovascular diseases. The present study aimed to explore the effect of endogenous SO2 on cardiomyocyte senescence and the underlying molecular mechanisms. Approach and resultsWe interestingly found a substantial reduction in the expression of AAT2 in the heart of aged mice in comparison to young mice. AAT2-knockdowned cardiomyocytes exhibited reduced SO2 content, elevated expression levels of Tp53, p21Cip/Waf, and p16INk4a, enhanced SA-β-Gal activity, and elevated level of γ-H2AX foci. Notably, supplementation with a SO2 donor ameliorated the spontaneous senescence phenotype and DNA damage caused by AAT2 deficiency in cardiomyocytes. Mechanistically, AAT2 deficiency suppressed the sulphenylation of signal transducer and activator of transcription 3 (STAT3) facilitated its nuclear translocation and DNA-binding capacity. Conversely, a mutation in the cysteine (Cys) 259 residue of STAT3 blocked SO2-induced STAT3 sulphenylation and subsequently prevented the inhibitory effect of SO2 on STAT3-DNA-binding capacity, DNA damage, and cardiomyocyte senescence. Additionally, cardiomyocyte (cm)-specific AAT2 knockout (AAT2cmKO) mice exhibited a deterioration in cardiac function, cardiomegaly, and cardiac aging, whereas supplementation with SO2 donors mitigated the cardiac aging and remodeling phenotypes in AAT2cmKO mice. ConclusionDownregulation of the endogenous SO2/AAT2 pathway is a crucial pathogenic mechanism underlying cardiomyocyte senescence. Endogenous SO2 modifies STAT3 by sulphenylating Cys259, leading to the inhibition of DNA damage and the protection against cardiomyocyte senescence.

Senescence marker p16INK4a expression in patients with multiple sclerosis

ObjectivesTelomere attrition is associated with disability accumulation and brain atrophy in multiple sclerosis (MS). Downstream of telomere attrition is cellular senescence. We sought to determine differences in the cellular senescence marker p16INK4a expression between MS and healthy control participants and the association of p16INK4a expression with MS disability and treatment exposure. MethodsPatients meeting diagnostic criteria for MS and healthy controls were recruited for a cross-sectional pilot study. RNA was extracted from peripheral blood mononuclear cells (PBMCs) and p16INK4a expression levels were measured using qRT PCR. Spearman correlation coefficients and regression models were applied to compare expression levels to chronological age, assess case control differences, and determine associations with clinical outcome measures. ResultsFifty-two participants with MS (67 % female, ages 25–70) and 38 healthy controls (66 % female, ages 23–65) were included. p16INK4a levels were not linearly correlated with chronological age in MS (rhos = −0.01, p = 0.94) or control participants (rhos = 0.02, p = 0.92). Higher median p16INK4a levels were observed in the >50-year age group for MS (0.25, IQR 0.14–0.35) vs. controls (0.12, IQR 0.05–0.15) and in this age group B cell depletion therapy was associated with lower expression levels. p16INK4a expression was not associated with any of the measured MS disability outcomes. DiscussionCaution is needed with using p16INK4a expression level from PBMCs as an aging biomarker in MS participants, given lack of correlation with chronological age or large associations with clinical outcomes.

TARGETING P21CIP1-HIGHLY-EXPRESSING SENESCENT CELLS TO IMPROVE HEALTHSPAN IN PROGEROID MICE

Abstract Aging is the leading risk factor for a variety of chronic diseases. Emerging evidence suggests that modulating fundamental aging processes such as cellular senescence may delay the onset of most chronic conditions as a group and increase healthy lifespan. Cellular senescence refers to the essentially irreversible growth arrest that occurs when cells experience stress. One common feature of senescent cells is the high expression level of p16Ink4a and/or p21Cip1. We and others have demonstrated the causal roles of p16Ink4a-highly-expressing (p16high) senescent cells in various age-related conditions. However, the role of p21high senescent cells in aging remains largely unknown. To address these questions, we have generated and validated a novel p21-Cre mouse model containing p21 promoter driving inducible Cre. By crossing with floxed mice, we managed to monitor, sort, image, eliminate, or modulate p21high cells in vivo. We investigated the maximal walking speed and grip strength of klotho (kl)-/- progeroid mice weekly starting from 5 weeks old until death. We showed that clearance of p21high cells can improve physical function in kl-/- mice for a long time period. Additionally, inactivation of NF-κB specifically in p21high cells also can improve tissue function in kl-/- mice. This study will provide invaluable research tools and pinpoint the role of cellular senescence in age-related tissue dysfunction.

The senolytic agent Dasatinib reduces fat accumulation in the heart and bone marrow of type 2 diabetic, obese mice and adipogenesis of human bone marrow pericytes

Abstract Background Inflammaging, a chronic inflammatory state associated with aging and obesity, participates in the pathogenesis of cardiovascular disease and type 2 diabetes (T2D). Adipocyte accumulation in bone marrow (BM) may contribute to this phenomenon. Senolytic drugs, like Dasatinib, reportedly eliminate senescent preadipocytes. However, the effects of Dasatinib on fat deposits in the heart and BM remain unknown. Purposes This study aimed to identify the cellular source of adipocytes in human BM and determine whether treatment with Dasatinib reverses adiposity and improves cardiovascular function in T2D obese mice. Methods and results Human CD34neg CD45neg CD146pos pericytes (hBM-PCs) were sorted from BM mesenchymal stem cells (hBM-MSCs) by immunomagnetic beads. HBM-PCs showed a higher propensity to differentiate into Oil red O positive (pos) adipocytes after 21 days of stimulation in a pro-adipogenic medium (P=0.0299 vs. hBMSCs (hBM-MSCs except for hBM-PCs)). Dasatinib treatment reduced the number of viable cells (P=0.0034 vs. Vehicle (DMSO)) and the rate of adipocyte differentiation (P= 0.0179 vs. Vehicle). In a randomized blind study, Dasatinib (5 mg/kg) or vehicle (10% DMSO+90% PEG-300) was given by oral gavage to T2D, obese 21-week-old male BKS.Cg-+Leprdb/+Leprdb/OlaHsd (Db/Db) mice. Groups of animals were sacrificed at 1 or 4 weeks. Dasatinib reduced the Oil red O pos cells in the heart (P= 0.001 and 0.045) and BM (P= 0.018 and 0.0253) at both time points. Senescence marker p16INK4a expression and plasma adipokines levels were similar between the two groups at one week. With long-term treatment, the expression of senescence marker p16INK4a was significantly reduced in the murine hearts (P=0.035) and BM (P=0.068), whereas adipokines levels were similar. In vitro treatment of murine BM-MSCs with Dasatinib reduced the expression of p21Waf1/Cip1 (P= 0.030). VisualSonics 3D echocardiography showed similar diastolic and systolic function in Dasatinib- or Vehicle-treated mice: stroke volume (P= 0.494); ejection fraction (P= 0.701); fractional shorten (P= 0.807); and cardiac output (P= 0.437). Conclusion Pericytes are the primary source of adipocytes in human BM. The senolytic agent Dasatinib reduces pericyte commitment to adipogenesis. The senolytic agent prevented fat accumulation and cell senescence in the BM and heart of T2D mice. However, more prolonged treatment may be necessary for the reduced adiposity to translate into improvements in heart function.

Korean Red Ginseng Attenuates Particulate Matter-Induced Senescence of Skin Keratinocytes.

Skin is a direct target of fine particulate matter (PM2.5), as it is constantly exposed. Herein, we investigate whether Korean red ginseng (KRG) can inhibit PM2.5-induced senescence in skin keratinocytes. PM2.5-treated human keratinocyte cell lines and normal human epidermal keratinocytes showed characteristics of cellular senescence, including flat and enlarged forms; however, KRG suppressed them in both cell types. Moreover, while cells exposed to PM2.5 showed a higher level of p16INK4A expression (a senescence inducer), KRG inhibited its expression. Epigenetically, KRG decreased the expression of the ten-eleven translocation (TET) enzyme, a DNA demethylase induced by PM2.5, and increased the expression of DNA methyltransferases suppressed by PM2.5, resulting in the decreased methylation of the p16INK4A promoter region. Additionally, KRG decreased the expression of mixed-lineage leukemia 1 (MLL1), a histone methyltransferase, and histone acetyltransferase 1 (HAT1) induced by PM2.5. Contrastingly, KRG increased the expression of the enhancer of zeste homolog 2, a histone methyltransferase, and histone deacetyltransferase 1 reduced by PM2.5. Furthermore, KRG decreased TET1, MLL1, and HAT1 binding to the p16INK4A promoter, corresponding with the decreased mRNA expression of p16INK4A. These results suggest that KRG exerts protection against the PM2.5-induced senescence of skin keratinocytes via the epigenetic regulation of p16INK4A.

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.

Background: It is well known that P16 INK4A , P14 ARF , P15 INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14 ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16 INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16 INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16 INK4A expression is mediated with CBX7. Results: In this study, we found that the upregulation of P16 INK4A , P14 ARF , P15 INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16 INK4A , P14 ARF and P15 INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells. Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16 INK4A , P14 ARF , P15 INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.

Hypoglycaemic and anti-ageing activities of green alga Ulva lactuca polysaccharide via gut microbiota in ageing-associated diabetic mice

Ageing-related type 2 diabetes is a significant public health problem. Particularly, the number of cases and fatality rates of ageing-associated diabetes increase with population ageing. This study aimed to investigate the structural characterisation of Ulva lactuca polysaccharide (ULP) and the hypoglycaemic effect on ageing-associated diabetic mice using gut microbiota variation. Sugar residuals analysis showed that the purified ULP (ULP-1) comprised β-D-Xylp-(1→3)-β-D-Arap-(1→6)-β-D-Galp-(1→6)-β-D-Glcp linked to [→α-L-Rhap-(1→4)-β-D-GlcpA→]n and α-D-Manp-(1→4)-α-L-Rhap(2SO3−)-(1→2)-α-L-Rhap(4SO3−)-(1→2)-α-L-Arap-(1→2)-α-L-Rhap-(1→ as its side chains at β-D-Glcp. Moreover, ULP modulated the expression levels of p16Ink4a, MMP2, FoxO1, GLP-1/GLP-1R, STAT3, and GLUT4 to improve the status of ageing and diabetes, which was concurrent with the increased abundance of Dubosiella, Enterococcus, Romboutsia, Bifidobacterium, Kurthia, Clostridium_sensu_stricto_1, Corynebacterium, Faecalibaculum, Aerococcus and Vibrio. Notably, Dubosiella, Romboutsia, Bifidobacterium, Turicibacter and Clostridium_sensu_stricto_1 could serve as important intermediates for delaying ageing and diabetes. Additionally, the ULP-1 structure is strongly binding interaction with the target protein through hydrogen bonding and Van der Waals force, especially for GLP-1 (−10.34 kcal/mol), p16Ink4a (−10.51 kcal/mol) and GLP-1R (−8.57 kcal/mol). Moreover, the average length of the hydrogen bond was observed to be 2.36 MPa, which is smaller than that of the traditional hydrogen bond. Therefore, ULP has the potential to function as a nutraceutical to delay or prevent the development of ageing-related type 2 diabetes.

Pharmacological Depletion of Microglia Leads to a Dose-Dependent Reduction in Inflammation and Senescence in the Aged Murine Brain

Chronic macrophage activation was implicated as one of the main culprits for chronical, low-grade inflammation which significantly contributes to development of age-related diseases. Microglia as the brain macrophages have been recently implicated as key players in neuroinflammation and neurodegeneration in the aged brain. Microglial cell functions are indispensable in early development, however, activation or senescence of microglia in aging cells may be detrimental. Depletion of microglia using genetical or pharmacological approaches leads to opposite results regarding effects on brain cognition. In this study we pharmacologically depleted microglia using orally delivered low and high doses of the CSF1R inhibitor PLX5622 and assessed the expression levels of known inflammation markers (TNF-α, IL1-β, IL-6, IL-10), glia markers (Iba-1 and Gfap) and specific senescence marker p16Ink4a in the aged murine brain. Our results indicate that treatment with low and high doses of PLX5622 leads to a dose-dependent depletion of microglial cells with similar levels in young and aged mice. We also show that treatment with low and high PLX5622 differentially affected cytokine levels in young and old brains. By using low doses we could achieve reduction in inflammation circumventing the astrocyte activation. Removal of microglia cells led to decreased expression of the senescence marker p16Ink4a in the aged brain, indicating a relevant contribution of these cells to the expression of this marker and their senescent status in the healthy aging brain. Our results indicate that increased and detrimental brain inflammation in aged murine brain can be impaired by selectively reducing the microglial cell population.

Sustained delivery efficiency of curcumin through ZSM-5 nanozeolites/electrospun nanofibers for counteracting senescence of human adipose-derived stem cells

In the present study, ZSM-5 nanozeolite was incorporated into the Poly-ε-Caprolactone/Gelatin nanofibers (PCL-GEL NFs) for controlled and sustained release of a natural polyphenol, curcumin (CUR) to enhance the prolonged in vitro expansion of human adipose-derived stem cells (hADSCs) without inducing cellular senescence. XRD, FTIR, BET, TGA, FE-SEM, TEM and water contact angle analysis were used to characterize the fabricated nanozeolites and NFs. A study on the drug discharge pattern from the composite CUR@ZSM-5/PCL-GEL electrospun NFs displayed that by loading CUR into the ZSM-5 nanozeolites, the initial burst release was somewhat eliminated, and a sustained and prolonged drug discharge from NFs was obtained. The data set obtained through FE-SEM, LSCM, MTT and PicoGreen assays, qPCR, and western blotting also revealed better cell adhesion and enhanced metabolic activity and proliferation rate for hADSCs cultured on the CUR@ZSM-5/PCL-GEL NFs compared to other types of NFs after 14 and 28 days of incubation. Besides, Utilizing ZSM-5/NFs for sustained and prolonged delivery of CUR not only remarkably reduced the expression levels of p16INK4A, a crucial modulator of cellular senescence but also led to a significant increase in the transcriptional level and activity of hTERT/telomerase and telomere extension, as well as osteoblastic differentiation markers. These findings demonstrated the potential of ZSM-5/NFs for sustained/controlled release of CUR therapeutic molecules in designing a novel platform toward counteracting cellular senescence and achieving sufficient quantities of fresh hADSCs for regenerative medicine applications.

Effect of vorinostat on INK4 family and HDACs 1, 2, and 3 in pancreatic cancer and hepatocellular carcinoma.

Background and purpose:In mammalian cells, several distinct surveillance systems, named cell cycle checkpoints, can interrupt normal cell-cycle progression. The cyclin-dependent kinases are negatively regulated by proteins of cyclin-dependent kinases inhibitors comprising INK4 and Cip/Kip families. Histone deacetylation induced by histone deacetylases (HDACs) inactivates the INK4 and Cip/Kip families lead to cancer induction. HDAC inhibitors (HDACIs) have been indicated to be potent inducers of differentiation, growth arrest, and apoptotic induction. Vorinostat (suberoylanilide hydroxamic acid, SAHA), as an HDACI, is reported to be useful in various cancers. Previously, we reported the effect of trichostatin A on hepatocellular carcinoma and also vorinostat on colon cancer cell lines. The current study was aimed to investigate the effect of vorinostat on p16INK4a, p14ARF, p15INK4b, and class I HDACs 1, 2, and 3 gene expression, cell growth inhibition, and apoptosis induction in pancreatic cancer AsPC-1 and hepatocellular carcinoma LCL-PI 11 cell lines.Experimental approach:The AsPC-1 and LCL-PI 11 cell lines were cultured and treated with vorinostat. To determine, viability, apoptosis, and the relative expression level of p16INK4a, p14ARF, p15INK4b, class I HDACs 1, 2, and 3 genes, MTT assay, cell apoptosis assay, and RT-qPCR were performed, respectively.Findings/Results:Vorinostat significantly inhibited cell growth, induced apoptosis, increased p16INK4a, p14ARF, p15INK4b, and decreased class I HDACs 1, 2, and 3 gene expression.Conclusion and implications:Vorinostat can reactivate the INK4 family through inhibition of class I HDACs 1, 2, and 3 genes activity.

The hypothalamic-pituitary-gonadal axis controls muscle stem cell senescence through autophagosome clearance.

BackgroundWith organismal aging, the hypothalamic–pituitary–gonadal (HPG) activity gradually decreases, resulting in the systemic functional declines of the target tissues including skeletal muscles. Although the HPG axis plays an important role in health span, how the HPG axis systemically prevents functional aging is largely unknown.MethodsWe generated muscle stem cell (MuSC)‐specific androgen receptor (Ar) and oestrogen receptor 2 (Esr2) double knockout (dKO) mice and pharmacologically inhibited (Antide) the HPG axis to mimic decreased serum levels of sex steroid hormones in aged mice. After short‐term and long‐term sex hormone signalling ablation, the MuSCs were functionally analysed, and their aging phenotypes were compared with those of geriatric mice (30‐month‐old). To investigate pathways associated with sex hormone signalling disruption, RNA sequencing and bioinformatic analyses were performed.ResultsDisrupting the HPG axis results in impaired muscle regeneration [wild‐type (WT) vs. dKO, P < 0.0001; Veh vs. Antide, P = 0.004]. The expression of DNA damage marker (in WT = 7.0 ± 1.6%, dKO = 32.5 ± 2.6%, P < 0.01; in Veh = 13.4 ± 4.5%, Antide = 29.7 ± 5.5%, P = 0.028) and senescence‐associated β‐galactosidase activity (in WT = 3.8 ± 1.2%, dKO = 10.3 ± 1.6%, P < 0.01; in Veh = 2.1 ± 0.4%, Antide = 9.6 ± 0.8%, P = 0.005), as well as the expression levels of senescence‐associated genes, p16 Ink4a and p21 Cip1, was significantly increased in the MuSCs, indicating that genetic and pharmacological inhibition of the HPG axis recapitulates the progressive aging process of MuSCs. Mechanistically, the ablation of sex hormone signalling reduced the expression of transcription factor EB (Tfeb) and Tfeb target gene in MuSCs, suggesting that sex hormones directly induce the expression of Tfeb, a master regulator of the autophagy–lysosome pathway, and consequently autophagosome clearance. Transduction of the Tfeb in naturally aged MuSCs increased muscle mass [control geriatric MuSC transplanted tibialis anterior (TA) muscle = 34.3 ± 2.9 mg, Tfeb‐transducing geriatric MuSC transplanted TA muscle = 44.7 ± 6.7 mg, P = 0.015] and regenerating myofibre size [eMyHC+tdTomato+ myofibre cross‐section area (CSA) in control vs. Tfeb, P = 0.002] after muscle injury.ConclusionsOur data show that the HPG axis systemically controls autophagosome clearance in MuSCs through Tfeb and prevents MuSCs from senescence, suggesting that sustained HPG activity throughout life regulates autophagosome clearance to maintain the quiescence of MuSCs by preventing senescence until advanced age.

The Association of Molecular Biomarkers in the Diagnosis of Cervical Pre-Cancer and Cancer and Risk Factors in Senegalese.

Background:Cervical intraepithelial neoplasia (CIN) grading is subjective and affected by substantial rates of discordance among pathologists. Although recent studies have suggested that p16INK4a may be a useful surrogate biomarker of cervical neoplasia, Ki-67 and human papillomavirus testing have also been shown to be useful in detecting neoplasia. The purpose of this study was to determine the expression of p16INK4a and Ki-67 in cervical neoplasia and its correlations with cofactors. Methods: The study involved 69 patients with and without cervical neoplasia who underwent colposcopic directed biopsy. On each patient, two samples were taken; the first was used for immunohistochemistry and the second for molecular testing, using HPV16and18 genotyping Real-Time PCR Kit. Results: The study revealed the expression level of p16INK4a and Ki-67 in a descending order, from invasive squamous cell carcinoma (SCC), CIN2/3, CIN1 and non-dysplastic lesions. Correlations showed an association between the staining of p16NK4a and Ki-67 with the increase of age (OR: 1.79 (95%IC: 0.49 – 6.55), p = 0.037) and marital status (OR: 0.17 (95%IC: 0.04 – 0.68), p = 0.003). We found that the expressions of p16INK4a and Ki-67 were significantly different between invasive SCC vs non-dysplasia (OR: 44.57 (95%IC: 4.91 – 403.91), p<0.0001). The study showed significant correlation between HPV 16and18 infection with p16 INK4a and Ki-67 expression (OR: 0.13 (95%IC: 0.03 – 0.52), p<0.0001). Strong expression of p16INK4a and Ki-67 were observed in invasive squamous cell carcinoma, moderate staining was found in CIN2/3, weak staining in CIN1 and normal histology. Conclusion:Our findings indicate that p16INK4a and Ki-67 expressions associated strongly with cervical pathology. Therefore, p16/Ki-67 could be considered as a suitable biomarker for cervical cancer screening, particularly in HPV-based screening programs.

Signatures of somatic mutations and gene expression from p16INK4A positive head and neck squamous cell carcinomas (HNSCC).

Human papilloma virus (HPV) causes a subset of head and neck squamous cell carcinomas (HNSCC) of the oropharynx. We combined targeted DNA- and genome-wide RNA-sequencing to identify genetic variants and gene expression signatures respectively from patients with HNSCC including oropharyngeal squamous cell carcinomas (OPSCC). DNA and RNA were purified from 35- formalin fixed and paraffin embedded (FFPE) HNSCC tumor samples. Immuno-histochemical evaluation of tumors was performed to determine the expression levels of p16INK4A and classified tumor samples either p16+ or p16-. Using ClearSeq Comprehensive Cancer panel, we examined the distribution of somatic mutations. Somatic single-nucleotide variants (SNV) were called using GATK-Mutect2 ("tumor-only" mode) approach. Using RNA-seq, we identified a catalog of 1,044 and 8 genes as significantly expressed between p16+ and p16-, respectively at FDR 0.05 (5%) and 0.1 (10%). The clinicopathological characteristics of the patients including anatomical site, smoking and survival were analyzed when comparing p16+ and p16- tumors. The majority of tumors (65%) were p16+. Population sequence variant databases, including gnomAD, ExAC, COSMIC and dbSNP, were used to identify the mutational landscape of somatic sequence variants within sequenced genes. Hierarchical clustering of The Cancer Genome Atlas (TCGA) samples based on HPV-status was observed using differentially expressed genes. Using RNA-seq in parallel with targeted DNA-seq, we identified mutational and gene expression signatures characteristic of p16+ and p16- HNSCC. Our gene signatures are consistent with previously published data including TCGA and support the need to further explore the biologic relevance of these alterations in HNSCC.

Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma

Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.

Abstract 336: Organotypic co-cultures as a novel 3D model for head and neck squamous cell carcinoma

Abstract Background. Head and neck squamous cell carcinomas (HNSCC) are histologically and molecularly heterogeneous tumors and frequently develop resistance mechanisms against treatment. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and how to overcome treatment failure. Methods. We developed a 3D organotypic co-culture (OTC) model for HNSCCs that maintains the architecture and cell composition of the patients' tumor. A dermal equivalent, composed of healthy, human-derived fibroblasts embedded into viscose fibers, grows in an insert for approximately 10 days in culture and serves as a scaffold for the patient sample. After outgrowth of fibroblast within the dermal equivalent, we cultivated vital HNSCC tumor explants from 13 patients (non-HPV driven, n=7; HPV-driven HNSCC, n=6) by carefully placing them on top of the scaffold. Fractionated irradiation (IR) was applied to 5 samples. To evaluate tumor proliferation and areas of apoptosis, we performed immunohistochemistry and immunofluorescence with antibodies against ki-67, cleaved caspase-3, pan-cytokeratin (PanCK), p16INK4a, and vimentin. Results. All 3D OTC models of non-HPV driven samples encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primary and 3D-OTC models were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth and invasion patterns. Radioresistant and clonally expanding tumor cells were enriched in 3D-OTC models after fractionated IR. HPV DNA was detectable in both primary and OTC (day 14) of HPV-driven tumors; However, p16INK4a expression levels were varying. Conclusion. Our 3D-dimensional organotypic co-culture model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth as well as clonal expansion of resistant cancer cells upon treatment. In the future, the introduced model might serve as a platform to perform sensitivity testing upon treatment to predict the patients' individual response to therapy. Citation Format: Julia C. Thierauf, Luca Engelmann, Natalia Koerich Laureano, Hans-Jürgen Stark, Elena-Sophie Prigge, Dominik Horn, Kolja Freier, Niels Grabe, Philippe Federspil, Peter K. Plinkert, Magnus von Knebel Doeberitz, Jochen Hess, Annette Affolter. Organotypic co-cultures as a novel 3D model for head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 336.

Multiparameter flow cytometric detection and quantification of senescent cells in vitro

It has been over half a century since cellular senescence was first noted and characterized, and yet no consensus senescent marker has been reliably established. This challenge is compounded by the complexity and heterogenic phenotypes of senescent cells. This necessitates the use of multiple biomarkers to confidently characterise senescent cells. Despite cytochemical staining of senescence associated-beta-galactosidase being a single marker approach, as well as being time and labour-intensive, it remains the most popular detection method. We have developed an alternative flow cytometry-based method that simultaneously quantifies multiple senescence markers at a single-cell resolution. In this study, we applied this assay to the quantification of both replicative and induced senescent primary cells. Using this assay, we were able to quantify the activity level of SA β-galactosidase, the expression level of p16INK4a and γH2AX in these cell populations. Our results show this flow cytometric approach to be sensitive, robust, and consistent in discriminating senescent cells in different cell senescence models. A strong positive correlation between these commonly- used senescence markers was demonstrated. The method described in this paper can easily be scaled up to accommodate high-throughput screening of senescent cells in applications such as therapeutic cell preparation, and in therapy-induced senescence following cancer treatment.

P16INK4A Immunohistochemistry as a Gold Standard for Cervical Cancer and Precursor Lesions Screening

High-risk (HR) Human papillomaviruses (HPVs) are known as the main factors implicated in the pathogenesis of cervical preinvasive and invasive lesions. Therefore, the presence or absence of HR-HPV can be followed for the prognosis of low-grade and high-grade squamous intraepithelial lesions. Since the overexpression of p16INK4a protein depends on the presence of transcriptionally-active HPV, and due to its availability and simple interpretation, it may be considered as a proper marker to diagnose cervical cancer. An immunohistochemical analysis of p16INK4a was performed in 72 cervical tissue specimens at Imam Khomeini Complex Hospital (Tehran, Iran) from 2016 to 2018. The performance parameters were calculated and compared using receiving operating characteristics curve (ROC) details. p16INK4a is significantly up-regulated in the cervical cancer samples in comparison with that in normal samples. Moreover, the ROC data showed the potential ability of p16INK4a under determined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. The molecular typing disclosed the attendance of HPV DNA in 44.4% of cases (32/72) with a predominance of HPV type 16. The molecular biomarker p16INK4a can be a good candidate for the early diagnosis and prognosis of cervical cancer in HPV-infected patients. Considering the increase in the expression level of p16INK4a in cancer and precancer tissues, p16INK4a may be used for early detection of cervical cancer.