P16 Ink4a Research Articles

MAFK Polymorphisms Located in 3'-UTR are Associated with Severity of Atrophy and CDKN2A Methylation Status in the Gastric Mucosa.

Objective: This study aimed to clarify the association of MAFK polymorphisms (rs4268033, rs3735656, and rs10226620) with the degree of gastric mucosal atrophy and CDKN2A CpG methylation status. Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Methods: A total of 491 subjects were enrolled in this study. Genotypes and methylation status were determined by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and methylation-specific PCR (Fujita Health University, HM18-094). Results: Either rs3735656 or rs10226620, located in the 3'-UTR of MAFK, was significantly associated with the severity of gastric mucosal atrophy using a dominant genetic model (odds ratio [OR], 2.10; p = 0.0012, and OR, 1.98; p = 0.0027, respectively). However, using a recessive genetic model, no significant association was found between three polymorphisms and gastric mucosal atrophy. The serum pepsinogen I/II ratio was significantly lower in subjects with minor alleles of rs3735656 and rs10226620 than in subjects with the wild homozygous allele (p = 0.018 and 0.013, respectively). In a subgroup including 400 of the 491 subjects, the CpG of p14ARF and p16 INK4a were methylated in 132 and 112 subjects, respectively. Fifty subjects had both CpG methylations and 206 subjects had neither methylation. When comparing the groups with both and neither methylations, there were no significant associations between three polymorphisms and CDKN2A methylation using a dominant genetic model. However, all polymorphisms investigated in this study (rs4268033, rs3735656, and rs10226620) were significantly associated with CDKN2A methylation in a recessive genetic model (OR, 3.58; p = 0.0071, OR, 4.49; p = 0.0004, and OR, 3.45; p = 0.0027, respectively). Conclusions: Our results indicate that carrying the minor allele of the MAFK polymorphisms, particularly when they are located in the 3'-UTR, has a high risk for the severity of gastric mucosal atrophy; furthermore, CDKN2A CpG methylation may develop in subjects with homozygous minor allele of these polymorphisms.

Accuracy of HPV E6/E7 mRNA examination using in situ hybridization in diagnosing cervical intraepithelial lesions

BackgroundThe consistency of pathologists in the diagnosis of cervical intraepithelial neoplasia (CINs) is not ideal, especially between low- and high-grade squamous intraepithelial lesions (LSIL and HSIL). This study was aimed to explore efficient strategies for the grading of CINs.MethodsThe medical records of patients with high risk human papillomavirus (HR-HPV) infections who had underwent cervical biopsy or conization from April 2018 to April 2019 in Beijing Chao-Yang Hospital were collected and examined. The HR-HPV E6/E7 mRNA in the tissues of patients with CINs was detected using RNAscope chromogenic in situ hybridization (RISH). Immunohistochemistry (IHC) was performed to evaluate the expression of p16INK4a (P16) and Ki67.ResultsHR-HPV E6/E7 mRNA signals were detected in 3/27 (11.1 %) of CIN 1, and in 32/33 (97.0 %) of CIN 2/3. Most of the staining patterns (27/32, 84.4 %) had a full-thickness epithelial layer staining with weak-to-strong nuclear and cytoplasmic dot-like signals in CIN 2/3, and there were also few special staining patterns that were significantly different from the others. A number of indicators were compared between LSIL and HSIL. There were statistically significant differences in E6/E7 mRNA, p16, Ki67 and cytology between the two groups (P < 0.05). According to the logistic regression analysis, merely E6/E7 mRNA positivity was significantly associated with CIN2/3 (OR: 52.53, 95 % CI, P < 0.05). In the detection of CIN 2/3, the sensitivity and specificity of HPV E6/E7 mRNA alone was not significantly inferior to that of its different combinations with Ki67, p16 and cytology (all, P > 0.05).ConclusionsRISH is efficient in grading of CINs. The HPV E6/E7 mRNA expression might reflect the phase HPV infections, and its positive pattern might predict the development direction of CINs, providing the possibility to realize more accurate treatments for patients.

9-Hydroxy-6,7-dimethoxydalbergiquinol suppresses hydrogen peroxide-induced senescence in human dermal fibroblasts through induction of sirtuin-1 expression

Objective: To investigate the potential anti-aging mechanism of 9-hydroxy-6,7-dimethoxydalbergiquinol (HDDQ) on hydrogen peroxide (H2O2)-induced oxidative stress in human dermal fibroblasts (HDFs). Methods: The effect of HDDQ on cell viability was assessed by MTT assay, and the effects of HDDQ on senescence-like phenotypes were determined by senescence-associated β-galactosidase (SA-β-gal) staining, Western blotting analysis, and a cell proliferation assay. The expression level and activity of sirtuin-1 (SIRT1) induced by HDDQ were also measured. Results: HDDQ reversed senescence-like phenotypes in the oxidant-challenged model, through reducing SA-β-gal activity and promoting cell growth. Meanwhile, decreases in ac-p53, p21 Cip1/WAF1 , and p16 Ink4a and an increase in pRb were observed. HDDQ induced the expression of SIRT1 in a concentration- and time-dependent manner. Moreover, HDDQ inhibited H2O2-induced phosphorylation of Akt by SIRT1 up-regulation and reduced SA-β-gal staining. Conclusions: HDDQ inhibits H2O2-induced premature senescence and upregulation of SIRT1 expression plays a vital role in the inhibition of the senescence phenotype in HDFs.

Intra-Articular Injection of (-)-Epigallocatechin 3-Gallate (EGCG) Ameliorates Cartilage Degeneration in Guinea Pigs with Spontaneous Osteoarthritis.

Osteoarthritis (OA) is the most prevalent joint disease that causes an enormous burden of disease worldwide. (−)-Epigallocatechin 3-gallate (EGCG) has been reported to reduce post-traumatic OA progression through its anti-inflammatory property. Aging is the most crucial risk factor of OA, and the majority of OA incidences are related to age and not trauma. In this study, we assess whether EGCG can ameliorate cartilage degradation in primary OA. In an in-vitro study, real-time PCR was performed to assess the expression of genes associated with human articular chondrocyte homeostasis. A spontaneously occurring OA model in guinea pigs was used to investigate the effect of EGCG in vivo. OA severity was evaluated using Safranin O staining and Osteoarthritis Research Society International (OARSI) scores, as well as by immunohistochemical (IHC) analysis to determine the protein level of type II collagen (Col II), matrix metalloproteinase 13 (MMP-13), and p16 ink4a in articular cartilage. In the in-vitro study, EGCG increased the gene expression of aggrecan and Col II and decreased the expression of interleukin-1, cyclooxygenase 2, MMP-13, alkaline phosphatase, Col X, and p16 Ink4a; EGCG treatment also attenuated the degraded cartilage with a lower OARSI score. Meanwhile, IHC results showed that EGCG exerted an anti-OA effect by reducing ECM degradation, cartilage inflammation, and cell senescence with a less-immunostained Col II, MMP-13, and p16 Ink4a. In conclusion, these findings suggest that EGCG may be a potential disease-modifying OA drug for the treatment of primary OA.

Senescence research from historical theory to future clinical application.

Historically, the findings from cellular lifespan studies have greatly affected aging research. The discovery of replicative senescence by Hayflick developed into research on telomeres and telomerase, while stress-induced senescence became known as a telomere-independent event. Senescence-inducing signals comprise several tumor suppressors or cell cycle inhibitors, e.g., p53, cyclin-dependent kinase inhibitor p16 Ink4a and others. Stress-induced senescence serves as a physiological barrier to oncogenesis in vivo, while it activates senescence-associated secretary phenotype, inducing chronic inflammation. Thus, beside telomere length, p16, p53 and inflammatory cytokines have been utilized as biomarkers for cellular senescence. Telomere lengths in human leukocytes correlate well with events of aging-related lifestyle diseases, indicating the importance of cellular senescence in organismal aging. As such, the development of senescence research will have significant future clinical applications, e.g., senolysis. Geriatr Gerontol Int 2021; 21: 125-130.

Apigenin 7-O-glucoside promotes cell apoptosis through the PTEN/PI3K/AKT pathway and inhibits cell migration in cervical cancer HeLa cells

Epidemiologic evidence promote the inclusion of flavones in diet due to their inhibitory effects on certain types of cancers, particularly in women. Among the naturally occurring plant flavonoids, Apigenin 7-O-glucoside (AGL) is endowed with anti-inflammatory, anti-oxidant, and anti-cancer activities. However, its mechanism of action on cervical cancer, the fourth largest cancer in women, has not yet been clarified. In the current study, we have determined the effect of AGL on human cervical cancer cells and studied its molecular mechanism against cervical cancer. The results showed that AGL inhibited the proliferation of HeLa cells (IC50 was 47.26 μM at 48 h) by inducing apoptosis. Furthermore, AGL treatment caused G0/G1 phase arrest, reduced mitochondrial membrane potential (MMP), and upgraded intracellular ROS production. AGL could promote the release of cytochrome c by regulating Bcl-2 family proteins, and then activated caspase 9/3 to promote cell apoptosis. Moreover, AGL treatment promoted the expression of p16 INK4A, while inhibited the expression of Cyclin A/D/E and CDK2/6. At the same time in HeLa cells treated with AGL, the PTEN/PI3K/AKT pathway was inhibited in a concentration-dependent manner, and cell migration was also impeded correspondingly through the matrix metalloproteinase 2 and 9. Our study may provide a new research direction for harnessing the novel natural compounds in cervical cancer treatment.

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.

Characterization of cervical biopsies of women with HIV and HPV co-infection using p16ink4a, ki-67 and HPV E4 immunohistochemistry and DNA methylation

This study aims to characterize cervical intraepithelial neoplasia (CIN) in women living with HIV using biomarkers. Immunohistochemical (IHC) staining for human papillomavirus (HPV) E4 protein indicates CIN with productive HPV infection, whereas Ki-67 and p16ink4a indicate CIN with transforming characteristics, which may be further characterized using DNA hypermethylation, indicative for advanced transforming CIN. Cervical biopsies (n = 175) from 102 HPV positive women living with HIV were independently reviewed by three expert pathologists. The consensus CIN grade was used as reference standard. IHC staining patterns were scored for Ki-67 (0–3), p16ink4a (0–3), and E4 (0–2) and correlated to methylation levels of four cellular genes in corresponding cervical scrapes. Reference standards and immunoscores were obtained from 165 biopsies:15 no dysplasia, 91 CIN1, 31 CIN2, and 28 CIN3. Ki-67 and p16ink4a scores increased with increasing CIN grade, while E4 positivity was highest in CIN1 and CIN2 lesions. E4 positive CIN1 lesions had higher Ki-67 and p16ink4a scores and higher methylation levels compared with E4 negative CIN1 lesions. E4 positive biopsies with low cumulative Ki-67/p16 ink4a immunoscores (0-3) had significantly higher methylation levels compared with E4 negative biopsies. No significant differences in Ki-67 and p16ink4a scores and methylation levels were observed between E4 negative and positive CIN2 or CIN3 lesions. The presence of high methylation levels in scrapes of CIN lesions with IHC characteristics of both productive (E4 positive) and transforming infections (increased Ki-67/p16ink4a expression) in women living with HIV might indicate a rapid aggressive course of HPV infections towards cancer in these women.

71TiP P16 protein expression as a predictive biomarker for response to neoadjuvant chemotherapy in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone in children and young adults. Necrotic response in the tumor specimen after preoperative chemotherapy is a reproducible and reliable prognostic marker for survival in patients with OS. Pathologic tumor necrosis 90% or greater in the surgical specimen has been correlated with a 5-year survival of 75% to 90%. Although several molecular markers, including P-glycoprotein, Ki-67, p53, ezrin and HER2 have been associated with OS, a clear consensus has not emerged regarding the prognostic and predictive significance. The downregulation of p16 INK4A proteins is found in around 60% of high-grade osteosarcomas and plays an important role in growth of osteosarcoma cells by deregulation of both the pRb and p53 cell growth control pathways. Based on previously published studies, p16 protein negative expression on IHC is a predictive biomarker to poor pathologic response to neoadjuvant chemotherapy and is associated with a worse prognosis. p16 expression is correlated to the tumor biology and is not influenced by chemotherapy treatment. Thus, given the paucity of Indian data and lack of prospective studies, we aimed to investigate the role of P16 protein expression as a predictive biomarker in osteosarcoma. Aim: To investigate the relationship of p16 expression in pretreatment osteosarcoma tumors to pathologic necrotic response after neoadjuvant chemotherapy in a prospective pilot study. Inclusion criteria: Histologically proven cases of newly diagnosed osteosarcoma planned for neoadjuvant chemotherapy. Exclusion criteria: 1. Metastatic and relapsed osteosarcoma; 2. Upfront operated cases of osteosarcoma; 3. Prior therapy. Study procedure: Immuno-histochemistry for p16 will be conducted in core biopsy samples upfront before starting neoadjuvant chemotherapy in newly diagnosed case of osteosarcoma tumor cell necrosis rate will be calculated after neoadjuvant chemotherapy on surgical specimen. The grade of tumour cell necrosis rate will be classified using the Huvos necrosis grading system P16 correlation with good response to chemotherapy in terms of tumour necrosis will be evaluated. The author.

Importance of HPV in Chinese Penile Cancer: A Contemporary Multicenter Study

Objective: To investigate the HPV DNA prevalence and genotype distribution among penile cancer in China. To identify association between HPV prevalence, different histological subtypes, tumor stage, tumor grade, demographics, comorbidity, and phimosis incidence trend. Standardized HPV DNA detection and p16INK4a expression were used in a multi-center series of 340 penile squamous cell carcinomas diagnosed from 2006 to 2017.Materials and Methods: HPV DNA detection and genotyping were examined by a validated kit for 23 different HPV subtypes (PCR-RDB HPV test). The cases with positive HPV DNA were additional tested for p16INK4a expression to confirm the HPV infection.Results: Using the PCR-RDB HPV test, overall HPV prevalence was 48.8% (166/340) and that of p16INK4a expression was 45.6%. In this studied population, HPV16 was the most frequent HPV type detected in HPV-positive cancers (76.5%). HPV18 was the second most common type in penile cancers (15.1%). After pathology review, 307 cases were confirmed as invasive penile cancer, and the other 33 were non-invasive caners. The histologic subtypes of warty, basaloid, clear cell papillary, adenosquamaous and pseudohyperplastic were showed high HPV DNA prevalence. Among invasive cancers, no statistically significant differences in prevalence were observed by tumor grade, tumor stage or lymphnode stage at diagnosis. HPV positive penile cancer incidence significantly increase and the phimosis incidence significantly decrease from 2006 to 2017.Conclusions: About a half of penile cancers were related to HPV infection. Our findings highlight the phimosis related penile cancers have been declining, the HPV related in the development of penile cancer and a fully aware of regional differences in HPV genotype distribution are tasks for penile cancer control and prevention.

ASSOCIATION BETWEEN THE PERCENTAGE OF p16-POSITIVE SENESCENT CELLS AND THE EXPRESSION OF ESTROGEN AND PROGESTERONE RECEPTORS IN HUMAN ENDOMETRIUM DURING THE MID-LUTEAL PHASE

Progesterone and estrogen secretion and the expression of their receptors are strongly connected with the cyclic changes in endometrium and its receptivity. We previously reported that the frequency of occurrence of p16-positive senescent cells in human endometrium appear to be also associated with endometrial receptivity and successful embryo implantation. The objective of this study was to analyze the relationship between the percentage of senescent cells and estrogen and progesterone receptors expression in human endometrium during the mid-luteal phase of the menstrual cycle. Prospective observational study. This study includes 383 women who had an endometrial biopsy during the mid-lutheal phase (7 days after LH surge) of the natural cycles. Tissue sections were stained immunohistochemically for ER, PR and p16 using the following monoclonal antibodies: progesterone receptor (PR) (NCL-PGR-312, Leica Microsystems), 1:100 dilution; estrogen receptor (ER) (NCL-L-ER-6F11, Leica Microsystems), 1:100 dilution; p16 ink4a antibody (MAD-000690QD–7, Master Diagnostica), ready-to-use. The percentage of positively stained p16 cells and the levels of ER/PR expression were evaluated by Image-J software (NIH, Maryland, USA) in multiple endometrial sections. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). No correlation was found between the frequencies of occurrence of senescent p16-positive stromal, luminal and glandular epithelial cells and PR (p>0.05). In contrast, the glandular expression of ER showed low, but a significant correlation with the percentage of p16+ glandular epithelial cells (R=0.34; p=0.03). In conclusion, we found that the increased cell senescence in endometrial glands during the mid-luteal phase is associated with increased expression of glandular ER in the mid-luteal phase of the cycle. These results suggest an estrogen related change of endometrial senescent cells during the period of embryo implantation.

Expression of p16 in cervical premalignant and malignant lesions- IHC study

Cervical cancer is the second leading cause of female cancer in India. Human papilloma virus (HPV) infection is the most important risk factor for uterine cervical neoplasms. HPV oncogene expression can be detected by cellular protein p16 (INK4a) using immunohistochemistry. The present study aims to assess the immunoexpression of protein p16INK4a in cervical dysplastic and neoplastic lesions. The study includes 86 cases of premalignant and malignant cervical lesions diagnosed on histopathology for two years. All cases were subjected to p16 INK4a immunohistochemical staining.72% of cases (62 out of 86) (including premalignant and malignant) were positive for p16. P16 was positive in 88% of invasive carcinoma, 70.83% of HSIL and 40% of LSIL. Cervical cancer is associated with HPV infection which can be identified by overexpression of p16 INK4a in premalignant and malignant lesions. The expression of p16 INK4a positivity increases with increasing grades of dysplasia and thus helps in the stratification of cervical dysplastic and neoplastic lesions. P16 INK4a can be used as a surrogate marker of HPV infection.

Inflammatory Drivers of Cardiovascular Disease: Molecular Characterization of Senescent Coronary Vascular Smooth Muscle Cells.

The senescence of vascular smooth muscle cells (VSMCs) has been implicated as a causal pro-inflammatory mechanism for cardiovascular disease development and progression of atherosclerosis, the instigator of ischemic heart disease. Contemporary limitations related to studying this cellular population and senescence-related therapeutics are caused by a lack of specific markers enabling their detection. Therefore, we aimed to profile a phenotypical and molecular signature of senescent VSMCs to allow reliable identification. To achieve this goal, we have compared non-senescent and senescent VSMCs from two in vitro models of senescence, replicative senescence (RS) and DNA-damage induced senescence (DS), by analyzing the expressions of established senescence markers: cell cycle inhibitors- p16 INK4a, p14 ARF, p21 and p53; pro-inflammatory factors-Interleukin 1β (IL-1β), IL-6 and high mobility group box-1 (HMGB-1); contractile proteins-smooth muscle heavy chain- (MYH11), smoothelin and transgelin (TAGLN), as well as structural features (nuclear morphology and LMNB1 (Lamin B1) expression). The different senescence-inducing modalities resulted in a lack of the proliferative activity. Nucleomegaly was seen in senescent VSMC as compared to freshly isolated VSMC Phenotypically, senescent VSMC appeared with a significantly larger cell size and polygonal, non-spindle-shaped cell morphology. In line with the supposed switch to a pro-inflammatory phenotype known as the senescence associated secretory phenotype (SASP), we found that both RS and DS upregulated IL-1β and released HMGB-1 from the nucleus, while RS also showed IL-6 upregulation. In regard to cell cycle-regulating molecules, we detected modestly increased p16 levels in both RS and DS, but largely inconsistent p21, p14ARF, and p53 expressions in senescent VSMCs. Since these classical markers of senescence showed insufficient deregulation to warrant senescent VSMC detection, we have conducted a non-biased proteomics and in silico analysis of RS VSMC demonstrating altered RNA biology as the central molecular feature of senescence in this cell type. Therefore, key proteins involved with RNA functionality, HMGB-1 release, LMNB-1 downregulation, in junction with nuclear enlargement, can be used as markers of VSMC senescence, enabling the detection of these pathogenic pro-inflammatory cells in future therapeutic studies in ischemic heart disease and atherosclerosis.

A Novel Biochemical Study of Anti-Ageing Potential of Eucalyptus Camaldulensis Bark Waste Standardized Extract and Silver Nanoparticles

Eucalyptus camaldulensis Dehnh belongs to family Myrtaceae. They are massive in Egypt. Although reputed for high phenolic content, barks are considered waste. Ageing is a natural phenomenon caused by apoptosis and senescence resulting in wrinkles. The phytochemical analysis of the 70% ethanolic Eucalyptus camaldulensis bark extract (EBE) and evaluation of its anti-ageing potential and as silver nanoparticles (AgNPs) were conducted in this study. Ultra performance liquid chromatography / electrospray ionization mass spectrometry of EBE fingerprint revealed twenty compounds, where Rutin was major. EBE was standardized to contain 1.26 % Rutin. AgNPs synthesized by green synthesis, were characterized by transmission electron microscope and zeta potential measurement. Both EBE and AgNPs were subjected to MTT assay in HFB4 cells and cell cycle arrest. Flow cytometry was used to assess apoptosis and p16 INK4a. Genetic expression of p53 and p21 and telomerase level were determined. Anti-wrinkle enzyme assays were done. AgNps were spherical, 468.7 nm in size and with Poly dispersity index of 0.817 ± 0.129. EBE and AgNPs with IC50 0.156 mg/mL ± 0.05 and 2.315 ± 0.07 μg/mL expressed significant difference in % of cells (DNA content) at G2/M, apoptotic cells numbers, p53 and p21expression and p16INK4a vs aged cells (P < 0.0001). Both expressed significant increase in telomerase (P < 0.0001). They exhibited elastase, collagenase and tyrosinase inhibition (75 ± 4.3 and 75.9 ± 6.8 % at 300 μg/mL, 58 ± 4.8 and 63 ± 2.3, at 500 μg/mL, 51 ± 4.8 and 65 ± 5.87, at 500 μg/mL, respectively. Although it is considered waste, EBE and Ag NPs are anti-ageing candidates as they inhibit apoptosis, senescence and prevent wrinkles formation.

Hallmarks of cellular senescence more abundant in damaged compared to intact cartilage in osteoarthritis

Purpose: Cellular senescence is a consequence of aging, which is associated with cell cycle arrest and has recently been identified as a major factor in the pathogenesis of osteoarthritis. Senescence could contribute to OA by several mechanisms including joint tissue increase in pro-inflammatory senescence-associated secretory proteins (SASPs). Our goal was to quantify the extent and characteristics of cellular senescence of human OA cartilage. To this end, we developed a short enzymatic digestion protocol for primary chondrocyte isolation and compared intact and damaged regions of cartilage within each patient to minimize potential confounding by cell stress that could induce senescence phenotypes during the cell isolation and manipulation procedures. We hypothesized that differences between damaged and intact regions of cartilage from each patient will more reliably identify disease associated senescence than evaluation of damaged regions alone. We characterized senescence based on β-galactosidase (β-gal) activity and p16 INK4a protein expression (two accepted markers for senescent cell identification), quantification of MMP3 protein expression (a SASP) and characterization of cell cycle phases. Methods: Human knee joints were obtained as surgical waste tissue under IRB approval from 4 OA patients during total knee arthroplasty. To isolate primary chondrocytes from macroscopically damaged vs intact regions, human articular cartilage from selected regions was finely diced and digested in 0.3% type II collagenase solution for three hours at 37 °C. Chondrocytes were isolated by centrifugation (300G) and directly stored as cell pellets in -80oC for β-gal activity (fluorimetric quantification) or fixed in 80% methanol and stored in -20oC for flow-cytometric analysis of p16 INK4a(ROCHE #9517 ), MMP3 (Thermo #701285) and cell cycle phase (by propidium iodide, Miltenyi Biotec). β-gal was assessed by SensoLyte ® MUG β - Galactosidase Assay Kit (Anaspec AS-72132) and normalized to DNA content (Biotium #31008). Results: Our protocol yielded a mean of 2.78 x 106 (range: 1.94-3.65x106) chondrocytes from 1 gram of human cartilage. Compared to intact cartilage, β-gal activity and p16INK4a protein expression were higher in damaged cartilage (Figure 1A, N=4, P=0.034; Figure 1B, N=4, P=0.031). The proportion of p16INK4a+/MMP3+ cells was 2.8-fold higher in damaged cartilage compared with intact cartilage (Figure 1C, N=4, P=0.082). Overall, β-gal activity was positively correlated with p16INK4aprotein expression. (Figure 1D, N=8, r=0.57, p=0.072). Compared with chondrocytes from intact cartilage, a higher proportion of chondrocytes from damaged cartilage were in the tetraploid G2 cell cycle phase (P=0.04) and a lower proportion were in the diploid G1 phase (P=0.02) (Figure 2A, N=4). Compared to diploid (G1 phase) chondrocytes,p16INK4a protein was much more highly expressed in tetraploid (G2 phase) chondrocytes (Figure 2B, N=8, P=0.001). Conclusions: We successfully isolated primary human chondrocytes using a short enzymatic digestion protocol to minimize phenotypic changes from cell manipulation, a stressor that might artifactually increase senescence cell markers in chondrocytes. β-gal activity was positively correlated with p16 INK4a expression and both markers were higher in damaged cartilage, which suggested an association of senescent chondrocytes with OA. Moreover, MMP3 expression was positively correlated with p16 INK4a expression. These results suggest at least one means, namely increased cartilage degradation by MMP3, by which senescent chondrocytes could contribute to the pathogenesis of OA. A previous study reported a higher percentage of tetraploid chondrocytes within damaged cartilage; we further identified increased p16INK4a expression in tetraploid chondrocytes. In conclusion, this study provides supportive evidence for the role of cellular senescence in the pathogenesis of human OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Targeted Reduction of Senescent Cell Burden Alleviates Focal Radiotherapy-Related Bone Loss.

Clinical radiotherapy treats life-threatening cancers, but the radiation often affects neighboring normal tissues including bone. Acute effects of ionizing radiation include oxidative stress, DNA damage, and cellular apoptosis. We show in this study that a large proportion of bone marrow cells, osteoblasts, and matrix-embedded osteocytes recover from these insults only to attain a senescent profile. Bone analyses of senescence-associated genes, senescence-associated beta-galactosidase (SA-β-gal) activity, and presence of telomere dysfunction-induced foci (TIF) at 1, 7, 14, 21, and 42 days post-focal radiation treatment (FRT) in C57BL/6 male mice confirmed the development of senescent cells and the senescence-associated secretory phenotype (SASP). Accumulation of senescent cells and SASP markers were correlated with a significant reduction in bone architecture at 42 days post-FRT. To test if senolytic drugs, which clear senescent cells, alleviate FRT-related bone damage, we administered the senolytic agents, dasatinib (D), quercetin (Q), fisetin (F), and a cocktail of D and Q (D+Q). We found moderate alleviation of radiation-induced bone damage with D and Q as stand-alone compounds, but no such improvement was seen with F. However, the senolytic cocktail of D+Q reduced senescent cell burden as assessed by TIF+ osteoblasts and osteocytes, markers of senescence (p16 Ink4a and p21), and key SASP factors, resulting in significant recovery in the bone architecture of radiated femurs. In summary, this study provides proof of concept that senescent cells play a role in radiotherapy-associated bone damage, and that reduction in senescent cell burden by senolytic agents is a potential therapeutic option for alleviating radiotherapy-related bone deterioration. © 2020 American Society for Bone and Mineral Research.

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation.

SummaryApproximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

Inclusion of an E7 DNA Amplification Test Improves the Robustness of Human Papillomavirus-Associated Oropharyngeal Squamous Cell Carcinoma Diagnosis.

BackgroundThe rise in human papillomavirus (HPV) infection rates over the last few decades in the USA has contributed to a significant increase in the overall incidence of patients diagnosed with squamous cell carcinoma of the head and neck. These head and neck carcinomas develop in the oropharynx, with more than 90% of them caused by infection with high-risk HPV type 16. Patients diagnosed with HPV-induced oropharyngeal squamous cell carcinomas (OPSCCs) have a better prognosis and treatment response than those diagnosed with head and neck cancers caused by alcohol consumption and tobacco use. To identify patients with HPV-positive OPSCC, new guidelines recommend positive staining of oropharyngeal tissues for p16 INK4a (p16) by immunohistochemistry (IHC). Herein we discuss the testing algorithm that was adopted to address discrepant results between p16 IHC and a DNA in situ hybridization (ISH) test used routinely to diagnose HPV-positive OPSCC patients.MethodsA DNA polymerase chain reaction (PCR) test that amplifies HPV16 and HPV18 E7 was developed to aid in the diagnosis of HPV-positive OPSCC in a subset of patients. Specimens from these patients stained positive for p16 by an IHC test, but negative for high-risk HPV by a commercial DNA ISH test. Moreover, these results did not match the histopathological characteristics of the specimens, nor the clinical presentations of the patients.ResultsOf 21 patients’ specimens that were tested for p16 by IHC, 11 specimens showed concordant results with the high-risk HPV 16/18 DNA ISH test. Whereas, in eight p16 IHC positive specimens, HPV viral DNA was not detected by HPV16/18 DNA ISH, and two specimens were not tested by DNA ISH. When these eight p16 IHC positive specimens with discrepant p16 IHC and DNA ISH results were further tested by DNA PCR, six specimens showed concordance with p16 IHC with positive results for HPV16 E7, while two specimens were negative for HPV16 E7 by DNA PCR. All tested specimens were negative for HPV18 E7 by DNA PCR. Thus, the addition of the HPV16 and HPV18 E7 DNA PCR test identified a significant number of false negative test results by the HPV16/18 DNA ISH test and likely several false positive results by p16 IHC.ConclusionsInclusion of an HPV16 E7 DNA PCR test improved the robustness of HPV-associated OPSCC diagnosis in patients with discrepant results from p16 IHC staining and a DNA ISH test, and identified patients for proper management with less misclassification.

Role of p16 INK4A Protein in Evaluation of Benign, Premalignant and Malignant Cervical Lesion in Iraqi Patients (Immunohistochemical Study)

Role of p16 INK4A Protein in Evaluation of Benign, Premalignant and Malignant Cervical Lesion in Iraqi Patients (Immunohistochemical Study)

Increased p16INK4a-expressing senescent bile ductular cells are associated with inadequate response to ursodeoxycholic acid in primary biliary cholangitis

Background & aimsSenescent biliary epithelial cells (BECs) may be involved in the pathophysiology of primary biliary cholangitis (PBC) by secreting senescence-associated secretory phenotypes. We examined an association of the extent of cellular senescence in BECs with clinicopathological features including response to ursodeoxycholic acid (UDCA) and a possibility of senolytic therapy in PBC. MethodsThe expression of senescent markers (p21WAF1/Cip1, p16INK4a) and B-cell lymphoma–extra large (Bcl-xL), a key regulator of senescent cell anti-apoptotic pathway, was immunohistochemically examined in livers from patients with PBC (n = 145) and 103 control livers. Senolytic effect of Bcl-xL inhibitors (A-1331852 and Navitoclax) was examined in senescent murine BECs. ResultsSenescent BECs were increased in small bile ducts in PBC, compared with control livers (p < 0.01). Senescent BECs were increased in ductular reactions in PBC, stage 3–4, compared with PBC, stage 1–2 and control livers (p < 0.01). The extent of senescent BECs in bile ductules was significantly correlated with stage and hepatitis activity (p < 0.01) and the expression of p16INK4a in bile ductules was significantly correlated to inadequate response to UDCA in PBC (p < 0.01). Double immunofluorescence revealed an increased expression of Bcl-xL in p16INK4a-positive senescent BECs in PBC. Bcl-xL inhibitors selectively induced apoptosis in senescent murine BECs (p < 0.01). ConclusionThe extent of senescent BECs in small bile ducts and bile ductules was closely related to stage and activity of PBC and the increased expression of p16 INK4a in bile ductules was correlated with inadequate response to UDCA.