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Abstract Background Approximately 50% of cases of penile carcinoma (PeCa), a rare neoplasm worldwide, are associated with human papillomavirus (HPV). However, the detection of HPV-DNA is not sufficient to consider it the etiological factor in the development of this type of cancer. Currently, the overexpression of P16INK4A is used as a surrogate biomarker of HPV carcinogenesis. Information on PeCa in Mexico is scarce, particularly regarding cases related to HPV and genotype frequency. Objective To evaluate the presence of HPV, its genotypes, and the presence of multiple genotypes, and the expression of P16INK4A, as well as its clinical and histopathological parameters. Methods For HPV-DNA detection and P16INK4A expression, we used the INNO-LiPA® test and immunohistochemistry, respectively. Results Sixty cases of PeCa were evaluated, of which 75% were HPV-non-related histological variants. We found that 58.9% (33/56) of PeCa cases were HPV-DNA positive, while 30.9% of the cases evaluated (17/55) were positive for P16INK4A. HPV16 was the main genotype in 42.9% of the cases, followed by HPV52 in 7.1% and HPV18 in 5.4%. Within the HPV-positive cases, 27.3% had multiple genotypes. All HPV-positive patients under the age of 45 years were positive only for HPV16. Conclusions HPV16 was the most commonly detected genotype in PeCa. HPV 31, 35 and 39 were infrequent; however, they were related to a single infection and P16INK4A overexpression; thus, they seem to be relevant in PeCa carcinogenesis. Our results suggest that P16INK4A overexpression could be useful for the classification of HPV-related PeCa. The role of multiple HPV genotypes in the development and prognosis of PeCa is still not completely understood. Thus, it is necessary to define criteria to establish reliable ways to classify HPV-related PeCa that could lead to optimal therapeutic approaches.

p16 INK4a and p21 WAF1/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 p16 INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21 WAF1/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 p21 WAF1/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., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Rocha et al.)

Cathepsin L (CTSL) has been implicated in aging and age-related diseases, such as cardiovascular diseases, specifically atherosclerosis. However, the underlying mechanism(s) is not well documented. Recently, we demonstrated a role of CUT-like homeobox 1 (CUX1) in regulating the p16 INK4a -dependent cellular senescence in human endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) via its binding to an atherosclerosis-associated functional SNP (fSNP) rs1537371 on the CDKN2A/B locus. In this study, to determine if CTSL, which was reported to proteolytically activate CUX1, regulates cellular senescence via CUX1, we measured the expression of CTSL, together with CUX1 and p16 INK4a , in human ECs and VSMCs undergoing senescence. We discovered that CUX1 is not a substrate that is cleaved by CTSL. Instead, CTSL is an upstream regulator that activates CUX1 transcription indirectly in a process that requires the proteolytic activity of CTSL. Our findings suggest that there is a transcription factor in between CTSL and CUX1, and cleavage of this factor by CTSL can activate CUX1 transcription, inducing endothelial senescence. Thus, our findings provide new insights into the signal transduction pathway that leads to atherosclerosis-associated cellular senescence.

Bladder cancer (BlCa) is an extensively heterogeneous disease that leads to great variability in tumor evolution scenarios and lifelong patient surveillance, emphasizing the need for modern, minimally invasive precision medicine. Here, we explored the clinical significance of copy number alterations (CNAs) in BlCa. CNA profiling was performed in 15 patient-derived xenografts (PDXs) and validated in The Cancer Genome Atlas BlCa (TCGA-BLCA; n  = 408) and Lindgren et al. ( n  = 143) cohorts. CDKN2A copy number loss was identified as the most frequent CNA in bladder tumors, associated with reduced CDKN2A expression, tumors of a papillary phenotype, and prolonged PDX survival. The study's screening cohort consisted of 243 BlCa patients, and CDKN2A copy number was assessed in genomic DNA and cell-free DNA (cfDNA) from 217 tumors and 189 pre-treatment serum samples, respectively. CDKN2A copy number loss was correlated with superior disease-free and progression-free survival of non-muscle-invasive BlCa (NMIBC) patients. Moreover, a higher CDKN2A index ( CDKN2A/LEP ratio) in pre-treatment cfDNA was associated with advanced tumor stage and grade and short-term NMIBC progression to invasive disease, while multivariate models fitted for CDKN2A index in pre-treatment cfDNA offered superior risk stratification of T1/high-grade and EORTC high-risk patients, enhancing prediction of treatment outcome. CDKN2A copy number status could serve as a minimally invasive tool to improve risk stratification and support personalized prognosis in BlCa., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Higher intensity exercise, despite causing more tissue damage, improved aging conditions. We previously observed decreased p16 INK4a mRNA in human skeletal muscle after high-intensity interval exercise (HIIE), with no change following equivalent work in moderate-intensity continuous exercise. This raises the question of whether the observed senolytic effect of exercise is mediated by inflammation, an immune response induced by muscle damage. In this study, inflammation was blocked using a multiple dose of ibuprofen (total dose: 1200 mg), a commonly consumed nonsteroidal anti-inflammatory drug (NSAID), in a placebo-controlled, counterbalanced crossover trial. Twelve men aged 20-26 consumed ibuprofen or placebo before and after HIIE at 120% maximum aerobic power. Multiple muscle biopsies were taken for tissue analysis before and after HIIE. p16 INK4a+ cells were located surrounding myofibers in muscle tissues. The maximum decrease in p16 INK4a mRNA levels within muscle tissues occurred at 3 h post-exercise (-82%, p < 0.01), gradually recovering over the next 3-24 h. A concurrent reduction pattern in CD11b mRNA (-87%, p < 0.01) was also found within the same time frame. Ibuprofen treatment attenuated the post-exercise reduction in both p16 INK4a mRNA and CD11b mRNA. The strong correlation (r = 0.88, p < 0.01) between p16 INK4a mRNA and CD11b mRNA in muscle tissues suggests a connection between the markers of tissue aging and pro-inflammatory myeloid differentiation. In conclusion, our results suggest that the senolytic effect of high-intensity exercise on human skeletal muscle is mediated by acute inflammation.

Background: Synchronous bilateral breast cancer (SBBC) provides a special condition where two independent breast tumors are exposed to cancer pharmacotherapy within a uniform pharmacokinetic milieu. Both senescence and apoptosis are established responses to therapy; however, they have potentially variable contributions to the overall outcome of treatment, which are yet to be determined. Methods: In this report, we describe the clinicopathological picture of two SBBC cases that received standard anticancer treatment and assess their expression profile of several molecular hallmarks of senescence and apoptosis. Results: Our analysis identified that synchronous tumors have variable expression profiles of both senescence- and apoptosis-associated biomarkers, despite comparable pathological responses to neoadjuvant chemotherapy and current survival rates. Conclusions: Our results highlight the variable expression of senescence- and apoptosis-associated markers in breast tumors (despite the shared somatic genetic background) and invites a large-scale assessment of both senescence and apoptosis in breast cancer tissue in vivo and their contribution to the pathological response and overall survival. [ABSTRACT FROM AUTHOR]

Identification of molecules and molecular pathways that can ameliorate aging-associated decline in cognitive function is crucial. Here we report that the protein levels of transcription factor EB (TFEB) were markedly reduced in both the cytosolic and nuclear fractions of the frontal cortex and hippocampus at 18-months of age relative to 6 months in the normal male wild-type mice. In the transgenic mice with ectopic expression of flag-TFEB in neurons, we observed that the levels of actin-normalized PGC1α and mtTFA were significantly increased in both the cortex and the hippocampus. Additionally, we confirmed increased mitochondria numbers in the flag-TFEB mice by transmission electron microscopy. Most importantly, TFEB expression in the 18-month-old transgenic mice mitigated markers of senescence including P16INK4a, γ-H2AX, and lamin B1, and improved memory skills implying that TFEB may exert an anti-aging effect by modulating neuronal senescence. Taken together these data strongly support that TFEB can be a useful therapeutic target for brain senescent cells to help overcome the age-related issues in cognition and possibly, achieve healthy aging. [ABSTRACT FROM AUTHOR]

Bizarre parosteal osteochondromatous proliferation (BPOP), or Nora's lesion, is a rare benign osteochondromatous lesion. At present, the molecular etiology of BPOP remains unclear. JMJD3(KDM6B) is an H3K27me3 demethylase and counteracts polycomb‐mediated transcription repression. Previously, Jmjd3 was shown to be critical for bone development and osteoarthritis. Here, we report that conditional deletion of Jmjd3 in chondrogenic cells unexpectedly resulted in BPOP‐like lesion in mice. Biochemical investigations revealed that Jmjd3 inhibited BPOP‐like lesion through p16Ink4a. Immunohistochemistry and RT‐qPCR assays indicated JMJD3 and p16INK4A level were significantly reduced in human BPOP lesion compared with normal subjects. This was further confirmed by Jmjd3/Ink4a double‐gene knockout mice experiments. Therefore, our results indicated the pathway of Jmjd3/p16Ink4a may be essential for the development of BPOP in human. © 2021 American Society for Bone and Mineral Research (ASBMR). [ABSTRACT FROM AUTHOR]

Prior studies have established the carcinogenic role of HPV16 and also demonstrated its unique biological behavior in cervical and oropharyngeal squamous cell carcinoma (OPSCC) but its role in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is not well explored. Therefore, in the present study, we assessed HPV16 prevalence using PCR and Anti-HPV16 antibodies for the first time and correlated its biological behavior using p16INK4a and Ki67 proliferation index (PI) in OL, OSCC, and OPSCC. This study included 63 subjects comprising of 25 OL, 26 OSCC, and 12 OPSCC cases. Exfoliated cells were collected and processed for PCR followed by immunohistochemistry with primary antibodies p16INK4a, Anti-HPV16, and Ki67. The expressions were evaluated and statistical analysis included Chi-square and Spearman's test. Cumulatively 37% (OL—7%, OSCC—14% & OPSCC—16%) of cases showed positive PCR expression. PCR positivity was observed to be significantly higher (p 0.00) in OPSCC (9/12) than OSCC (9/26) and OL (5/25) cases. Overall immunohistochemical expression of p16INK4a, Anti-HPV16, and Ki67 were significantly (p 0.02) higher in HPV16 (PCR) positive cases. HPV16 + OSCC cases showed higher grades of p16INK4a and Ki67 expression. We have demonstrated a prevalence of HPV16 in OL, OSCC, and OPSCC through PCR, which may be concluded as a gold standard for the detection of HPV16 DNA. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer screening with cytology suffers from low sensitivity, whereas the efficiency of human papillomavirus (HPV)‐based screening is limited by low specificity. The authors evaluated a novel p16INK4a immunocytology approach in cervical cancer screening compared with HPV‐based and cytology‐based screening. Methods: In total, 2112 women aged 49 to 69 years from Shanxi, China were screened from March to July 2019. HPV testing, liquid‐based cytology (LBC), and p16INK4a immunocytology were performed on samples from all women. Any positive result triggered a referral to colposcopy with biopsy, if indicated. Screening performance for detecting cervical intraepithelial neoplasia grade 2 and 3 or worse (CIN2+/CIN3+) was evaluated using multiple algorithms. Results: p16INK4a had a lower positive rate (10.0%) than LBC abnormality (vs 12.1%; P =.004) and a high‐risk HPV positivity (21.4%; P <.001). For the detection of CIN3+, the relative sensitivity of p16INK4a compared with HPV and LBC was 0.93 (95% CI, 0.82‐1.07) and 1.12 (95% CI, 0.95‐1.32), respectively. The specificity of p16INK4a was significantly higher than that for HPV and LBC, with a relative specificity of 1.13 (95% CI, 1.11‐1.16) and 1.02 (95% CI, 1.01‐1.04), respectively. In addition, p16INK4a alone yielded a clinical performance very similar to that of the current mainstream strategy of using HPV16/18 with reflex cytology (ASC‐US+, atypical squamous cells of undetermined significance or worse). The immediate risk of CIN3+ was 14.6% if p16INK4a results were positive and 0.2% if p16INK4a results were negative. Conclusions: With minimal colposcopy referrals, p16INK4a screening demonstrated promising utility for risk stratification and yielded a better balance between sensitivity and specificity compared with HPV and LBC primary screening. Moreover, with accuracy and efficiency similar to what is achieved using mainstream cotest algorithms, p16 may simplify the screening practice. More evidence will be required before clinical recommendation. This evaluation of a novel p16INK4a test demonstrates that p16INK4a may be a promising alternative to cytology for primary screening. Moreover, with screening efficacy similar to that of mainstream human papillomavirus‐cytology cotest algorithms, p16INK4a screening has great potential to simplify screening practice. [ABSTRACT FROM AUTHOR]

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy. [ABSTRACT FROM AUTHOR]

High‐grade cervical intraepithelial neoplasia (CIN2 and CIN3) represents a heterogeneous disease with varying cancer progression risks. Biomarkers indicative for a productive human papillomavirus (HPV) infection (HPV E4) and a transforming HPV infection (p16ink4a, Ki‐67 and host‐cell DNA methylation) could provide guidance for clinical management in women with high‐grade CIN. This study evaluates the cumulative score of immunohistochemical expression of p16ink4a (Scores 0‐3) and Ki‐67 (Scores 0‐3), referred to as the "immunoscore" (IS), in 262 CIN2 and 235 CIN3 lesions derived from five European cohorts in relation to immunohistochemical HPV E4 expression and FAM19A4/miR124‐2 methylation in the corresponding cervical scrape. The immunoscore classification resulted in 30 lesions within IS group 0‐2 (6.0%), 151 lesions within IS group 3‐4 (30.4%) and 316 lesions within IS group 5‐6 (63.6%). E4 expression decreased significantly from CIN2 to CIN3 (P <.001) and with increasing immunoscore group (Ptrend <.001). Methylation positivity increased significantly from CIN2 to CIN3 (P <.001) and with increasing immunoscore group (Ptrend <.001). E4 expression was present in 9.8% of CIN3 (23/235) and in 12.0% of IS group 5‐6 (38/316). Notably, in a minority (43/497, 8.7%) of high‐grade lesions, characteristics of both transforming HPV infection (DNA hypermethylation) and productive HPV infection (E4 expression) were found simultaneously. Next, we stratified all high‐grade CIN lesions, based on the presumed cancer progression risk of the biomarkers used, into biomarker profiles. These biomarker profiles, including immunoscore and methylation status, could help the clinician in the decision for immediate treatment or a "wait and see" policy to reduce overtreatment of high‐grade CIN lesions. What's new? Treating all high‐grade cervical intraepithelial neoplasia (CIN2/3) with excisional therapy leads to overtreatment, as these lesions have varying cancer progression risks. Here, the authors evaluated expression patterns of p16ink4a, Ki‐67 and the HPV E4 protein, and methylation of FAM19A4/miR124‐2 in high‐grade CIN. The biomarker expression patterns revealed the high degree of heterogeneity among CIN2/3 lesions. Biomarker profiles based on the presumed cancer progression risks were established and could guide clinicians in choosing whether to treat immediately or wait and see. [ABSTRACT FROM AUTHOR]

This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1–deficient (Bmi-1–/–), Bmi-1 and p16INK4a double-knockout (Bmi-1–/–p16INK4a–/–), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)–dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1–/– and Bmi-1–/–p16INK4a–/– mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1–/– mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α–dependent barrier permeability and aging. Accumulated p16INK4a combined with occludin at the 1st–160th residue in cytoplasm of intestinal epithelium cells from Bmi-1–/– mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16INK4a deletion could maintain barrier function and microbiota balance in Bmi-1–/– mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16INK4a accumulation. The clearance of p16INK4a-positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1–160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16INK4a with occludin for protecting TJ.

T-cell dysfunction arising upon repeated antigen exposure prevents effective immunity and immunotherapy. Using various clinically and physiologically relevant systems, we show that a prominent feature of PD-1-expressing exhausted T cells is the development of cellular senescence features both in vivo and ex vivo. This is associated with p16INK4a expression and an impaired cell cycle G1 to S-phase transition in repeatedly stimulated T cells. We show that these T cells accumulate DNA damage and activate the p38MAPK signaling pathway, which preferentially leads to p16INK4a upregulation. However, in highly dysfunctional T cells, p38MAPK inhibition does not restore functionality despite attenuating senescence features. In contrast, p16INK4a targeting can improve T-cell functionality in exhausted CAR T cells. Collectively, this work provides insights into the development of T-cell dysfunction and identifies T-cell senescence as a potential target in immunotherapy.

Problem: Cervical cancer screening strategies in the United States include cotesting (human papillomavirus (HPV) with cytology), primary HPV with genotyping and reflex cytology, and cytology alone. An ongoing challenge is the appropriate triage of patients to colposcopy to those at highest risk. We investigated whether incorporation of p16INK4a immunodetection by enzyme‐linked immunosorbent assay (ELISA) on fresh cervical samples obtained at the time of screening could improve appropriate referral to colposcopy. Method of Study: A derivation group comprised of cervical swabs collected from subjects with high‐grade dysplasia or cancer (positive control) and from subjects with negative screening history (negative control). Samples collected from colposcopy were used to evaluate the existing screening strategies individually and with incorporation of p16INK4a ELISA. Histology was used as the gold standard. Results: Among 163 subjects recruited, 138 were included. In the derivation group, mean p16INK4a level was 2.86 ng/mL (n = 31) and 0.58 ng/mL (n = 20) among positive and negative controls respectively (p = 0.002) with an area under the receiver operator characteristic curve of 0.79 (p < 0.001). Among colposcopy subjects, sensitivity/specificity for cotesting, primary HPV, and cytology were 94%/42%, 88%/45%, and 88%/49%, respectively. Incorporation of p16INK4a resulted in similar sensitivity and improved specificity (cotesting+p16 88%/58%, primary HPV+p16 88%/57%, cytology+p16 81%/62%; p = 0.23/p = 0.008) with decrease in colposcopy referrals by 15% to 22% (p = 0.01). Conclusions: These results demonstrate the feasibility of quantifying p16INK4a by ELISA in fresh cervical samples, and its potential as an adjunct to existing screening strategies in the identification of high grade‐dysplasia while reducing the number of colposcopic referrals. [ABSTRACT FROM AUTHOR]

Contrast-induced acute kidney injury (CI-AKI) is the third leading cause of hospital-acquired acute kidney injury. Cellular senescence is associated with CI-AKI. P16INK4a (p16) is a cell cycle regulator and link to aging and senescence. We found that the expression of p16 was elevated in CI-AKI renal tissues, however its role in CI-AKI remains insufficiently understood. In this study, we used p16 knockout (p16 KO) mice and wild-type (WT) littermates to establish CI-AKI mice model to elucidate the impact of p16 on CI-AKI. The results showed that serum creatinine (SCr), blood urea nitrogen (BUN), and serum neutrophil gelatinase-associated lipocalin (NGAL) levels were markedly reduced in p16 KO CI-AKI mice. Both immunohistochemistry and western blot analyses confirmed that p16 knockout alleviated renal cell apoptosis. Furthermore, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were attenuated by downregulating NLRP3 and NF-κB inflammasomes. Additionally, ROS levels were diminished via activating Nrf2/Keap-1 pathway in p16 KO CI-AKI mice. Collectively, our findings suggest that p16 deletion exerts protective effects against apoptosis, inflammation, and oxidative stress in CI-AKI mice model, p16 deletion might be a potential therapeutic strategy for ameliorating CI-AKI. • p16INK4a overexpression in contrast-induced acute kidney injury (CI-AKI). • p16INK4a knockout alleviated CI-AKI renal function • p16INK4a knockout suppressed pro-inflammatory response and oxidative stress in CI-AKI mice. [ABSTRACT FROM AUTHOR]

Human Werner syndrome (WS) is an autosomal recessive progeria disease. A mouse model of WS manifests the disease through telomere dysfunction-induced aging phenotypes, which might result from cell cycle control and cellular senescence. Both p21Waf1/Cip1 (p21, encoded by the Cdkn1a gene) and p16Ink4a (p16, encoded by the Ink4a gene) are cell cycle inhibitors and are involved in regulating two key pathways of cellular senescence. To test the effect of p21 and p16 deficiencies in WS, we crossed WS mice (DKO) with p21–/– or p16–/– mice to construct triple knockout (p21-TKO or p16-TKO) mice. By studying the survival curve, bone density, regenerative tissue (testis), and stem cell capacity (intestine), we surprisingly found that p21-TKO mice displayed accelerated premature aging compared with DKO mice, while p16-TKO mice showed attenuation of the aging phenotypes. The incidence of apoptosis and cellular senescence were upregulated in p21-TKO mice tissue and downregulated in p16-TKO mice. Surprisingly, cellular proliferation in p21-TKO mice tissue was also upregulated, and the p21-TKO mice did not show telomere shortening compared with age-matched DKO mice, although p16-TKO mice displayed obvious enhancement of telomere lengthening. Consistent with these phenotypes, the SIRT1-PGC1 pathway was upregulated in p16-TKO but downregulated in p21-TKO compared with DKO mouse embryo fibroblasts (MEFs). However, the DNA damage response pathway was highly activated in p21-TKO, but rescued in p16-TKO, compared with DKO MEFs. These data suggest that p21 protected the stem cell reservoir by regulating cellular proliferation and turnover at a proper rate and that p21 loss in WS activated fairly severe DNA damage responses (DDR), which might cause an abnormal increase in tissue homeostasis. On the other hand, p16 promoted cellular senescence by inhibiting cellular proliferation, and p16 deficiency released this barrier signal without causing severe DDR.

Cervical cancer remains one of the preventable cause of women’s death, the second most common cause of death in Europe. Several screening tests for precancerous lesions are available, including cervicovaginal cytology and screening of high-risk human papillomavirus (HPV) strains – 16, 18, 31, 33. Cervical cytology (Babeş-Papanicolau smear test) remains the elected method for cervical cancer screening in many countries. Last decades, HPV DNA sequence was identified, including gene number and encoded proteins. HPV DNA gene expression involves the use of multiple promoters and complex patterns of splicing, which served as a model for the elaboration of cellular and molecular biomarkers for the identification. Herein, we present the usefulness and efficiency of viral and cellular biomarkers in improving the diagnosis and managing precursor lesions of cervical cancer. Viral and cellular biomarkers can help differentiating low-grade lesion leading to a better diagnosis. HPV DNA tests, although a primary screening method in actual guidelines, can serve as a better screening test method. Other viral biomarkers that include E6 and E7 mRNA testing and determining the methylation of viral genes have good test screening results. Cellular biomarkers such as the immunocytochemical staining of p16INK4a, alone or combined with the proliferation marker Ki-67, can reveal anomalies in the cell cycle. The E4 protein, strongly expressed in productive infections, can prove a transforming infection when it is absent. In recent years, detection of the TERC gene and oncogenetic microRNAs expressed early in carcinogenesis before clinical findings show great promise in differentiating lesions likely to progress to cancer. In conclusion, biomarkers are more powerful instruments to enhance early pre-cervical cancer lesions diagnosis, which allow better prognosis and more reliable treatment. [ABSTRACT FROM AUTHOR]

Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including epithelial ovarian cancer (EOC). Previously published data on gene methylation of EOC focused mainly on single gene or on cancer tissues. Objectives of the study were to estimate the promoter hypermethylation status of DAPK1 and p16INK4a genes in circulating blood of EOC patients and to determine their association with clinicopathological features of EOC. This case–control study included 50 EOC patients and 20 apparently healthy and age matched female controls. Isolation of genomic DNA was carried out from peripheral venous blood. Methylation in promoter region of DAPK1 and p16INK4a genes was determined by methylation-specific PCR. Methylation of DAPK1 was occurred in 42 out of 50 cases (84.0%) and methylation of p16INK4a gene was occurred in 34 out of 50 cases (68.0%). Methylation of both genes was occurred in 25 cases (50.0%). Occurrence of methylation in DAPK1 and p16INK4a genes was statistically significant (p < 0.0001) in cases compared to controls. Methylation of both genes was not statistically associated with age at diagnosis, menopausal status, histopathological types and FIGO staging of EOC. Identification of the peculiar promoter hypermethylation of DAPK1 and p16INK4a genes might be a successful approach for ancillary diagnosis of EOC at early stage in blood sample. [ABSTRACT FROM AUTHOR]

Although CDKN2A is well-known as a susceptibility gene for melanoma and pancreatic cancer, germline variants have also been anecdotally associated with a broader range of neoplasms including neural system tumors, head and neck squamous cell carcinomas, breast carcinomas, as well as sarcomas. The CDKN2A gene encodes for two distinct tumor suppressor proteins, p16INK4A and p14ARF, however, the independent association of germline alterations affecting these two proteins with cancer is under-appreciated. Here, we reviewed CDKN2A germline alterations reported among individuals and families with cancer in the literature, specifically addressing the cancer phenotypes in relation to the molecular consequence on p16INK4A and p14ARF. While melanoma is observed to associate with variants affecting both p16INK4A and p14ARF transcripts, it is noted that variants affecting p14ARF are more frequently observed with a heterogenous range of cancers. Finally, we reflected on the implications of this inferred genotype-phenotype association in clinical practice and proposed that clinical management of CDKN2A germline variant carriers should involve dedicated cancer genetics services, with multidisciplinary input from various healthcare professionals. [ABSTRACT FROM AUTHOR]

Human Werner syndrome (WS) is an autosomal recessive progeria disease. A mouse model of WS manifests the disease through telomere dysfunction-induced aging phenotypes, which might result from cell cycle control and cellular senescence. Both p21Waf1/Cip1 (p21, encoded by the Cdkn1a gene) and p16Ink4a (p16, encoded by the Ink4a gene) are cell cycle inhibitors and are involved in regulating two key pathways of cellular senescence. To test the effect of p21 and p16 deficiencies in WS, we crossed WS mice (DKO) with p21 –/– or p16 –/– mice to construct triple knockout (p21-TKO or p16-TKO) mice. By studying the survival curve, bone density, regenerative tissue (testis), and stem cell capacity (intestine), we surprisingly found that p21-TKO mice displayed accelerated premature aging compared with DKO mice, while p16-TKO mice showed attenuation of the aging phenotypes. The incidence of apoptosis and cellular senescence were upregulated in p21-TKO mice tissue and downregulated in p16-TKO mice. Surprisingly, cellular proliferation in p21-TKO mice tissue was also upregulated, and the p21-TKO mice did not show telomere shortening compared with age-matched DKO mice, although p16-TKO mice displayed obvious enhancement of telomere lengthening. Consistent with these phenotypes, the SIRT1-PGC1 pathway was upregulated in p16-TKO but downregulated in p21-TKO compared with DKO mouse embryo fibroblasts (MEFs). However, the DNA damage response pathway was highly activated in p21-TKO, but rescued in p16-TKO, compared with DKO MEFs. These data suggest that p21 protected the stem cell reservoir by regulating cellular proliferation and turnover at a proper rate and that p21 loss in WS activated fairly severe DNA damage responses (DDR), which might cause an abnormal increase in tissue homeostasis. On the other hand, p16 promoted cellular senescence by inhibiting cellular proliferation, and p16 deficiency released this barrier signal without causing severe DDR. [ABSTRACT FROM AUTHOR]

Senescent cells with replicative arrest can be generated during genotoxic, oxidative, and oncogenic stress. Long-term retention of senescent cells in the body, which is attributed to highly expressed BCL-family proteins, chronically damages tissues mainly through a senescence-associated secretory phenotype (SASP). It has been documented that accumulation of senescent cells contributes to chronic diseases and aging-related diseases. Despite the fact that no unique marker is available to identify senescent cells, increased p16INK4a expression has long been used as an in vitro and in vivo marker of senescent cells. We reviewed five existing p16INK4a reporter mouse models to detect, isolate, and deplete senescent cells. Senescent cells express high levels of anti-apoptotic and pro-apoptotic genes compared to normal cells. Thus, disrupting the balance between anti-apoptotic and pro-apoptotic gene expression, such as ABT-263 and ABT-737, can activate the apoptotic signaling pathway and remove senescent cells. Mitochondrial abnormalities in senescent cells were also discussed, for example mitochondrial DNA mutation accumulation, dysfunctional mitophagy, and mitochondrial unfolded protein response (mtUPR). The mitochondrial-targeted tamoxifen, MitoTam, can efficiently remove senescent cells due to its inhibition of respiratory complex I and low expression of adenine nucleotide translocase-2 (ANT2) in senescent cells. Therefore, senescent cells can be removed by various strategies, which delays chronic and aging-related diseases and enhances lifespan and healthy conditions in the body.

p16INK4a (CDKN2A) is a central tumor-suppressor and activator of senescence. We recently found that prolonged expression of p16INK4a in epidermal cells induces hyperplasia and dysplasia through Wnt-mediated stimulation of neighboring keratinocytes. The study suggests a pro-tumorigenic function of p16INK4a in early epidermal lesions, which could potentially be targeted by senolytic therapy.

It is now definitively established that a large part of the human genome is transcribed. However, only a scarce percentage of the transcriptome (about 1.2%) consists of RNAs that are translated into proteins, while the large majority of transcripts include a variety of RNA families with different dimensions and functions. Within this heterogeneous RNA world, a significant fraction consists of sequences with a length of more than 200 bases that form the so-called long non-coding RNA family. The functions of long non-coding RNAs range from the regulation of gene transcription to the changes in DNA topology and nucleosome modification and structural organization, to paraspeckle formation and cellular organelles maturation. This review is focused on the role of long non-coding RNAs as regulators of cyclin-dependent kinase inhibitors’ (CDKIs) levels and activities. Cyclin-dependent kinases are enzymes necessary for the tuned progression of the cell division cycle. The control of their activity takes place at various levels. Among these, interaction with CDKIs is a vital mechanism. Through CDKI modulation, long non-coding RNAs implement control over cellular physiology and are associated with numerous pathologies. However, although there are robust data in the literature, the role of long non-coding RNAs in the modulation of CDKIs appears to still be underestimated, as well as their importance in cell proliferation control.

In this review, we seek a novel strategy for establishing a rejuvenating microenvironment through senescent cells specific reprogramming. We suggest that partial reprogramming can produce a secretory phenotype that facilitates cellular rejuvenation. This strategy is desired for specific partial reprogramming under control to avoid tumour risk and organ failure due to loss of cellular identity. It also alleviates the chronic inflammatory state associated with ageing and secondary senescence in adjacent cells by improving the senescence-associated secretory phenotype. This manuscript also hopes to explore whether intervening in cellular senescence can improve ageing and promote damage repair, in general, to increase people’s healthy lifespan and reduce frailty. Feasible and safe clinical translational protocols are critical in rejuvenation by controlled reprogramming advances. This review discusses the limitations and controversies of these advances’ application (while organizing the manuscript according to potential clinical translation schemes) to explore directions and hypotheses that have translational value for subsequent research.

Background: To improve the efficiency of early diagnosis systems for cervical cancer, the use of cellular and viral markers for identifying precancerous lesions with a greater probability to progress to cancer has been proposed. Several cellular proteins and markers of oxidative DNA damage have been suggested as possible biomarkers of cervical carcinogenesis; however, they have not been evaluated together. In this study, we analyzed the expression of the cellular markers p16INK4a, Ki-67, CyclinE1, TOP2A/MCM2, and telomerase, as well as the DNA oxidative damage markers ROS and 8-OHdG. The analyses were performed in liquid-based cervical cytology samples or biopsies with premalignant lesions or cervical cancer diagnosis, with the purpose of selecting a panel of biomarkers that allow the identification of precursor lesions with greater risk of progression to cervical cancer.Methods: We analyzed 1485 liquid-based cytology samples, including 239 non-squamous intraepithelial lesions (NSIL), 901 low-grade squamous intraepithelial lesions (LSIL), 54 high-grade squamous intraepithelial lesions (HSIL), and 291 cervical cancers (CC). The biomarkers were analyzed by immunocytochemistry and Human Papilloma Virus (HPV) genotyping with the INNO-LiPA genotyping Extra kit.Results: We found that all tested cellular biomarkers were overexpressed in samples with high risk-HPV infection, and the expression levels increased with the severity of the lesion. TOP2A/MCM2 was the best biomarker for discriminating between LSIL and HSIL, followed by p16INK4a and cyclinE1. Statistical analysis showed that TOP2A/MCM2 provided the largest explanation of HSIL and CC cases (93.8%), followed by p16INK4a (91%), cyclin E1 (91%), Ki-67 (89.3%), and telomerase (88.9%).Conclusions: We propose that the detection of TOP2A/MCM2, p16INK4a and cyclin E1 expression levels is useful as a panel of biomarkers that allow identification of cervical lesions with a higher risk for progression to CC with high sensitivity and precision; this can be done inexpensively, in a single and non-invasive liquid-based cytology sample. [ABSTRACT FROM AUTHOR]

Introduction: Cervical adenocarcinoma is the second most common type of cervical cancer. The marker to predict the risk of this cancer is still need to be improved. This study aimed to evaluate the correlation between expression of E6 HPV with p53 and p16INK4A in cervical adenocarcinoma and cervical normal. Methods: This study was a cross sectional study conducted at the Department of Anatomic Pathology, Dr. Soetomo General Academic Hospital Surabaya Indonesia using 49 paraffin blocks during period January-December 2017. The E6 HPV, p53, and p16INK4A expression were detected by immunohistochemistry staining. Results: There were significant differences between the expression of E6 HPV and p16INK4A in cervical adenocarcinoma and cervical normal, but the expression of p53 was no significant difference with p=0.060 (P>0.05). There was a positive correlation between the expression of E6 HPV with p53 and p16INK4A (p<0.05) in cervical adenocarcinoma and cervical normal, and in cervical adenocarcinoma group, there was a significant correlation between the expression of E6 HPV with p53 (p=0.004) and p16INK4A (p=0.028), but no correlation was found between p53 with p16INK4A expression (p=0,092). Conclusion: It suggests that p16INK4A might serve as marker for predicting the risk of developing cervical cancer from the epithelium of cervical normal. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, but its pathology has not been fully characterized and the optimal treatment strategy remains unclear. Cellular senescence is a permanent state of cell-cycle arrest that can be induced by multiple stresses. Senescent cells contribute to the pathogenesis of various diseases, owing to an alteration in secretory profile, termed 'senescence-associated secretory phenotype' (SASP), including with respect to pro-inflammatory cytokines. Senolytics, a class of drugs that selectively eliminate senescent cells, are now being used clinically, and a combination of dasatinib and quercetin (DQ) has been extensively used as a senolytic. We aimed to investigate whether cellular senescence is involved in the pathology of PCOS and whether DQ treatment has beneficial effects in patients with PCOS. We obtained ovaries from patients with or without PCOS, and established a mouse model of PCOS by injecting dehydroepiandrosterone. The expression of the senescence markers p16INK4a, p21, p53, γH2AX, and senescence-associated β-galactosidase and the SASP-related factor interleukin-6 was significantly higher in the ovaries of patients with PCOS and PCOS mice than in controls. To evaluate the effects of hyperandrogenism and DQ on cellular senescence in vitro, we stimulated cultured human granulosa cells (GCs) with testosterone and treated them with DQ. The expression of markers of senescence and a SASP-related factor was increased by testosterone, and DQ reduced this increase. DQ reduced the expression of markers of senescence and a SASP-related factor in the ovaries of PCOS mice and improved their morphology. These results indicate that cellular senescence occurs in PCOS. Hyperandrogenism causes cellular senescence in GCs in PCOS, and senolytic treatment reduces the accumulation of senescent GCs and improves ovarian morphology under hyperandrogenism. Thus, DQ might represent a novel therapy for PCOS., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Introduction: Tissue expression of P16ink4A is correlated with cervical lesions. In this study we determined the association between serum P16ink4A concentrations and cervical lesions among women attending the cervical cancer clinic at Mbarara Regional Hospital (MRRH) South Western Uganda., Material and Methods: We recruited 90 cervical intraepithelial neoplasia (CIN) cases, 90 cervical cancer (CC) cases before treatment and 90 controls. Clinical and demographic data were recorded. Serum P16ink4A concentrations were measured by quantitative Elisa. Cases were confirmed with cytology and/or histology. Descriptive statistics and logistic regression were done with STATA 17 and P-values of <0.05 were considered statistically significant., Results: The mean serum P16ink4A concentration among CIN cases, CC cases and controls was 1.11(+/-0.66) ng/ml, 1.45(+/-1.11) ng/ml and 1.13(+/-0.61) ng/ml respectively (p = 0.008). 50 % of CIN cases and controls as well as 60 % of CC cases had P16ink4A concentration above 0.946 ng/ml. There were increased odds of CIN for serum P16ink4A though statistically insignificant (AOR: 1.11, p-value: 0.70). There was also a statistically significant reduction in odds of CC for serum P16ink4A (AOR: 0.55, p-value: 0.01)., Conclusion: Serum P16ink4A may likely be associated with cervical lesions especially CC in our study population and this may aid detection of such lesions. Diagnostic utility studies for circulating P16ink4A in detection of cervical cancer are recommended., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

The proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) are involved in the corneal inflammatory response and wound healing following corneal injuries. However, the mechanism by which proinflammatory cytokines modulate corneal epithelial wound healing remains unclear. In this study, we found that IL‐1β or TNF‐α was transiently elevated during corneal epithelial wound healing in mice. After corneal epithelial debridement, persistent treatment with IL‐1β or TNF‐α restrained the level of phosphorylated signal transducer and activator of transcription 3 (p‐STAT3) and boosted the level of cell cycle inhibitor p16Ink4a, resulting in impaired corneal epithelial repair. When p16Ink4a was deleted, the p‐STAT3 level in corneal epithelium was enhanced and corneal epithelial wound healing was clearly accelerated. In diabetic mice, IL‐1β, TNF‐α, and p16Ink4a appeared a sustained and strong expression in the corneal epithelium, and p16Ink4a knockdown partially reverted the defective diabetic corneal epithelial repair. Furthermore, immunoprecipitation proved that p16Ink4a interacted with p‐STAT3 and thus possibly suppressed the STAT3 activity. Our findings revealed a novel mechanism that the proinflammatory cytokines modulate corneal epithelial wound healing via the p16Ink4a‐STAT3 signaling. [ABSTRACT FROM AUTHOR]

Understanding the impact of inactivating mutations in SIRT1 on the p53 and p16 tumor suppressor genes may yield new insight into the oncogenic mechanisms underlying Bowen's disease. [ABSTRACT FROM AUTHOR]

Senescent cells with replicative arrest can be generated during genotoxic, oxidative, and oncogenic stress. Long-term retention of senescent cells in the body, which is attributed to highly expressed BCL-family proteins, chronically damages tissues mainly through a senescence-associated secretory phenotype (SASP). It has been documented that accumulation of senescent cells contributes to chronic diseases and aging-related diseases. Despite the fact that no unique marker is available to identify senescent cells, increased p16INK4a expression has long been used as an in vitro and in vivo marker of senescent cells. We reviewed five existing p16INK4a reporter mouse models to detect, isolate, and deplete senescent cells. Senescent cells express high levels of anti-apoptotic and pro-apoptotic genes compared to normal cells. Thus, disrupting the balance between anti-apoptotic and pro-apoptotic gene expression, such as ABT-263 and ABT-737, can activate the apoptotic signaling pathway and remove senescent cells. Mitochondrial abnormalities in senescent cells were also discussed, for example mitochondrial DNA mutation accumulation, dysfunctional mitophagy, and mitochondrial unfolded protein response (mtUPR). The mitochondrial-targeted tamoxifen, MitoTam, can efficiently remove senescent cells due to its inhibition of respiratory complex I and low expression of adenine nucleotide translocase-2 (ANT2) in senescent cells. Therefore, senescent cells can be removed by various strategies, which delays chronic and aging-related diseases and enhances lifespan and healthy conditions in the body. [ABSTRACT FROM AUTHOR]

Background: Cellular senescence, measured by expression of the cell cycle kinase inhibitor p16INK4a, may contribute to accelerated aging in survivors of childhood, adolescent, and young adult cancer. The authors measured peripheral blood T‐lymphocyte p16INK4a expression among pediatric and young adult cancer survivors, hypothesizing that p16INK4a expression is higher after chemotherapy and among frail survivors. Methods: A cross‐sectional cohort of young adult survivors and age‐matched, cancer‐free controls were assessed for p16INK4a expression and frailty. Newly diagnosed pediatric patients underwent prospective measurements of p16INK4a expression before and after cancer therapy. Frailty was measured with a modified Fried frailty phenotype evaluating sarcopenia, weakness, slowness, energy expenditure, and exhaustion. Results: The cross‐sectional cohort enrolled 60 survivors and 29 age‐matched controls with a median age of 21 years (range, 17‐29 years). The prospective cohort enrolled 9 newly diagnosed patients (age range, 1‐18 years). Expression of p16INK4a was higher among survivors compared with controls (9.6 vs 8.9 log2 p16 units; 2‐sided P =.005, representing a 25‐year age acceleration in survivors) and increased among newly diagnosed patients from matched pretreatment to posttreatment samples (7.3‐8.9 log2 p16 units; 2‐sided P =.002). Nine survivors (16%) were frail and had higher p16INK4a expression compared with robust survivors (10.5 [frail] vs 9.5 [robust] log2 p16 units; 2‐sided P =.055), representing a 35‐year age acceleration among frail survivors. Conclusions: Chemotherapy is associated with increased cellular senescence and molecular age in pediatric and young adult cancer survivors. Frail survivors, compared with robust survivors, exhibit higher levels of p16INK4a, suggesting that cellular senescence may be associated with early aging in survivors. The expression of p16INK4a, a biomarker of cellular aging and senescence, increases among childhood, adolescent, and young adult cancer survivors after chemotherapy and is higher among frail survivors, indicating advanced cellular aging related to cancer treatment. [ABSTRACT FROM AUTHOR]

This aim of this study was to observe the effect of Yang Yan Qing E Wan (YYQEW) on senescent phenotypes and the expression of β-catenin and p16INK4a in the hydrogen peroxide (H 2 O 2)-induced premature senescence of normal human skin fibroblasts (NHSFs). Primary normal human skin fibroblasts were randomly divided into a normal group, a blank group, a model group, and a YYQEW group. The cells of the model group and the YYQEW group were exposed to 150 μmol/L H 2 O 2 for 2 h. The morphological changes of the cells were analyzed by microscopy and by kits used to estimate the activities of the senescence-associated β-galactosidase (SA-β-gal), reactive oxygen species (ROS), and superoxide dismutase (SOD). The outcomes revealed that dyeing rate proportion of SA-β-gal was 2.78% ± 0.22% in the normal group, 2.83% ± 0.29% in the blank group, 37.58% ± 2.56% in the model group, and 28.39% ± 0.93% in the YYQEW group. The number of SA-β-gal positive cells was thus significantly higher in the model group than in the normal or blank group. There were also fewer SA-β-gal positive cells in the YYQEW group compared with the model group. The expression of ROS and p16INK4a in the model group increased significantly compared with that in the normal or blank groups, while the expression of ROS and p16INK4a in the YYQEW group decreased significantly compared with that in the model group. The expression of SOD and β-catenin in the model group decreased significantly compared with that in the normal or blank group, and the expression of SOD and β-catenin in the YYQEW group increased significantly compared with that in the model group. Overall, it was found that YYQEW was able to delay the senescence of NHSFs induced by H 2 O 2 treatment by alleviating oxidative stress and regulating a number of senescence-related molecules, such as β-catenin and p16INK4a. • This aim of this study was to observe the Yang Yan Qing E Wan is able to inhibit skin cell senescence induced by hydrogen peroxide, regulate oxidative stress damage, up-regulate β-catenin expression, and inhibit the expression level of p16INK4a. • YYQEW is able to inhibit skin cell senescence induced by hydrogen peroxide, regulate oxidative stress damage, up-regulate β-catenin expression, and inhibit the expression level of p16INK4a. • The exact mechanism of YYQEW in delaying skin cell aging requires further investigation. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the effect of Guilu Erxian Glue (龟鹿二仙胶, GEG) on cyclophosphamide (CTX)-induced bone marrow hematopoietic stem cells (HSCs) senescence in mice and explore the underlying mechanism. Methods: The H22 liver cancer ascites lump model was established in male Kunming mice by injecting intraperitoneally (i.p.) with 5 × 106/mL H22 cells per mouse. Fifty tumor-bearing mice were divided into the control, model, pifithrin-α, GEG, and GEG+pifithrin-α groups using a random number table, 10 mice in each group. CTX (100 mg/kg i.p.) was administrated to mice from day 1 to day 3 (d1–d3) continuously except for the control group. The mice in the pifithrin-α, GEG and GEG+pifithrin-α groups were treated with pifithrin-α (2.2 mg/(kg·d) i.p.) for 6 consecutive days (d4–d9), GEG (9.5 g/(kg·d) i.p.) for 9 consecutive days (d1–d9), and GEG plus pifithrin-α, respectively. HSCs were collected after 9-d drug treatment. The anti-aging effect of GEG was studied by cell viability, cell cycle, and β -galactosidase (β -gal) assays. The mRNA and protein expressions of cyclin-dependent kinase 2 (CDK2), CDK4, inhibitor of cyclin-dependent kinase 4a encoding the tumor suppressor protein p16 (p16INK4a), p21Cip1/Waf1, p53, and phosphorylated retinoblastoma (pRb) were evaluated by quantitative real-time reverse transcription-polymerase chain reaction and semi-quantitative Western blot, respectively. Results: Compared with the model group, GEG increased cell viability as well as proliferation (P<0.05 or P<0.01) and reduced β -gal expression. Furthermore, GEG significantly decreased the expressions of p16INK4a, p53 and p21Cip1/Waf1 proteins, and increased the expressions of CDK2, CDK4 and pRb proteins compared with the model group (P<0.05 or P<0.01). Conclusion: GEG can alleviate CTX-induced HSCs senescence in mice, and the p16INK4a-Rb signaling pathway might be the underlying mechanism. [ABSTRACT FROM AUTHOR]

We assessed the prevalence of HPV DNA in a large series of Chinese penile cancer and examine its association with the histological subtype, p16INK4a expression, and prognosis. We pathologically categorized 226 invasive penile squamous cell carcinomas and assessed HPV genotyping by real-time PCR and p16INK4a immunohistochemistry. The results were correlated with histopathological and clinical parameters and disease-specific survival (DSS). HPV DNA was detected in 32.7% (74/226) of penile cancer cases. The most frequent genotype was HPV 16 (64/74, 86.5%), followed by HPV 18 (6/74, 8.1%). Fifty-nine (26.1%) cases were positive for the p16INK4a expression, and p16INK4a expression had a sensitivity of 56.8% (95% CI, 45.2–68.3%) and a specificity of 88.8% (95% CI, 83.8–93.9%) for defining HPV status. HPV DNA (P = 0.019), p16INK4a (P = 0.038), age (P = 0.018), grade of differentiation (P = 0.001), lymph nodes (P < 0.001), T stage (P < 0.001), M stage (P < 0.001), and lymphovascular invasion (LVI, P = 0.001) were prognostic factors for DSS. HPV-positivity (HR 0.334; 95% CI, 0.158–0.705, P = 0.004) was still a significant prognostic factor for DSS in the multivariate Cox regression model. HPV DNA was observed in one third of Chinese penile carcinoma cases. The p16INK4a expression can indicate high-risk human papillomavirus (HR-HPV). HPV-positive penile tumors confer a survival benefit over HPV-negative tumors. [ABSTRACT FROM AUTHOR]

Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite sharing the name and similar morphological features with cutaneous melanoma (CM), it is an entirely different neoplasia with a particular genetic background and clinical behavior. CDKN2A is a gene located at chromosome 9p21, encoding for P16INK4a and P14(ARF) proteins, whose role as a tumor suppressor has been clearly defined in many malignant tumors. CDKN2A frequently presents germline mutations in familial CM and epigenetic downregulation in a considerable percentage of sporadic CM. It has been hypothesized that CDKN2A alterations are early events in CM development, playing a central role in the malignant transformation of melanocytes. Alterations of the CDKN2A gene reduce the expression of P16INK4a in most CM subtypes. Immunohistochemical evaluation of P16INK4a is currently used, in association with Ki67 and HMB45, in pathology practice to discriminate between dysplastic nevi and melanoma. On the other hand, CKDN2A is rarely mutated in UM, and the immunohistochemical expression of P16INK4a has only been reported in small case series. We tested P16INK4a expression on paraffin-embedded tissue sections from 9 tissue microarrays (TMAs), built with 2 mm cores derived from 133 uveal melanoma FFPE blocks, collected from 1990 to 2018, and from selected paraffin-blocks of 3 UM liver metastases. The immunohistochemical expression of P16INK4a was assessed with a visual evaluation by light microscopy and then with a digital approach. Both approaches, with an acceptable concordance rate, revealed P16INK4a expression in a large proportion of UM cases and all liver metastases, opening new possibilities of using it in the differential diagnosis between cutaneous and uveal melanoma metastases in cases of unknown primary tumor or patients with two different primary melanomas.

Introduction Prophylactic vaccines are already available for prevention of human papillomavirus (HPV) infection. However, we still await development of therapeutic vaccines with high efficiency for stimulating specific T lymphocytes to clear HPV infection. Objective This study investigates the potential for subunits of human p16INK4a protein and E2 protein of HPV16 to stimulate dendritic cells and enhance the specific response of T lymphocytes against HPV-infected cells. Methodology Immunogenic epitopes of HPV16 E2 and p16INK4a proteins were predicted through the common HLA class I and II alleles present in the Thai population. Then, monocyte-derived dendritic cells (MDCs) were pulsed with HPV16 E2 and/or p16INK4a protein s and their maturity assessed. MDCs pulsed with either or both of these proteins at optimal concentrations were used for activation of autologous T lymphocytes and IFN-γ production was measured for specific response function. Results HPV16 E2 and p16INK4a proteins contain various immunogenic epitopes which can be presented by antigen-presenting cells via both HLA class I and II molecules. The stimulation of MDCs with either HPV16 E2 or p16INK4a proteins increased percentages and mean fluorescence intensity (MFI) of CD83+ MDCs in a dose-dependent manner. An optimum concentration of 250 ng/mL and 150 ng/mL of HPV16 E2 and p16INK4a proteins, respectively, stimulated MDCs via the MAPK pathway (confirmed by use of MAPK inhibitors). T lymphocytes could be activated by MDCs pulsed with these proteins, leading to high percentages of both CD4+ IFN-γ+ T lymphocytes and CD8+ IFN-γ+ T lymphocytes. The production of IFN-γ was higher in co-cultures containing MDCs pulsed with HPV16 E2 protein than those pulsed with p16INK4a. Interestingly, MDCs pulsed with a combination of HPV16 E2 and p16INK4a significantly increased IFN-γ production of T lymphocytes. The IFN-γ production was inhibited by both HLA class I and II blockade, particularly in co-cultures with MDCs pulsed with a combination of HPV16 E2 and p16INK4a. Conclusions This suggests that MDCs pulsed with both proteins enhances specific response of both CD4+ and CD8+ T lymphocytes. This study might provide a strategy for further in vivo study of stimulation of T lymphocytes for therapy of HPV-associated cancer.

Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Senescence is the irreversible arrest of cell proliferation that has now been shown to play an important role in both health and disease. With increasing age senescent cells accumulate throughout the body, including the bone marrow and this has been associated with a number of age-related pathologies including malignancies. It has been shown that the senescence associated secretory phenotype (SASP) creates a pro-tumoural environment that supports proliferation and survival of malignant cells. Understanding the role of senescent cells in tumor development better may help us to identify new treatment targets to impair tumor survival and reduce treatment resistance. In this review, we will specifically discuss the role of senescence in the aging bone marrow (BM) microenvironment. Many BM disorders are age-related diseases and highly dependent on the BM microenvironment. Despite advances in drug development the prognosis particularly for older patients remains poor and new treatment approaches are needed to improve outcomes for patients. In this review, we will focus on the relationship of senescence and hematological malignancies, how senescence promotes cancer development and how malignant cells induce senescence.

Abstract Background A pharmacologic strategy for age‐related muscle weakness is desired to improve mortality and disability in the elderly. Angiotensin‐converting enzyme 2 (ACE2) cleaves angiotensin II into angiotensin 1‐7, a peptide known to protect against acute and chronic skeletal muscle injury in rodents. Since physiological aging induces muscle weakness via mechanisms distinct from other muscle disorders, the role of ACE2‐angiotensin 1‐7 in age‐related muscle weakness remains undetermined. Here, we investigated whether deletion of ACE2 alters the development of muscle weakness by aging and whether angiotensin 1‐7 reverses muscle weakness in older mice. Methods After periodic measurement of grip strength and running distance in male ACE2KO and wild‐type mice until 24 months of age, we infused angiotensin 1‐7 or vehicle for 4 weeks, and measured grip strength, and excised tissues. Tissues were also excised from younger (3‐month‐old) and middle‐aged (15‐month‐old) mice. Microarray analysis of RNA was performed using tibialis anterior (TA) muscles from middle‐aged mice, and some genes were further tested using RT‐PCR. Results Grip strength of ACE2KO mice was reduced at 6 months and was persistently lower than that of wild‐type mice (p

Latar Belakang: Kondilomata akuminata (KA) merupakan penyakit infeksi menular seksual yang disebabkan oleh Human papillomavirus (HPV) dengan gejala berupa pertumbuhan tunggal atau multipel pada daerah anogenital. HPV risiko tinggi mempunyai kemampuan untuk berkembang menjadi keganasan, sedangkan HPV risiko rendah sangat jarang menimbulkan keganasan. Pemeriksaan p16ink4a digunakan untuk mendeteksi HPV yang berpotensi kearah keganasan. Deteksi genotipe HPV dengan menggunakan PCR memiliki sensitivitas yang sangat tinggi. Tujuan: Mengevaluasi profil ekspresi p16INK4a pada lesi KA pada wanita dengan infeksi HPV tipe risiko rendah, risiko tinggi, dan infeksi multipel HPV. Metode: Penelitian deskriptif, observasional, cross sectional dengan melakukan pemeriksaan p16INK4a dan genotyping HPV dengan teknik PCR lesi KA pada wanita. Hasil: Satu pasien dengan HPV risiko rendah (HPV 6 dan 11) memiliki gambaran p16ink4a negatif, 4 pasien memiliki gambaran p16ink4a sporadis, dan 2 pasien memiliki gambaran p16ink4a fokal, 1 pasien dengan HPV 18 memiliki gambaran p16ink4a difus, 1 pasien dengan infeksi multipel HPV 6,81,82,89 memiliki gambaran p16ink4a fokal, 4 pasien dengan infeksi multipel HPV memiliki gambaran p16ink4a difus. Simpulan: Terdapat 5 pasien dengan gambaran p16INK4a yang difus. p16ink4a yang difus menunjukkan bahwa pada pasien tersebut memiliki kemampuan untuk berkembang menjadi keganasan.

Abstract Background Cartilage degradation would result in osteoarthritis (OA). p16INK4awas found in some age-related diseases. In this study, we aimed to determine the role of p16INK4a during OA and to investigate the underlying mechanisms. Methods Enzyme-linked immunosorbent assay (ELISA) was performed to test the activity of senescence-associated secretory phenotype (SASP). Real-time PCR (RT-PCR) and Western blot were used to determine the expressions of target genes. Results The increased expressions of p16INK4a and E2F2 were accompanied with cartilage degradation induced by IL-1β. Over-expression of p16INK4a enhanced the secretion of SASP markers (TGFβ, IL-6, IL-8, IL-1α, MMP3 and MMP13), reduced the expression of type II procollagen (COL2A1).Thus, the over-expression of p16INK4a lead to cartilage injury. Moreover, we found that the expression of E2F2 was enhanced in p16INK4a over-expression group, and that cartilage injury caused by p16INK4a was alleviated by depleting E2F2. Conclusions p16INK4a was up-regulated during the cartilage injury in OA. p16INK4a promoted cartilage injury by increasing the expression of E2F2. Thus, this study extends the molecular regulation network for understanding pathological progression of OA, and provides potential therapeutic target for OA.

The aim of the study - to determine the dynamics of expression of р16, Ki-67 in patients with HPV-associated cervical intraepithelial neoplasia (CIN) in the complex treatment with inosine pranobex. Materials and methods. The study included 62 patients with cervical HPV infection, the main group consisted of 47 patients with confirmed CIN 1 and CIN 2-3 associated to HPV high-risk, the comparison group - patients with latent HPV infection and 13 women of the control group. At the 2nd stage, a group of women with CIN 2-3 was divided into two groups: 2A (n=17) included patients who received excision treatment in combination with inosine pranobex; 2B group (n=9) patients after excision treatment (LEEP or LLETZ) without inosine pranobex. Traditional methods of examination of cervical pathology were used, including HPV genotyping, and dynamic immunocytochemical examination to determine p16, Ki-67. Results. Comparative analysis (n=75) allowed to establish the absence of differences in the expression of р16, Ki-67 and co-expression in HPV-associated CIN 1 relative to the comparison and control group. CIN 2-3 was characterized by a predominance of the levels of biomarkers in comparison with CIN 1 and latent infection (p

Abstract Background The preferential use of aerobic glycolysis by tumor cells lead to high accumulation of lactate in tumor microenvironment. Clinical evidence has linked elevated lactate concentration with cancer outcomes. However, the role and molecular mechanisms of lactate in cellular senescence and tumor progression remain elusive. Methods The function of Snail in lactate-induced EMT in lung cancer cells was explored by wound healing assay and cell invasion assay. The qRT-PCR and dual luciferase reporter assay were performed to investigate how lactate regulates Snail expression. The level of TGF-β1 in culture supernatant of cells was measured by ELISA for its correlation with extracellular levels of lactate. Ras activity assay and SA-β-gal activity assay were established to determine the effect of lactate on oncogene-induced senescence in human lung epithelial cells. ChIP assays were conducted to determine the binding of snail to p16INK4a promoter. Two TCGA data sets (TCGA-LUAD and TCGA-LUSC) were used to explore the correlations between SNAI1 and CDKN2A expression. Results In this study, we showed the invasive and migratory potential of lung cancer cells was significantly enhanced by lactate and was directly linked to snail activity. We also demonstrated that extracellular acidification itself is a direct cause of the increased snail expression and physiologically coupled to LDHA-dependent conversion of pyruvate to lactate. Mechanistically, lactate exerts its central function in induction of snail and EMT by directly remodeling ECM and releasing activated TGF-β1. We also demonstrated that Snail help premalignant cells to escape the oncogene-induced senescence by directly targeting and inhibiting p16INK4a expression. Conclusions Our study extends the understanding of EMT in tumorigenesis by uncovering the role of snail in cellular senescence. This study also reveals lactate may be a potent tumor-promoting factor and provides the basis for the development of lactate-targeted therapy.

Introduction: Human papilloma virus (HPV) which is causative factor for cervical cancer may interact with p16 leading to malignant transformation of cervical epithelial cells. The present study was conducted to assess the immunoexpression of p16 INK4a in premalignant and malignant lesions of cervix and to correlate it with HPV 16 expression. It was also intended to study the various risk factors which may be associated with cervical cancer in this north Himalayan region of India. Material and Methods: The study included 50 cases of premalignant and malignant cervical lesions and 50 controls diagnosed on histopathology over a period of one year. All the relevant clinical details were noted and both cases and controls were subjected to HPV 16 and p16 INK4a immunohistochemical staining. Results: 67% of subjects (including cases and controls) and 94% of the cases were positive for HPV 16 expression. p16 INK4a expression was negative in all the controls, positive in 96% of invasive cancer, 66.6% in HSIL and 37.5% in LSIL. Conclusion: Cervical cancer is associated with low socio economic status, illiteracy, smoking, early age of marriage and conception in north Himalayan region of India. HPV 16 infection is positive in both cases and controls indicating high prevalence of HPV 16 in this region. Neoplastic transformation by HPV is identified by over expression of p16 INK4a in premalignant and malignant cases. The immunopositivity of p16 INK4a increases with the severity of cervical lesions and thus may play an important role in stratification of premalignant and malignant lesions.

Background: Immunohistochemical (IHC) expressions of p63, Ki-67, cyclin-D1, and p16 have been evaluated to assess the malignant potential of oral potentially malignant disorders (OPMDs); most of these studies are subjective and lack a definite quantitative approach. Further, H score of IHC expression has produced promising results and this has not been explored in any marker to predict malignant transformation in OPMDs. Aim: To evaluate the IHC expressions of p63, Ki-67, cyclin-D1, and p16INK4a in predicting the malignant transformation of the most common OPMDs. Materials and Methods: The IHC expressions of p63, Ki-67, cyclin-D1, and p16INK4a on paraffin-embedded sections of 10 cases each in normal oral mucosa, mild, moderate, and severe oral epithelial dysplasia, oral submucous fibrosis (OSMF), and oral squamous cell carcinoma (OSSC) were evaluated using mean H score for every group. Mean H score was compared within and between the groups using analysis of variance. Pairwise comparison was made using Tukey's multiple post hoc procedure and strength of association was assessed using Karl's Pearson's correlation. Results: Mean H scores of p63, Ki-67, and cyclin-D1 increased significantly (P = 0.0001) with increasing grades of dysplasia to OSCC, whereas mean H score of p16 significantly (0.041) decreased with increasing grades of dysplasia to malignancy. OSMF indicated a variation in its expression pattern. Only mean H scores of Ki-67 (0.0239) and cyclin-D1 (0.0044) significantly differentiated severe dysplasia from OSCC. Conclusion: Combination of mean H score of Ki-67 and mean H score of cyclin-D1 could be effectively used to identify transformation potential of OPMDs.