p16INK4a Protein Expression Research Articles

SLPI and AnxA2 expression in neoplasm-free palatine tonsils is associated with smoking habit of individuals

In order to confirm the inverse correlation between secretory leucocyte protease inhibitor (SLPI) expression, and human papillomavirus (HPV) infection previously observed in head and neck squamous-cell carcinoma, the present study retrospectively investigated the association between SLPI and Annexin A2 (AnxA2) expression, and HPV status in non-neoplastic chronic tonsillitis (n=118), and tonsillar hyperplasia (n=96) tissue. We hypothesised that smoking induces the upregulation of SLPI, resulting in reduced binding of HPV to AnxA2, a known modulator of HPV entry into the cell. SLPI and cyclin-dependent kinase inhibitor 2A (p16INK4A) protein expression was measured using immunohistochemistry in 214 specimens; SLPI and AnxA2 gene expression was measured using reverse transcription-quantitative polymerase chain reaction in 213 cases; and DNA was isolated from all the specimens to determine HPV status. The association between the results of the aforementioned analyses and the smoking habits of patients was analysed. The samples were HPV-negative. p16INK4A expression demonstrated moderate and strong staining in 38, and 0 cases, respectively. SLPI expression presented negative, weak and moderate signals in 163, 45, and 6 cases, respectively. A positive correlation was identified between smoking and SLPI (P=0.0001). Gene expression analysis (n=213) revealed that smoking (n=48) resulted in a significant increase in SLPI and AnxA2 expression. A significant positive correlation between AnxA2 and SLPI, indicating a surplus of AnxA2 in relation to SLPI, was exclusively identified in non-smokers. The data demonstrated that smoking results in increased SLPI and AnxA2 expression also in non-neoplastic tonsillar tissue. The observed surplus of AnxA2 in relation to SLPI identified exclusively in the tonsillar tissue of non-smokers indicates a higher possibility of a successful HPV infection of the tonsillar tissue of non-smokers, given the properties of AnxA2 to function as an infection modulator.

The inhibitor of cyclin-dependent kinase 4a signaling pathway induces aging in human skeletal muscle myoblasts and decreases the mitochondrial membrane potential

Objective To study the effect of the inhibitor of cyclin-dependent kinase 4a (INK4a) signaling pathway on myoblastic aging. Methods We transfected human skeletal muscle myoblasts with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were used to identify p16INK4a gene transcription and protein expression. Senescence-associated β-galactosidase staining was used to assess the degree of cell senescence. The mitochondrial membrane potential was analyzed by JC-1 staining and flow cytometry. Results We demonstrated that a senescence phenotype was evident in myoblasts transfected with p16INK4a, the analysis of mitochondrial membrane potential after JC-1 staining by flow cytometry showed a marked decrease in p16 group (-40.287±12.663) mV, while it was (-9.267±1.332) mV in CV group and (-6.967±2.031) mV in NC group. The relative amount of p16 protein and mRNA in p16 group was 45.25±8.21 and 33.89±7.87, which was more than that in CV (10.18±2.69 and 5.26±1.92)and NC (9.41±1.70 and 7.16±1.86) groups (P=0.000). Conclusion These findings indicated that upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activates the mitochondrial pro-aging pathway by reducing the mitochondrial membrane potential, which indirectly accelerates aging in myoblasts. Key words: Aging; Human skeletal muscle myoblast; Inhibitor of cyclin-dependent kinase 4a; Mitochondrial membrane potential

Deregulation of hsa-miR-20b expression in TNF-α-induced premature senescence of human pulmonary microvascular endothelial cells

miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17–92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17–92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.

Astemizole protects against human umbilical vein endothelial cell injury induced by hydrogen peroxide via the p53 signaling pathway

Astemizole has gained attention as an antineoplastic drug that targets important ion channels. The present study aimed to investigate the protective effects of astemizole against hydrogen peroxide (H2O2)‑induced oxidative damage to human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with astemizole (0.5 and 1µM) for 12h, then exposed to H2O2 (200µM) for 12h. Cell viability was measured using the MTT assay. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px), reactive oxygen species (ROS) and apoptotic percentage were determined. Additionally, the protein expression of p53, p21Cip1/Waf1 and p16INK4a was measured by western blot analysis The results demonstrated that astemizole (0.5‑1µM) was able to significantly restore the viability of HUVECs under oxidative stress and scavenge intracellular ROS induced by H2O2. Astemizole also suppressed the production of lipid peroxides, such as MDA, and restored the activities of endogenous antioxidants, including SOD and GSH‑Px, indicating that cell apoptosis may be inhibited. In addition, astemizole significantly increased p53, p21Cip1/Waf1 and p16INK4a protein expression. In conclusion, astemizole effectively protected endothelial cells against oxidative stress induced by H2O2, a function that may involve ROS/p53/p21Cip1/Waf1/ p16INK4a signaling pathways. The present study therefore served as a preliminary investigation into the ROS‑protective effects of astemizole, and may pave the way for future studies into the development of this compound as a novel therapy for atherosclerosis.

Methylation Status of P16Ink4a in Human Papillomavirus-Associated Cancer of Oral Cavity and Oropharynx in Northeastern Thailand

Background:Over-expression of p16INK4a protein is a biomarker for human papillomavirus (HPV)-associated cervical cancer. However, absence of p16INK4a protein expression in HPV-associated cancer of the oral cavity and oropharynx has been reported. Among a number of possible reasons for this is methylation, which is frequently noted in the promoter region of p16INK4a and is associated with silencing of the gene and disease severity.Methods:We investigated the relationships between p16INK4a protein expression, HPV infection and methylation status of the p16INK4a promoter in cancers of the oral cavity and oropharynx. Fifty-three formalin-fixed paraffin-embedded (FFPE) cancer tissue samples from the oral cavity (49 cases) and oropharynx (4 cases) were studied. P16INK4a protein expression was determined using immunohistochemical staining (IHC). Additional oral tissues lacking squamous intraepithelial lesions (SILs), and cervical tissues with high-level SILs, were used as negative and positive controls, respectively. High-risk HPV infection was detected using HPV E6/E7 mRNA in situ hybridization. Methylation status of the p16INK4a promoter was investigated using sodium bisulfite treatment and methylation-specific PCR (MS-PCR).Results:HPV infection was found in 40.8% (20/49) and 50.0% (2/4) of oral cavity and oropharynx cancers, respectively. Promoter methylation of p16INK4a occurred in 73.6 % of all cases and differed significantly in frequency between HPV-positive (90.9%, 20/22) and HPV-negative (61.3%, 19/31) samples. Expression of p16INK4a was found in 35.8% (19/53) and commonly detected in samples with p16INK4a unmethylation (79.5%). Interestingly, the silencing of p16INK4a (64.2%, 34/53) was significantly associated with methylation status (91.2%, 31/34), especially in HPV-infected samples in which the p16INK4a promoter was methylated (52.9%, 18/34).Conclusions:This result demonstrated high frequency of p16INK4a promoter methylation status in HPV-associated HNSCC subsets that could influence the silent p16INK4a expression and might promote disease severity.

Expression of p16INK4A Protein in Cervical Intraepithelial Neoplasia and Invasive Carcinoma of Uterine Cervix.

Cervical cancer still ranks as the most frequent cancer among women in India. Infection with Human Papilloma Virus (HPV) results in precancerous lesions and invasive cervical cancer. Interaction of HPV oncogenic proteins with cellular regulatory proteins leads to upregulation of p16INK4A, a cyclin dependent kinase inhibitor. Therefore, p16INK4A overexpression is a surrogate biomarker of HPV infection making it useful in evaluating HPV associated premalignant and malignant lesions of gynaecological tract. This study was conducted to evaluate the usefulness of p16INK4A as a biomarker in dysplastic and malignant alteration of cervical epithelium.Materials and Methods: A total of 57 specimens diagnosed histopathologically as cervical dysplasia or malignancy were included in this prospective study of two year duration. Immunohistochemical (IHC) study of p16INK4A expression was performed on normal cervical tissue samples, Cervical Intraepithelial Neoplasia (CIN) 1, CIN 2, CIN 3, squamous cell carcinoma, adenocarcinoma. Expression was scored by two methods. One was simple positive versus negative p16INK4A staining and the other method was a semi-quantitative IHC scoring (0 to 8 points) according to the intensity of staining and the proportion of stained cells. The collected data was entered in excel sheet and analysed using Statistical Package For Social Science (SPSS) 16.0 software and the descriptive statistics, Chi-square test. The p-value of <0.05 was considered statistically significant. Nuclear and/or cytoplasmic p16INK4A immunoreactivity was absent in normal cervical tissue, while immunopositivity was 25% in CIN 1, 50% in CIN 2, 75% in CIN 3, 75% and 100% in squamous cell carcinoma and adenocarcinoma respectively. All normal cervical samples had low scores (0 to 2 points), while number of cases with high scores was proportional to the degree of cervical dysplasia or invasive carcinoma. p16INK4A immunostaining with higher expression (score>5) was observed in 75% cases of carcinoma cervix. IHC expression of p16INK4A is related to degree of histological dysplasia and malignancy, suggesting its prognostic and predictive value in the management of cervical cancers.

Immunohistochemical Expression of the Tumor Suppressor Protein p16INK4a in Cervical Adenocarcinoma.

Objective To evaluate the diagnostic utility of the p16ink4a protein expression as a marker for adenocarcinoma of the cervix. Methods In a cross-sectional study, p16ink4a expression was evaluated in 30 cervical biopsies from patients diagnosed with invasive adenocarcinoma from 2 reference clinics in Brazil, and compared with 18 biopsies of endocervical polyps (control cases). The performance of the tests for p16ink4a was evaluated using a conventional contingency table, and the Kappa (κ) index was used to evaluate the agreement of the marker with the tissue diagnosis. Results In total, 66% of the invasive adenocarcinoma cases were positive for p16ink4a. All of the adenomatous polyps cases used as negative controls were shown to be negative for p16ink4a. The marker showed a high sensitivity and a high negative predictive value. The Kappa index was good for p16ink4a (κ = 0.6). Conclusion Considering the strong association between the p16ink4a marker and the cervical adenocarcinoma, its use represents an important tool for reducing incorrect diagnoses of adenocarcinoma and thereby avoiding overtreatment.

Prognostic significance of p16 in squamous cell carcinoma of the larynx and hypopharynx

PurposeTo evaluate the prognostic significance of p16 expression among patients with squamous cell carcinoma of the larynx (LSCC) and hypopharynx (HSCC). MethodsThe medical records of all patients with locally advanced, non-metastatic LSCC/HSCC were reviewed. p16INK4A (p16) protein expression was evaluated on pathological specimens by immunohistochemistry (IHC), and the Kaplan–Meier method was used to estimate overall survival (OS) and locoregional control (LRC). In select cases, p16 expression was correlated to high-risk and low-risk HPV genotypes using in situ hybridization (ISH). ResultsThirty-one patients (23 LSCC; 8 HSCC) were identified. Seventeen (54.8%) patients were p16 negative; 14 (45.2%) were p16-positive. The primary treatment modality was radiation therapy for 22 (71.0%) patients and surgery for 9 (29.0%). Nineteen (61.3%) patients were evaluated for high-risk HPV and low-risk HPV genotypes by IHC, of whom 2 (10.5%) patients were positive for high-risk HPV and 1 (5.3%) was positive for low-risk HPV. For high-risk HPV, the positive predictive value (PPV), sensitivity, and specificity of p16 was 20.0%, 100%, and 52.9%. There was no significant difference in the 2-year actuarial rates of OS (91% vs. 64%, p=0.34) or LRC (51% vs. 46%, p=0.69) between the p16-positive and p-16 negative patients. ConclusionIn this small cohort of 31 LSCC and HSCC patients, p16 was not a significant predictive of either LRC or OS. Furthermore, p16 was poorly correlated with HPV genotyping as identified by ISH.

TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16INK4a was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16INK4a post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis.

Expression of Ki-67 and P16 INK4a in chemically-induced perioral squamous cell carcinomas in mice.

to evaluate the influence of Ki-67 and P16INK4a proteins immunohistochemical expressions on the clinical and morphological parameters of perioral squamous cell carcinoma induced with 9,10-dimethyl-1,2-benzanthracene (DMBA) in mice. we topically induced the lesions in the oral commissure of ten Swiss mice for 20 weeks, determining the time to tumors onset and the average tumor volume up to 26 weeks. In histopathological analysis, the variables studied were histological malignancy grade and the immunohistochemical expression of Ki-67 and P16INK4a proteins. The correlation between variables was determined by application of the Spearman correlation test. the mean time to onset of perioral lesions was 21.1 ± 2.13 weeks; mean tumor volume was 555.91 ± 205.52 mm3. Of the induced tumors, 80% were classified as low score and 20% high score. There was diffuse positivity for Ki-67 in 100% of lesions - Proliferation Index (PI) of 50.1 ± 18.0. There was a strong direct correlation between Ki-67 immunoreactivity and tumor volume (R = 0.702) and a low correlation with the malignancy score (R = 0.486). The P16INK4a protein expression was heterogeneous, showing a weak correlation with tumor volume (R = 0.334). There was no correlation between the immunohistochemical expression of the two proteins studied. in an experimental model of DMBA-induced perioral carcinogenesis, tumor progression was associated with the tumor proliferative fraction (Ki-67 positive cells) and with tumor histological grading, but not with P16INK4a expression. avaliar a influência da expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a sobre parâmetros clínico-morfológicos em carcinomas espinocelulares periorais quimicamente induzidos com 9,10-dimetil-1,2-benzantraceno (DMBA) em modelo murino. as lesões foram induzidas topicamente na comissura labial de dez camundongos Swiss durante 20 semanas, sendo determinado o momento de surgimento dos tumores e volume tumoral médio até 26 semanas. Na análise histopatológica, as variáveis estudadas foram gradação histológica de malignidade tumoral e expressão imuno-histoquímica das proteínas Ki-67 e p16INK4a. A correlação entre as variáveis estudadas foi determinada pela aplicação do teste de correlação de Spearman. o tempo médio de surgimento das lesões periorais foi 21,1±2,13 semanas. Volume tumoral médio foi de 555,91±205,52mm3. Dos tumores produzidos, 80% foram classificados como de baixo escore e 20%, alto escore. Evidenciou-se positividade difusa para Ki-67 em 100% das lesões - índice de marcação (PI) de 50,1±18,0. Verificou-se correlação direta forte entre a imunoexpressão do Ki-67 e o volume tumoral (R=0,702) e fraca correlação com o escore de malignidade (R=0,486). A expressão da proteína p16INK4a foi heterogênea, mostrando fraca correlação com o volume tumoral (R=0,334). Não houve correlação entre a expressão imuno-histoquímica das duas proteínas estudadas. Em modelo experimental de carcinogênese perioral DMBA-induzida, a progressão tumoral está associada à fração proliferativa do tumor (células ki-67 positivas) e com a gradação histológica tumoral, porém não com a expressão da p16INK4a.

Abstract 5155: Status and clinical relavence of G1/S cell cycle regulatory proteins in Ocular Surface Squamous Neoplasia

Abstract Purpose: Ocular Surface Squamous Neoplasia (OSSN) is a common tumour of ocular surface arising from the limbus, cornea or conjunctiva. It includes conjunctival intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC). Although confined to ocular surface, they have capability to recur (5-53%), metastasize (0-16%) and even cause death (8%).One of the most important issues still unresolved in the treatment and management of OSSN is the inability to determine factors causing disease progression. Identification of reliable biomarkers which could detect progression as well as identify high risk cases is an urgent necessity. The Cyclin D1/p16INK4a/pRb pathway plays a critical role in tumorigenesis and alterations in these proteins have been reported in various malignancies. This study was designed to explore the status of pRb, p16INK4a and cyclin D1 in various clinical stages of OSSN patients. Methods:Sixty-four histopathologically confirmed OSSN cases (44 squamous cell carcinomas and 20 conjunctival intraepithelial neoplasias) were included in this study. AJCC TNM staging was done and patients were followed up for 36 to 58 months (median 46 months). Immunohistochemical expression of total pRb (clone-13A10), phosphorylated Rb (clone-Phospho-Ser780), p16INK4a (clone-6H12), and cyclin D1 (clone-SP4) protein was evaluated. Kaplan-Meier survival and Cox regression analysis was done to assess the prognostic significance of these proteins. Results: Immunoexpression of the targeted proteins demonstrated loss /inactive form of pRb in 87% OSSN cases, p16INK4a and cyclin D1 overexpression in 28% in 63% cases respectively. From T1 to T3 AJCC category there was a progressive increase of p16INK4a (0%in T1 to 100% in T3) (P = 0.007) and cyclin D1 overexpression (25% in T1 to 88% in T4). Loss of pRb (75% in T1 to 100% in T4) (P = 0.03) was also seen. Loss of pRb expression was the earliest change which was detected in 75% cases in T1.The Kaplan Meier survival curves revealed loss of pRb (P = 0.0002, log rank analysis) as well as p16INK4a(P = 0.11, log rank analysis) and cyclin D1 over expression (P = 0.06, log rank analysis) adversely affected the disease free survival. pRb loss however emerged as the single most sensitive poor prognostic indicator in all AJCC stages of OSSN patients. Conclusion: G1/S cell cycle proteins (retinoblastoma, p16INK4aand cyclin D1) have prognostic relevance in OSSN patients. Of these pRb loss is the most useful indicator of aggressive behavior and is therefore recommended for detecting high risk OSSN patients. The dysregulation of p16INK4a, cyclin D1 and pRb proteins also plays an important role in pathogenesis of OSSN patients. Note: This abstract was not presented at the meeting. Citation Format: Sheetal Chauhan, Seema Sen, Anjana Sharma, Seema Kashyap, M. Vanathi, Neelam Pushker, Radhika Tandon. Status and clinical relavence of G1/S cell cycle regulatory proteins in Ocular Surface Squamous Neoplasia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5155. doi:10.1158/1538-7445.AM2015-5155

Frequency and prognostic significance of p16(INK4A) protein overexpression and transcriptionally active human papillomavirus infection in laryngeal squamous cell carcinoma.

Background:Human papillomavirus (HPV) infection is a powerful prognostic biomarker in a subset of head and neck squamous cell carcinomas, specifically oropharyngeal cancers. However, the role of HPV in non-oropharyngeal sites, such as the larynx, remains unconfirmed.Methods:We evaluated a cohort of 324 laryngeal squamous cell carcinoma (LSCC) patients for the expression of p16INK4A (p16) protein by immunohistochemistry (IHC) and for high-risk HPV E6 and E7 mRNA transcripts by RNA in situ hybridisation (ISH). p16 expression and HPV status were correlated with clinicopathological features and outcomes.Results:Of 307 patients assessable for p16 IHC, 20 (6.5%) were p16 positive. Females and node-positive patients were more likely to be p16 positive (P<0.05). There were no other significant clinical or demographic differences between p16-positive and -negative cases. There was no difference in overall survival (OS) between p16-positive and -negative patients with 2-year survival of 79% in each group (HR=0.83, 95% CI 0.36–1.89, P=0.65). There was no statistically significant difference in failure-free survival (FFS) with 2-year FFS of 79% and 66% for p16-positive and -negative patients, respectively (HR=0.60, 95% CI 0.26–1.36, P=0.22). Only seven cases were found to be HPV RNA ISH positive, all of which were p16 IHC positive. There was no statistically significant difference in OS between patients with HPV RNA ISH-positive tumours compared with -negative tumours with 2-year survival of 86% and 71%, respectively (HR=0.76, 95% CI 0.23–2.5, P=0.65). The 2-year FFS was 86% and 59%, respectively (HR=0.62, 95% CI 0.19–2.03, P=0.43).Conclusions:p16 overexpression is infrequent in LSCC and the proportion of cases with high-risk HPV transcripts is even lower. There are no statistically significant correlations between p16 IHC or HPV RNA ISH status and OS or disease outcomes.

P16INK4a as an adjunct test in cervical cytology.

BACKGROUND This study investigated correlation between cervical cytology, expression of P16INK4a, and human papilloma virus (HPV) infection. MATERIALS AND METHODS The study included 100 subjects with suspected pathological cervical lesions. Cervical smears were analyzed for malignancy and p16INK4a. Histological finding represented the golden standard. RESULTS Immunocytochemical analysis of protein p16INK4a expression on epithelial cells of cervical smear demonstrated increased p16lNK4a expression in 36.0% of subjects. There was statistically significant positive correlation (Spearman r = 0.70; p < 0.001) between pathological findings and intensity of p16INK4a protein expression inside epithelial cells, as well as with histological finding (Spearman r = 0.71; p < 0.001). The intensity of p16INK4a protein expression in cytology finding was significantly higher in HPV16 positive patients (Mann- Whitney test, p = 0.0065). CONCLUSION Good correlation between expression rate and severity of lesions indicates that this test might improve results of cytology and HPV screening, as well as results of predicting prognosis of disorder of cervix.

High OCT4 and Low p16(INK4A) Expressions Determine In Vitro Lifespan of Mesenchymal Stem Cells.

After long-term culture, mesenchymal stem cells alter their biological properties and enter into a state of replicative senescence. Although several classical biomarkers have been used for quantitative assessment of cellular senescence, no hallmark has been proven completely unique to the senescent state in cells. We used bone marrow-derived MSCs (BM-MSCs) from different healthy young donors and an in vitro model with well-defined senescence end points to identify a set of robust markers that could potentially predict the expansion capacity of MSCs preparations before reaching senescence. For each early passage BM-MSC sample (5th or 6th passages), the normalized protein expression levels of senescence-associated markers p16INK4A, p21WAF1, SOD2, and rpS6S240/244; the concentration of IL6 and IL8 in cell culture supernatants; and the normalized gene expression levels of pluripotency markers OCT4, NANOG, and SOX2 were correlated with final population doubling (PD) number. We revealed that the low expression of p16INK4A protein and a high OCT4 gene expression, rather than other evaluated markers, might be potential hallmarks and predictors of greater in vitro lifespan and growth potential, factors that can impact the successful therapeutic use of MSCs preparations.

Quantitative analysis of CDKN2A methylation, mRNA, and p16INK4a protein expression in children and adolescents with Burkitt lymphoma: Biological and clinical implications

CDKN2A is a tumor suppressor gene critical in the cell cycle regulation. Little is known regarding the role of CDKN2A methylation in the pathogenesis of Burkitt lymphoma (BL). CDKN2A methylation was investigated using pyrosequencing in 51 tumor samples. p16INK4a mRNA and protein levels were measured using real-time PCR and immunohistochemistry, respectively. CDKN2A methylation was detectable in 72% cases. Nuclear expression of p16INK4a was not detected in 41% cases. There was an association between methylation and absence of CDKN2A mRNA (P=0.003). In conclusion, CDKN2A methylation occurs at a high frequency suggesting a role in BL pathogenesis and potential therapeutic implications.

Magnolol Causes Alterations in the Cell Cycle in Androgen Insensitive Human Prostate Cancer Cells In Vitro by Affecting Expression of Key Cell Cycle Regulatory Proteins

Prostate cancer, one of the most common cancers in the Western world, affects many men worldwide. This study investigated the effects of magnolol, a compound found in the roots and bark of the magnolia tree Magnolia officinalis, on the behavior of 2 androgen insensitive human prostate cancer cell lines, DU145 and PC3, in vitro. Magnolol, in a 24-h exposure at 40 and 80 μM, was found to be cytotoxic to cells. Magnolol also affected cell cycle progression of DU145 and PC3 cells, resulting in alterations to the cell cycle and subsequently decreasing the proportion of cells entering the G2/M-phase of the cell cycle. Magnolol inhibited the expression of cell cycle regulatory proteins including cyclins A, B1, D1, and E, as well as CDK2 and CDK4. Protein expression levels of pRBp107 decreased and pRBp130 protein expression levels increased in response to magnolol exposure, whereas p16INK4a, p21, and p27 protein expression levels were apparently unchanged post 24-h exposure. Magnolol exposure at 6 h did increase p27 protein expression levels. This study has demonstrated that magnolol can alter the behavior of androgen insensitive human prostate cancer cells in vitro and suggests that magnolol may have potential as a novel anti-prostate cancer agent.

387: Quantitative analysis of CDKN2A methylation, mRNA, and p16INK4a protein expression in children and adolescents with Burkitt lymphoma: Biological and clinical implications

387: Quantitative analysis of CDKN2A methylation, mRNA, and p16INK4a protein expression in children and adolescents with Burkitt lymphoma: Biological and clinical implications

High‐risk human papillomavirus infection in different histological subtypes of renal cell carcinoma

Limited data exist regarding whether a high-risk human papillomavirus (HR-HPV) infection increases the risk of developing renal cell carcinoma. The aim of this study was to investigate whether HPV infection has a role in the pathogenesis or development of a certain histological subtype of renal cell carcinoma. Formalin-fixed paraffin-embedded (FFPE) specimens of 122 patients with histopathologically proven renal cell carcinoma and their respective peritumoral tissues were examined. The presence of HPV-DNA was determined by a combination of MY/GP+ consensus primers and HPV-16/18 type specific nested PCRs followed by direct sequencing. Catalyzed signal-amplified colorimetric in situ hybridization (CSAC-ISH) technique was applied to determine the physical status of viral genome. The expression of p16INK4a and HPV L1 capsid proteins was evaluated using immunohistochemistry. HPV genome was detected in 37 (30.3%) tumor specimens and their four (4.1%) corresponding peritumoral tissues. HPV-18 was the most common viral type identified followed by HPV-16 and 58. Immunoexpression of p16INK4a was detected in 24 (20.3%) cases. Data analysis showed a significant correlation between p16INK4a expression and the presence of HR-HPV DNA (P < 0.001). CSAC-ISH analysis confirmed HR-HPV infection in 45% of tumors, which were previously tested positive for HPV-DNA. Diffuse signal pattern was identified in 15 (83.3%) samples whereas a mixed pattern of diffuse and punctate signals was only detectable in three cases. The results indicate an association of HR-HPV types with renal cell carcinoma. It is proposed that HPV infection in high-grade tumors might precede disease progression in a number of tumors, particularly of the papillary subtype.

Expression of Bcl-2, P16INK4a, and Cyclin D1 Proteins in Squamous Cell Carcinoma of the Lip

The protein expression of Bcl-2, p16INK4a, and cyclin D1 in squamous cell carcinoma of the lip (SCCL) was evaluated. Study Design: Sixteen cases of SCCL were studied by immunohistochemistry and evaluated according to histological grading and histological features. Results: The expression of p16INK4a was the highest among proteins studied, with 68.7% of positive cases, especially in well-differentiated SCCL and in keratin pearls, followed by Bcl-2 expression in 42.8% of cases, especially in moderately differentiated SCCL and in cords.

The Severity of Internal Carotid Artery Stenosis is Associated with the Cyclin-Dependent Kinase Inhibitor 2A Gene Expression

The INK4b-ARF-INK4a locus in the chromosome 9p21 region is known to play an important role in the development of atherosclerosis. The INK4/ARF transcript p16(INK4a) inhibits the activity of the cyclin-dependent kinases CDK4/CDK6 and arrests cell-cycle progression. CDK inhibitors also regulate G1/S phase progression in vascular smooth muscle cells(VSMCs) and may modulate the early stages of atherosclerosis. Therefore, we aimed to study the expression of the INK4/ARF locus genes CDKN2A and CDKN2BAS in order to examine the p16(INK4a) protein expression and the level of cell proliferation in carotid plaques and saphenous tissue samples. A total of 50 patients(33 symptomatic subjects and 17 asymptomatic subjects) with carotid atherosclerosis CA) were studied. The CDKN2A and CDKN2BAS gene expression levels were determined using quantitative real-time polymerase chain reaction(qRT-PCR). All tissue sections were also analyzed for the p16(INK4a) and proliferating cell nuclear antigen(PCNA) protein expression using immunohistochemistry(IHC). The CDKN2A gene expression was significantly higher in the carotid plaques than in the saphenous tissues(p=0.009), whereas no such differences were observed in the CDKN2BAS transcripts(p=0.157). The carotid plaque CDKN2A mRNA levels were higher in the symptomatic patients than in the asymptomatic patients(p=0.050); this finding was also associated with the severity of internal carotid artery(ICA) stenosis(p=0.034). The p16(INK4a) immune(＋) cell counts in the carotid plaques were higher in the symptomatic patients than in the asymptomatic patients (p=0.056), as was the cell proliferation index(p=0.001). An increased CDKN2A gene expression in carotid plaques may increase the severity of ICA stenosis, thus raising the risk of atherosclerosis and contributing to the development of symptoms. In addition, the p16(INK4a) expression is associated with carotid atherosclerosis in various patient subgroups.