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To investigate the protective effect of p14ARF in a nitric acid (NA) aerosol inhalation-induced bronchiolitis obliterans (BO) mouse model and its potential regulatory mechanism. A BO mouse model was established by NA aerosol inhalation. The expressions of p14ARF, phosphatidylinositol-3-kinase (PI3K), and protein kinase B (AKT) were detected by quantitative reverse transcription PCR (qRT-PCR) and western blot (WB). Hematoxylin (HE) staining, Masson staining, and periodic acid-Schiff (PAS) staining observed pulmonary histological changes. TdT-mediated dUTP nick end labeling (TUNEL) staining detected pulmonary cell apoptosis, and enzyme-linked immunosorbent assay (ELISA) measured matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), interleukon-6 (IL-6), and transforminh growth factor-β (TGF-β) levels in lung tissue and bronchoalveolar lavage fluid (BALF). The expressions of p14ARF, PI3K, and AKT showed a time gradient change, with a decrease trend (*P < 0.05 and **P < 0.01). Severe inflammatory infiltration and tracheal fibrosis were found in lung tissue in the modeling group (BO group) compared with the control group (Con group). The pH, PaO2, and PaO2/FiO2 values significantly reduced, while the PaCO2 value and the number of TUNEL-positive cells increased in BO group (P < 0.05). In addition, MMP-2, MMP-9, IL-6, and TGF-β levels remarkably increased, with an increase in the number of white blood cells, neutrophils, and lymphocytes in BO group (P < 0.05). Furthermore, p14ARF up-regulation reversed the trend of the aforementioned indexes in BO mice. p14ARF ameliorated the inflammatory response and airway remodeling in a BO mouse model via the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study is to determine the relationship between APC gene mutations and subcellular differentiation levels of E2F1, P14ARF and KI67 proteins. Furthermore, if such connections can be used in the area of preventive health care. Materials and Methods: The 30 hours Immunohistochemistry protocol used, had samples preparation, antigen retrieval, background blocking, target detection and sample visualization. Samples were viewed and captured by light microscopy. Results: The conducted research concern 88 patients with a range age of 56 years. 57,7% had no tobacco addiction, 3,84% were obese and 19,23% had the tendency to consume alcohol. About 31% had at least one family member with a history of cancer. Intensity and positivity of the genes vary as seen in tables. Conclusions: By targeting specific and simultaneously multiple pathways based on molecular signatures, enables cases to be detected at an earlier stage, when there are greater chances of cure as treatment is more effective. A plan for early detection of cancer is a key component within an overall cancer control plan. An early diagnosis program is far more cheap and easy leading to appropriate treatments which finally reduce death rates and suffering due to cancer. [ABSTRACT FROM AUTHOR]

The goal of this study was to clarify the role of p16INK4a, p14ARF, and p53 protein expressions in carcinogenesis in squamous cell carcinomas of the lip. The expressions of the p53, p16INK4a, and p14ARF proteins were examined in 46 formaline-fixed paraffin embedded tissue specimens, which included 19 cases of squamous cell carcinoma of the lip, 14 cases of actinic cheilitis, and 13 cases of normal mucosa. Immunoreactivity in the peritumoral epithelium adjacent to squamous cell carcinomas was also evaluated. p16INK4a expression was increased in actinic cheilitis in comparison with normal mucosa (p=0.001). p14ARF expression progressively increased from normal mucosa to actinic cheilitis (p=0.001) and was observed to decrease significantly during the process of transition from actinic cheilitis to carcinoma (p=0.003). p53 values progressively increased from normal mucosa to actinic cheilitis (p=0.001) and carcinoma (p=0.008). A significant positive correlation was found between p14ARF and p53 in the peritumoral epithelium adjacent to carcinomas. Our findings indicated that p16INK4a and p14ARF immunohistochemistry does not determine whether or not actinic cheilitis has the potential to develop carcinoma. The p14ARF/p53 pathway is activated in the peritumoral epithelium adjacent to carcinoma; however, this activation would not be adequate to prevent carcinogenesis. [ABSTRACT FROM AUTHOR]

Current management of locally advanced rectal carcinoma (LARC) involves preoperative chemoradiotherapy (preCRT) before surgery. Despite improved local control rate, the response to preCRT of individual patients is variable and may reflect heterogeneous biological properties among tumors of the same clinical stage. Identifying novel molecular parameters with predictive and/or prognostic value is of great clinical importance for a personalized therapeutic approach. In this study, KRAS mutation status was analyzed by direct sequencing, while methylation-specific polymerase chain reaction (MSP) was used to examine p16INK4a and p14ARF gene methylation status in pretreatment tumor biopsies of 60 patients with LARC. The examined molecular changes of KRAS, p16INK4a and p14ARF genes were mutually independent (p16INK4a/KRAS, P=0.272; p14ARF/KRAS, P=0.923; p16INK4a/p14ARF, P=0.715). However, the simultaneous presence of p14ARF methylation and KRAS mutation was associated with a more frequent appearance of local recurrences and distant metastasis (P=0.027). Moreover, patients with the simultaneous presence of p16INK4a and p14ARF methylation and KRAS mutation had significantly shorter overall survival (P=0.011). The obtained results strongly suggest that combined analyses of examined genetic and epigenetic molecular alterations could contribute to the identification of LARC patient subgroups with more aggressive tumor behavior and worse disease outcome. [ABSTRACT FROM AUTHOR]

p27Kip1 is known as a major cyclin‐dependent kinase inhibitor and a tumor suppressor, and often functionally hampered at protein level. p27 protein expression levels are frequently low in various cancers and negatively correlated with malignancy of cancer. However, in our previous study, we discovered that p27 overexpression does not inhibit the proliferation of two cancer cell lines due to a functional suppression of p27 by nucleophosmin isoform 1 (NPM1); that is, a qualitative, not quantitative, suppression of p27 function occurs in these cancer cell lines. To clarify the regulation of p27 in several types of cancer, we investigated p27 function in other cancer cell lines, based on proliferation assays in those cell lines carrying doxycycline‐inducible p27, and found that MDAH041 cells which express p14ARF, an antagonist of NPM1, show growth inhibition depending on p27 induction. Moreover, to investigate p27 function under anchorage‐independent culture conditions, we performed soft agar colony formation assay and observed that the colony formation of some cell lines carrying wild‐type p53, a major tumor suppressor, was inhibited depending on p27 induction. These results suggest that p27 function is regulated differentially among cancer cell types under anchorage‐dependent and anchorage‐independent culture conditions. [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]

Background: Jab1 has been reported to regulate various proteins in signal transduction pathways and be implicated in carcinogenesis or tumor progression. However, the precise role and molecular mechanism of Jab1 in gastric tumorigenesis have not yet been fully elucidated. Methods: Jab1 staining in gastric cancer tissues and paired non-cancerous tissues was measured using tissue microarray (TMA) technology. The impact of Jab1 on tumor growth in vivo was analyzed using xenotransplantation experiments in Balb/c mice. The expression of Jab1 and p14ARF in gastric cancer cells was analyzed by western blot and confocal immunofluorescence. CCK-8 and cell cycle experiment were used to evaluate the cell proliferation. Ubiquitination assay was performed to validate whether ubiquitination is involved in Jab1-mediated p14ARF degradation. Results: The expression level of protein p14ARF was inversely correlated with the protein level of Jab1. Then, we investigated the mechanism that how Jab1 induced p14ARF depletion. Mechanistic studies showed that Jab1 induced ubiquitin-independent proteasomal p14ARF degradation in gastric cancer cells. Our data demonstrated that Jab1 protein was a vital upstream negative modulation factor of p14ARF, and Jab1 could promote cell proliferation and tumor growth via inhibiting the expression of p14ARF in vivo and in vitro. Moreover, silencing Jab1 protein expression declined tumor growth and further increased the apoptosis rate of gastric cancer cells. In further studies of gastric cancer specimens, we found the increased level of Jab1 protein shortened the overall survival. Conclusion: Jab1 is upstream of p14ARF and promote gastric cancer cell proliferation in vitro and in vivo. Furthermore, Jab1 decreased the expression of p14ARF though ubiquitination independent proteasomal degradation. Therefore, the connection of Jab1 and p14ARF may provide new methods for the treatment of gastric cancer. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is characterized by a unique dependence on optimal androgen receptor (AR) activity where physiological androgen concentrations induce proliferation but castrate and supraphysiological levels suppress growth. This feature has been exploited in bipolar androgen therapy (BAT) for castrate resistant malignancies. Here, we investigated the role of the tumor suppressor protein p14ARF in maintaining optimal AR activity and the function of the AR itself in regulating p14ARF levels. We used a tumor tissue array of differing stages and grades to define the relationships between these components and identified a strong positive correlation between p14ARF and AR expression. Mechanistic studies utilizing CWR22 xenograft and cell culture models revealed that a decrease in AR reduced p14ARF expression and deregulated E2F factors, which are linked to p14ARF and AR regulation. Chromatin immunoprecipitation studies identified AR binding sites upstream of p14ARF. p14ARF depletion enhanced AR-dependent PSA and TMPRSS2 transcription, hence p14ARF constrains AR activity. However, p14ARF depletion ultimately results in apoptosis. In PCa cells, AR co-ops p14ARF as part of a feedback mechanism to ensure optimal AR activity for maximal prostate cancer cell survival and proliferation. [ABSTRACT FROM AUTHOR]

The objective of the present study was to evaluate the epigenetic changes occurring in early stages of breast cancer. The present study investigated the methylation profile of the ATM, p14ARF and p16INK4a promoters in total blood and plasma cell-free DNA (cfDNA) from women with impalpable breast lesions compared with in total blood of a control cohort of women without breast lesions. The samples were evaluated using the methylation-specific PCR method. The Fisher's exact test was used to evaluate statistical significance between the methylation and clinical variables. A total of 111 women were evaluated, including 56 women with impalpable breast cancer (39/56 also had paired plasma cfDNA) and 55 women in the control cohort (55 blood DNA). For blood DNA from women with malignant impalpable breast lesions, p16INK4a exhibited the greatest percentage of methylation (48%), followed by ATM (37.5%) and p14ARF (27%) promoters, regardless of age variation. For plasma cfDNA, the methylation rates for ATM, p14ARF and p16INK4a were 26, 26 and 10%, respectively. The methylation rates for the blood DNA of controls were the lowest for ATM (9%), p14ARF (7%) and p16INK4a (7%). The women with impalpable breast lesions (benign and malignant lesions) exhibited the highest methylation rate, regardless of age, compared with the paired plasma cfDNA and controls. This epigenetic change was statistically significant for the promoters of ATM (P=0.009) and p16INK4a (P=0.001) (impalpable breast lesions vs. control). The present study demonstrated that epigenetic changes occurring in the ATM and CDKN2A genes detectable in liquid biopsy were associated with the development of impalpable breast lesions. [ABSTRACT FROM AUTHOR]

In this study, we identified P14 alternate reading frame (P14ARF) as a novel regulator of inflammation and vascularization in intervertebral disk degeneration (IVDD). We collected IVD tissues from IVDD patients and normal individuals for analysis of P14ARF expression. We also induced experimental IVDD by needle puncture injuries in the caudal intervertebral disks of Sprague- Dawley (SD) rats and achieved recombinant adenovirus-mediated P14ARF overexpression in experimental IVDD rats. Regulation relationships between P14ARF and tissue inhibitors of metalloproteinases- 3 (TIMP3) were confirmed in P14ARF-overexpressed and TIMP3-depleted nucleus pulposus (NP) cells. Tube formation in vitro was evaluated in coculture systems of human umbilical vein endothelial cells (HUVECs) and rat degenerated NP cells (DNPCs). Inflammatory response was assessed from levels of TNF-γ, IL-1, and IL-6 and neovascularization from expression of endothelial growth factor (VEGF). The P14ARF and TIMP3 were downregulated in degenerated IVD tissue derived from patients and experimental IVDD rats. Overexpressed P14ARF suppressed inflammatory cytokine levels and vascularization. There was decreased in vitro tube formation in response to P14ARF overexpression and TIMP3 elevation. Finally, attenuated inflammatory responses and suppression of VEGF were achieved by P14ARF-mediated promotion of TIMP3 in rat DNPCs. Taken together, the present study reveals that P14ARF/TIMP3 modulation of inflammatory response and vascularization in the context of IVDD highlights a potential target for future therapeutic strategies. [ABSTRACT FROM AUTHOR]

The article offers information regarding a study which aims at evaluating p14 expression patterns in nevi and melanoma, and this analysis identifies p14 as a candidate biomarker for melanoma diagnosis and prognosis. It mentions information on presence or absence of a particular protein within melanocytes. It presents information on clinical and histologic features of benign nevi, cutaneous melanoma, and metastatic melanoma.

The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that ensures the fidelity of chromosomes segregation. Reduced expression of some of its components weakens the SAC and induces chromosome instability and aneuploidy, which are both well-known hallmarks of cancer cells. Centromere protein-E (CENP-E) is a crucial component of the SAC and its function is to facilitate kinetochore microtubule attachment required to achieve and maintain chromosome alignment. The present study investigates the possible role of p14ARF as a controller of aneuploid cells proliferation. We used RNA interference to induce aneuploidy by partial depletion of CENP-E in human primary fibroblasts (IMR90) and in near diploid tumor cells (HCT116). In contrast to IMR90 aneuploid cell number, which was drastically reduced and leaned towards the WT condition, HCT116 aneuploid cell numbers were slightly decreased at later time points. This euploidy restoration was accompanied by increased p14ARF expression in IMR90 cells and followed ectopic p14ARF re-expression in p14ARF-null HCT116 cells. Collectively, our results suggest that hampering proliferation of aneuploid cells could be an additional role of the p14ARF tumor suppressor. [ABSTRACT FROM AUTHOR]

Background/aim: Urothelial bladder cancer arises from the accumulation of multiple epigenetic and genetic changes. We aimed to investigate the specificity and sensitivity of gene-specific promoter methylation of CDH1 and p14ARF genes in the early diagnosis of bladder cancer and compare those with other diagnostic tests in our population. Patients and methods: In the current study, 65 patients with urothelial bladder cancer and 35 controls without any history of cancer were recruited. Methylation profiles of CDH1 and p14ARF genes from tumor and urine samples were determined by methylation-specific polymerase chain reaction method. Results: Methylation of CDH1 and p14ARF genes in tumor samples was 95.4% and 78.5%, respectively. The methylation frequencies were found to be 68.8% for CDH1 gene and 72.9% for p14ARF gene in urine samples. Sensitivities of CDH1, p14ARF and urine cytology were found to be 67.4%, 72.1% and 34.9%, respectively, while their specificities were 93.9%, 63.6% and 93.9%, respectively. Conclusion: Aberrant promoter methylation of CDH1 and p14ARF genes can be used to detect urothelial bladder cancer. In low-grade tumors, when compared with urine cytology, combined methylation analysis of CDH1 and p14ARF genes may not increase the sensitivity to identify malignant cells in urine samples. [ABSTRACT FROM AUTHOR]

ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired. [ABSTRACT FROM AUTHOR]

Background: Endometrial cancer is a common gynecological malignancy with good prognosis in the early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we have examined the methylation status of the p16INK4a and p14ARF genes in endometrial cancer and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk, and clinicopathological parameters. Methods: We collected 28 formalin fixed paraffin embedded samples and 26 blood samples from endometrial cancer patients and 22 controls. Methylation-specific PCR was applied to analyze the promoter methylation status of the p16INK4a and p14ARF genes in the studied population. The results were analyzed with SPSS software version 20. Results: There was a significant difference between the study groups and the presence of promoter CpG hypermethylation status in the p14 (P<0.0001) and p16 (P<0.05) genes. p14 hypermethylation in the blood samples was associated with depth of myometrial invasion in endometrial cancer (P=0.03). A significant association existed between p16 methylation in tissue with endometrial cancer grade (P=0.06). No statistically significant difference existed between thep16INK4a andp14ARF promoter hypermethylations in blood (P=0.177) and formalin fixed paraffin embedded (P=0.221) samples. An association existed between p16INK4a and p14ARF gene hypermethy-lations in blood and tissue with diabetes. Conclusion: Our results have confirmed that epigenetic mechanisms play an important role in endometrial cancer incidence. They can be utilized as prognostic biomarkers for endometrial cancer. The lack of a significant difference between the p16INK4a and p14ARF promoter hypermethylations in blood and formalin fixed paraffin embedded samples has indicated that methylation status of a blood sample can be an early, non-invasive diagnostic marker in endometrial cancer. [ABSTRACT FROM AUTHOR]

p14ARF, a tumor suppressor protein, encoded by the p16 tumor suppressor gene, has been reported to be associated with the clinicopathological features of lung cancer. However, the evaluated outcomes were inconsistent and remained inconclusive. In this study, we conducted a meta-analysis to clarify the significance of p14ARF expression in lung cancer pathogenesis. Materials and methods: Electronic databases, PubMed, Web of Knowledge, Embase, and CNKI, were retrieved to collect relevant articles with inclusion and exclusion criteria. Using Stata 12.0 software, 95% confidence intervals (CIs) and odds ratios (ORs) were calculated. Results: A total of 15 eligible case-control studies that evaluated the relationship between p14ARF expression and lung cancer were included in the meta-analysis. The results demonstrated that there were significant associations between p14ARF expression and the risk of non-smallcell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous carcinoma (for NSCLC, OR =11.02, 95% CI =5.30-22.92; for lung adenocarcinoma, OR =7.28, 95% CI =3.92-13.50; and for lung squamous carcinoma, OR =14.40, 95% CI =2.83-73.24). In the stratified analysis based on race, significant associations between p14ARF expression and lung cancer risk were found in Chinese population and Caucasians (for Chinese population, OR = 7.02, 95% CI =4.48-11.00 and for Caucasians, OR =4.19, 95% CI =1.42-12.38). Furthermore, the expression of p14ARF was significantly associated with the TNM-stage of lung cancer in Chinese population (OR =2.07, 95% CI =1.38-3.10). Conclusion: p14ARF expression was significantly associated with the risk of lung cancer. In addition, the data of the meta-analysis showed that there was a significant correlation between p14ARF expression and the TNM-stage of lung cancer in Chinese population. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the effects of CDKN2A (p16INK4A/p14ARF) overexpression on the proliferation and migration of human melanoma A375 cells. Methods: Melanoma tissues and pigmented nevi tissues were collected. Human melanoma A375 cells were transfected by CDKN2A (p16INK4A) and CDKN2A (p14ARF) over-expressing vectors and then assigned into blank, negative control (NC), p16INK4A and p14ARF groups. The expression of CDKN2A (p16INK4A) and CDKN2A (p14ARF) mRNA and protein was detected by qRT-PCR and Western blotting. CCK-8, flow cytometry and Transwell assays were applied to observe cell proliferation, the cell cycle and apoptosis, and migration and invasion, respectively. The model of subcutaneous xenografts in nude mice was established to measure cell growth in vivo. Results: Compared with pigmented nevi tissues, CDKN2A (p16INK4A) and CDKN2A (p14ARF) mRNA and protein expression were significantly decreased in melanoma tissues. CDKN2A (p16INK4A) and CDKN2A (p14ARF) over-expression inhibited proliferation, migration, invasion and progression from G0/G1 to S phase of A375 cells and xenograft tumor growth, but promoted apoptosis. Conclusion: Our study demonstrated that over-expression of CDKN2A (p16INK4A) and CDKN2A (p14ARF) suppressed proliferation and migration of human melanoma A375 cells. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the effects of CDKN2A (p16INK4A/p14ARF) over-expression on the proliferation and migration of human melanoma A375 cells. Methods: Melanoma tissues and pigmented nevi tissues were collected. Human melanoma A375 cells were transfected by CDKN2A (p16INK4A) and CDKN2A (p14ARF) over-expressing vectors and then assigned into blank, negative control (NC), p16INK4A and p14ARF groups. The expression of CDKN2A (p16INK4A) and CDKN2A (p14ARF) mRNA and protein was detected by qRT-PCR and Western blotting. CCK-8, flow cytometry and Transwell assays were applied to observe cell proliferation, the cell cycle and apoptosis, and migration and invasion, respectively. The model of subcutaneous xenografts in nude mice was established to measure cell growth in vivo. Results: Compared with pigmented nevi tissues, CDKN2A (p16INK4A) and CDKN2A (p14ARF) mRNA and protein expression were significantly decreased in melanoma tissues. CDKN2A (p16INK4A) and CDKN2A (p14ARF) over-expression inhibited proliferation, migration, invasion and progression from G0/G1 to S phase of A375 cells and xenograft tumor growth, but promoted apoptosis. Conclusion: Our study demonstrated that over-expression of CDKN2A (p16INK4A) and CDKN2A (p14ARF) suppressed proliferation and migration of human melanoma A375 cells. [ABSTRACT FROM AUTHOR]

Background: The cyclin-dependent kinase inhibitors p14ARF and p15INK4B are tumor suppressor genes that have been reported to be silenced through promoter methylation in many human cancers. However, the strength of association between p14ARF or p15INK4B promoter methylation and lung cancer remains unclear. Thus, we first determined whether p14ARF and p15INK4B promoter methylation played a key role in the carcinogenesis of lung cancer. Methods: Eligible studies were selected from the online electronic databases. The pooled odds ratios or hazard ratios and 95% confidence intervals were calculated and summarized. Results: Finally, 12 studies with 625 lung cancer samples and 488 nontumor samples were included under the fixed-effects model. The pooled odds ratio showed that p14ARF promoter methylation was observed to be significantly higher in non-small-cell lung cancer (NSCLC) than in nontumor samples (P<0.001). No significant correlation was found between p15INK4B promoter methylation and lung cancer (P=0.27). Subgroup analysis of ethnicity revealed that p14ARF promoter methylation was significantly related to the risk of NSCLC in Asian and Caucasian populations. Subgroup analysis of sample type demonstrated that p14ARF promoter methylation was correlated with the risk of NSCLC in tissue samples (P<0.001), but not in bronchoalveolar lavage fluid and blood samples. P14ARF promoter methylation from one study was not significantly correlated with overall survival of patients with NSCLC. Promoter methylation of p14ARF and p15INK4B was not correlated with clinicopathological characteristics, such as gender status, smoking status, tumor differentiation, and tumor stage (P>0.05). Conclusion: Our findings suggested that p14ARF promoter methylation may play an important role in the carcinogenesis of lung cancer, but not p15INK4B promoter methylation. Promoter methylation of p14ARF and p15INK4B was not associated with clinicopathological parameters. However, more extensive large-scale studies are essential to further validate our study. [ABSTRACT FROM AUTHOR]

Aims Breast cancer is a hormonally driven disease. Cellular senescence is an age-related irreversible cell cycle arrest at the G1 phase upon induction. The aim of this study was to characterize the expression patterns of the senescence markers p14ARF, p16INK4a and p21WAF1/Cip1 during breast cancer progression in a large patient cohort. Methods and results We conducted a retrospective study of 1080 patients with invasive ductal carcinoma, no special type, over an 11-year period. We performed immunohistochemical staining on tissue microarrays that included normal, benign hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma tissue from each patient. Invasive ductal carcinomas showed higher expression of p14ARF and p16INK4a but lower expression of p21WAF1/Cip1 than non-malignant tissues. There were significant correlations of normal, benign, preinvasive and malignant tissues with p14ARF, p16INK4a and p21WAF1/Cip1 expression ( P < 0.05). Univariate comparison showed a correlation between high p16INK4a expression and poor survival ( P = 0.000) and an increased risk of relapse ( P = 0.000), whereas high p14ARF expression correlated only with an increased risk of relapse ( P = 0.038). Multivariate analysis showed p16INK4a to be an important prognostic factor for overall survival ( P = 0.011) and disease-free survival ( P = 0.004), with p14ARF also being a significant prognostic factor for disease-free survival ( P = 0.043). Moreover, patients showing both high p16INK4a expression and and high p14ARF expression had an adjusted three-fold increased risk of disease recurrence ( P < 0.05) and a two-fold increased risk of all-cause-related death ( P < 0.05). Conclusions These finding suggest p16INK4a expression and p14ARF expression may play an important role in the progression of proliferative breast tissue to invasive cancer, and may be useful as prognostic factors. [ABSTRACT FROM AUTHOR]

Recent studies have shed considerable light into schwannomas. To date, only merlin has been identified as a hallmark or pathogenesis of both sporadic and NF2-related schwannomas. Here, we show, by immunoblot and immunohistochemical analyses of 58 sporadic vestibular schwannomas, that upregulation of p53 was observed in 90 % of tumors examined. No p53 mutations were found in 12 % tumors analyzed. Expression of p14ARF was negative in 95 % of tumors, while overexpression of MDM2 was found in all specimens. Aberrant DNA hypermethylation of the p14ARF promoter was observed in three of seven tumors examined (43 %), associated with remarkably decreased mRNA levels. The very high degree of concordance in the aberrations of the p14ARF/MDM2/p53 pathway in this tumor may be considered to be a new player in the pathogenesis of sporadic vestibular schwannomas. Moreover, expression of p21 (waf1) was negative in all tumors, suggesting that the aberration of this pathway is associated with greater attenuation of p21-mediated signals that are critical for growth inhibition. These data are in agreement with the model in RT-4 rat schwannoma cells: i.e., overexpression of ARF was associated with accumulation of p21 expression both at protein and mRNA levels. ShRNA knock-down of p53 expression attenuated p21-mediated increase in cellular arrest in the G1-phase, suggesting that p14ARF regulates p21 protein levels through a p53-dependent pathway. Thus, this study reveals a high degree of concordance in the aberrations of the p14ARF/MDM2/p53 pathway with the development of sporadic vestibular schwannomas. [ABSTRACT FROM AUTHOR]

Repetitive sequences are among the most unstable regions in the eukaryotic genome and defects in their maintenance correlate with premature aging and cancer development. Promyelocytic leukemia protein (PML) induces accumulation of proteins at distinct nuclear sites, thereby affecting a plethora of processes including DNA repair or maintenance of telomeres. Doxorubicin, the broadly used chemotherapeutic compound, induces formation of PML-nucleolar associations (PNAs). Nevertheless, molecular factors affecting formation of PNAs are still largely unknown. Here we show that PNAs can accumulate ribosomal DNA (rDNA) and, after restoration of RNA polymerase I activity, these structures transfer a fraction of rDNA outside the nucleolus. Mutagenesis of PML isoforms revealed that this process depends on the SUMO-interacting motif and adjacent serine-rich region, and is enhanced by exon8b present exclusively in PML IV isoform. Moreover, we demonstrate that PNAs formation is also regulated by p14ARF/p53 tumor suppressors and casein kinase 2. Our data elucidate how PML nucleolar compartment is assembled, bring the first evidence of PML interacting with rDNA, and show the PML-dependent translocation of rDNA away from the nucleolus. • Unfinished DNA damage repair induces formation of PML-nucleolar biocondensates. • PML-nucleolar associations accumulate rDNA and sequester it outside nucleolus. • PML-nucleolar interaction depends on PML phospho-SIM domain and exon8b. • PML-nucleolar interaction is regulated by casein kinase 2 and p14ARF/p53. [ABSTRACT FROM AUTHOR]

Carcinosarcomas (CSs; malignant mixed Müllerian tumors) of the uterus are highly malignant neoplasms characterized by an unfavorable outcome. They represent less than 5% of all uterine malignancies, and the median patient survival rate is only 21 months. p53 pathway alterations have been studied in CSs originating from the uterus, supporting the monoclonal nature of most but not all of these neoplasms. This paper gives an overview of the current knowledge of p53 pathway distortions in patients with uterine CSs. The survival of patients with uterine CSs in relation to p53 pathway alterations is also briefly summarized. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The objective of this study is to evaluate the promoter methylation status of the p14ARF in esophageal squamous cell carcinoma ( ESCC). Cell lines were treated with the demethylation agent 5-aza-2′-deoxycytidine, and p14ARF messenger RNA ( mRNA) expression was detected by reverse transcription-polymerase chain reaction. We analyzed the methylation status of p14ARF promoter by methylation-specific polymerase chain reaction in 50 ESCC and their noncarcinoma tissues. Then demethylation caused by 5-aza-2′-deoxycytidine increased the p14ARF mRNA expression level in esophagus cancer cell lines. p14ARF methylation was found in 48% (24 of 50) of ESCC patients but only in 18% (9 of 50) corresponding noncarcinoma tissues ( P = 0.001). There was a statistically significant correlation between the presence of methylation and tumor metastasis ( P < 0.001). The p14ARF mRNA was lower in ESCC tissues than nontumor tissues (mean ± standard deviation, 0.47 ± 0.32 vs. 1.40 ± 0.58; P = 0.002). Meanwhile, a signification association was found between the methylation status of p14ARF promoter and p14ARF mRNA expression in tissues ( P < 0.05). The aberrant promoter methylation of p14ARF is a common phenomenon in ESCC, which may be an important mechanism of downregulating p14ARF mRNA expression. [ABSTRACT FROM AUTHOR]

Osteosarcoma (OS) is the eighth most common form of childhood and adolescence cancer. Approximately 10%-20% of patients present metastatic disease at diagnosis and the 5-year overall survival remains around 70% for nonmetastatic patients and around 30% for metastatic patients. Metastatic disease at diagnosis and the necrosis grade induced by preoperative treatment are the only well-established prognostic factors for osteosarcoma. The DNA aberrant methylation is a frequent epigenetic alteration in humans and has been described as a molecular marker in different tumor types. This study evaluated the DNA aberrant methylation status of 18 genes in 34 OS samples without previous chemotherapy treatment and in four normal bone specimens and compared the methylation profile with clinicopathological characteristics of the patients. We were able to define a three-gene panel (AIM1, p14ARF, and ESR1) in which methylation was correlated with OS cases. The hypermethylation of p14ARF showed a significant association with the absence of metastases at diagnoses, while ESR1 hypermethylation was marginally associated with worse overall survival. This study demonstrated that aberrant promoter methylation is a common event in OS and provides evidence that p14ARF and ESR1 hypermethylation could be useful as a prognostic indicator for this disease. [ABSTRACT FROM AUTHOR]

Genome-Wide Association studies (GWAS) offer an unbiased means to understand the genetic basis of traits by identifying single nucleotide polymorphisms (SNPs) linked to causal variants of complex phenotypes. GWAS have identified a host of susceptibility SNPs associated with many important human diseases, including diseases associated with aging. In an effort to understand the genetics of broad resistance to age-associated diseases (i.e., 'wellness'), we performed a meta-analysis of human GWAS. Toward that end, we compiled 372 GWAS that identified 1775 susceptibility SNPs to 105 unique diseases and used these SNPs to create a genomic landscape of disease susceptibility. This map was constructed by partitioning the genome into 200 kb 'bins' and mapping the 1775 susceptibility SNPs to bins based on their genomic location. Investigation of these data revealed significant heterogeneity of disease association within the genome, with 92% of bins devoid of disease-associated SNPs. In contrast, 10 bins (0.06%) were significantly ( P < 0.05) enriched for susceptibility to multiple diseases, 5 of which formed two highly significant peaks of disease association ( P < 0.0001). These peaks mapped to the Major Histocompatibility (MHC) locus on 6p21 and the INK4/ARF ( CDKN2a/b) tumor suppressor locus on 9p21.3. Provocatively, all 10 significantly enriched bins contained genes linked to either inflammation or cellular senescence pathways, and SNPs near regulators of senescence were particularly associated with disease of aging (e.g., cancer, atherosclerosis, type 2 diabetes, glaucoma). This analysis suggests that germline genetic heterogeneity in the regulation of immunity and cellular senescence influences the human healthspan. [ABSTRACT FROM AUTHOR]

Abstract: Recently, PIWIL4 has been identified as a functional protein involved in tumorigenesis. Cervical cancer is the second most prevalent form of cancer worldwide. The relationship between PIWIL4 and cervical cancer is still unknown. Here, we found that PIWIL4 is up-regulated in human cervical cancer tissues in comparison to adjacent normal tissues, and it promotes cell growth and proliferation by inhibiting apoptosis through the p14ARF/p53 pathway. PIWIL4 can also promote the invasion of cervical cancer cells. These results suggest that PIWIL4 might play an oncogenic role in cervical cancer and be useful as a new therapeutic target in the future. [Copyright &y& Elsevier]

Abstract: EGFR family members are tyrosine kinase transmembrane receptors that, in response to specific extracellular ligands, activate cytoplasmic pathways involved in cell proliferation, migration and differentiation. More recently, a pivotal role for EGF receptors has emerged, through the description of their nuclear localization.We report here the characterization of a nuclear variant of the kinase-defective ErbB3 receptor, ErbB380kDa, spanning the intracytoplasmic domain of the receptor. We assessed the putative transcriptional functions of ErbB380KDa in cancer cells, through the regulation of the proliferative Cyclin D1 gene, an already known target of the ErbB3 cytoplasmic signaling. We demonstrate here that the binding of ErbB380KDa on the promoter activates Cyclin D1 transcription and subsequent protein expression, leading to an increased cell proliferation. This mechanism can be balanced in response to the ectopic expression of the tumor suppressor p14ARF that physically interacts with ErbB3100kDa and sequesters it into nucleoli. Our data also show that ErbB380kDa increases the transcription of proliferative genes even though the cytoplasmic pathways are not activated. This nuclear ErbB3 pathway and the target genes concerned need to be further studied. Indeed, such mechanism could explain the tumor relapse observed in response to treatments aimed at blocking the receptor activation in response to ligand binding. [Copyright &y& Elsevier]

Abstract: The protein p14ARF is a known tumor suppressor protein controlling cell proliferation and survival, which mainly localizes in nucleoli. However, the regulatory mechanisms that govern its activity or expression remain unclear. Here, we report that a novel proapoptotic nucleolar protein, PANO, modulates the expression and activity of p14ARF in HeLa cells. Overexpression of PANO enhances the stability of p14ARF protein by protecting it from degradation, resulting in an increase in p14ARF expression levels. Overexpression of PANO also induces apoptosis under low serum conditions. This effect is dependent on the nucleolar localization of PANO and inhibited by knocking-down p14ARF. Alternatively, PANO siRNA treated cells exhibit a reduction in p14ARF protein levels. In addition, ectopic expression of PANO suppresses the tumorigenicity of HeLa cells in nude mice. These results indicate that PANO is a new apoptosis-inducing gene by modulating the tumor suppressor protein, p14ARF, and may itself be a new candidate tumor suppressor gene. [Copyright &y& Elsevier]

Cutaneous melanoma is a highly aggressive cancer with still limited, but increasingly efficacious, standard treatment options. Recent preclinical and clinical findings support the notion that cutaneous melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma already has great implications for the management of cutaneous melanoma. Herein, we review our rapidly growing understanding of the molecular biology of cutaneous melanoma, including the pathogenic roles of the mitogen-associated protein kinase (MAPK) pathway, the phosphatidylinositol 3 kinase [PI3K]/phosphatase and tensin homologue deleted on chromosome 10 [PTEN]/Akt/mammalian target of rapamycin [mTOR])PTEN (phosphatase and tensin homolog) pathway, MET (hepatocyte growth factor), Notch signaling, and other key molecules regulating cell cycle progression and apoptosis. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with clinical benefit from agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Cutaneous melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. These findings suggest that prospective genotyping of patients with melanoma, combined with the growing availability of targeted agents, which can be used to rationally exploit these findings, should be used increasingly as we work to develop new and more effective treatments for this devastating disease. [ABSTRACT FROM AUTHOR]

BACKGROUND: p14ARF, an alternate reading frame (ARF) product of the cyclin-dependent kinase inhibitor 2A locus, plays a critical role in crosstalk between the tumor protein 53 (p53) and retinoblastoma (Rb) pathways and in cellular anticancer mechanisms. Therefore, the authors of this report investigated the association between single nucleotide polymorphisms (SNPs) of the p14ARF gene and the risk of developing a second primary malignancy (SPM) after an index squamous cell carcinoma of the head and neck (SCCHN). METHODS: The log-rank test and Cox proportional hazards models were used to assess the association of 2 p14ARF SNPs (reference SNP [rs]3731217 and rs3088440) with SPM-free survival and with the risk of developing an SPM among 1287 patients who had SCCHN. RESULTS: Patients with either p14ARF variant genotypes of the 2 polymorphisms had a significantly reduced SPM-free survival compared with patients with no variant genotypes (log-rank test; P = .006). Compared with the p14ARF thymine-thymine (TT) and guanine-guanine (GG) genotypes, the variant genotypes of p14ARF TG/GG and guanine-adenine (GA)/ AA were associated with a significantly moderately increased risk of developing an SPM (p14ARF rs3731217: adjusted hazard ratio [aHR], 1.48; 95% confidence interval [CI], 1.00-2.19; p14ARF rs3088440: aHR, 1.6, 95% CI, 1.07-2.43). Moreover, after combining the variant genotypes of the 2 SNPs, patients who had variant genotypes were at significantly greater risk of developing an SPM compared with patients who had no variant genotypes (aHR, 3.07; 95% CI, 1.546.12), and the risk was particularly pronounced in several subgroups. CONCLUSIONS: The current results suggested that there is a modestly increased risk of developing an SPM after an index SCCHN with each p14ARF polymorphism, and there is an even greater risk of developing an SPM for patients with combined variant genotypes of the 2 SNPs. Therefore, p14ARF polymorphisms may be susceptible markers of the risk of developing an SPM in patients with SCCHN. [ABSTRACT FROM AUTHOR]

Induction of cell death by p14ARF is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21CDKN1. Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21CDKN1 or p53-reconstituted DU145 prostate cancer cells, we show that p14ARF-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14ARF in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14ARF-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14ARF-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21CDKN1 strongly enhances p14ARF-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14ARF-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14ARF-triggered apoptosis is enhanced by loss of p21CDKN1-mediated cell cycle checkpoint control. [ABSTRACT FROM AUTHOR]

Introduction: Amongst the genitourinary cancers, carcinoma of the urinary bladder is one of the leading causes of death in India. Hypermethylation of the CpG islands of gene promoter is one of the earliest and most frequent epigenetic alterations leading to cancer as well as in its development. Several studies have suggested that tumour suppressor genes play a key role in the development of cancer. Methylation in the CDKN2A has been associated with various malignant diseases, but information with respect to urinary bladder cancer is lacking in north Indian population. Materials and methods: We analyzed the methylation of P16INK4a and P14ARF in 80 tissues and matched blood samples of patients suffering from bladder cancer and 80 blood samples of cancer-free individuals by MS-PCR. Results: In tissue and matched blood samples of bladder cancer patients, the incidence of P14ARF hypermethylation significantly increased (OR=0.31, 95%CI =0.12–0.8, P=0.01) and (OR=0.0, 95%CI=0.0–0.62, P=0.006) respectively with an increase in age. Clinicopathological analysis revealed that P14ARF hypermethylation in tissue and blood samples was significantly associated with invasive stage (&ges; T2) (OR=0.21, 95%CI = 0.08–0.51, P=0.0002) and (OR = 0.09, 95%CI = 0.03–0.37, P= 0.00001) respectively. Muscle invasive tumour stage (&ges;T2) showed significant association with increased risk of P16INK4α promoter hypermethylation in tissue and blood samples of patients (OR = 0.38, 95%CI = 0.17–0.82, P= 0.01) and (OR = 0.13, 95%CI = 0.05–0.36, P= 0.00005) respectively. Conclusion: These results suggest that the CpG island hypermethylation status of the defined panel of genes may be a useful biomarker in patients suffering from bladder cancer. [ABSTRACT FROM AUTHOR]

Although Myc-interacting zinc-finger protein-1 (Miz-1) is known to be a poxvirus and zinc-finger (POZ) transcription factor required for Myc transcriptional repression, additional regulatory function of Miz-1 is less well understood. Using a yeast two-hybrid screen, we identified human alternate reading frame (ARF) protein as a novel interaction partner of Miz-1. The zinc-finger domain of Miz-1 is involved in its binding to ARF. In addition, we found that Miz-1 was able to interact with p53 through its DNA-binding domain, thus to diminish the binding of p53 to its target promoter and inhibit p53-mediated gene transcription. Interestingly, the Miz-1-regulated p53 transcriptional suppression does not require the presence of ARF or Mdm2. Importantly, ARF and p53 were found to competitively bind to Miz-1 in regulating p53-mediated transcription, and this conclusion was verified by both in vitro binding assay and competitive chromatin immunoprecipitation assay using a bona fide p53 endogenous Bax and Puma promoters. Thus, our study reveals that Miz-1 acts as a p53 suppressor by interfering with p53 DNA-binding ability, and ARF is able to counteract the suppression of Miz-1 on p53 by direct binding to Miz-1, suggesting that Miz-1 is a novel mediator in the ARF-p53 pathway. [ABSTRACT FROM AUTHOR]

A primary failsafe program against unrestrained proliferation and oncogenesis is provided by the p53 tumor suppressor protein, inactivation of which is considered as a hallmark of cancer. Intriguingly, mutations of the TP53 gene are rarely encountered in neuroblastoma tumors, suggesting that alternative p53-inactivating lesions account for escape from p53 control in this childhood malignancy. Several recent studies have shed light on the mechanisms by which neuroblastoma cells circumvent the p53-driven antitumor barrier. We review here these mechanisms for evasion of p53-mediated growth control and conclude that deregulation of the p14ARF-MDM2-p53 axis seems to be the principal mode of p53 inactivation in neuroblastoma, opening new perspectives for targeted therapeutic intervention. [ABSTRACT FROM AUTHOR]

Nucleophosmin (NPM) is a multifunctional protein involved in both proliferation and apoptosis. Importantly, NPM negatively regulates p53 and is frequently overexpressed in a wide variety of cancers. To identify inhibitory molecules of NPM, we used an in vitro selection method termed systematic evolution of ligands by exponential enrichment (SELEX) to select RNA aptamers that bind to NPM with high affinity and specificity. The selected RNA aptamers bind to the central acidic region of NPM and affect its oligomerization both in vitro and in vivo. Remarkably, expression of NPM-specific aptamers causes mislocalization of NPM in the nucleoplasm rather than in the nucleolus, suggesting that NPM oligomerization is important for its proper localization. Moreover, p14ARF is mislocalized in the nucleoplasm and p53 is upregulated in cells expressing NPM aptamers. In addition, cancer cells expressing NPM aptamers not only undergo apoptosis on their own, but are more susceptible to apoptosis induced by DNA-damaging agents as well. These results suggest that interfering with NPM oligomerization can inhibit NPM function and aptamers targeting NPM can serve as potential lead for developing anticancer drugs. [ABSTRACT FROM AUTHOR]

To determine the interrelationships of p53, MDM2, and p14ARF protein expression in primary esophageal squamous cell carcinoma (ESCC) and their prognostic value in ESCC. In total, 119 patients treated for ESCC with esophagectomy were enrolled in this study. Demographic and clinical data including gender, age, depth of tumor invasion, lymph node involvement, and 5-year survival rate were collected by chart review. p53, MDM2, and p14ARF were detected immunohistochemically in the resected tumors to evaluate their usefulness as biomarkers of clinical outcome. p53, MDM2, and p14ARF were expressed in 61 (51.3%), 34 (28.6%), and 22 (18.5%) of 119 tumor specimens, respectively. Overall, p53 protein expression was positively correlated with MDM2 ( P = 0.024) and p14ARF expression ( P = 0.026). In addition, p14ARF expression was most often found in specimens that were positive for both p53 and MDM2. Changes in the p53, MDM2, and p14ARF protein levels were not correlated with 5-year survival rate. Expression of p53 protein correlates with increased MDM2 and p14ARF protein levels in ESCC. In addition, status of p53 (wild-type versus mutant) rather than expression level of p53, MDM2, or p14ARF is likely to be the more critical determinant of clinical outcome. [ABSTRACT FROM AUTHOR]

It has been reported that the induction of cellular senescence through p53 activation is an effective strategy in tumor regression. Unfortunately, however, tumors including adult T-cell leukemia/lymphoma (ATL) have disadvantages such as p53 mutations and a lack of p16(INK4a) and/or p14(ARF). In this study we characterized Nutlin-3a-induced cell death in 16 leukemia/lymphoma cell lines. Eight cell lines, including six ATL-related cell lines, had wild-type p53 and Nutlin-3a-activated p53, and the cell lines underwent apoptosis or cell-cycle arrest, whereas eight cell lines with mutated p53 were resistant. Interestingly, senescence-associated-beta-galactosidase (SA-beta-gal) staining revealed that only ATL-related cell lines with wild-type p53 showed cellular senescence, although they lack both p16(INK4a) and p14(ARF). These results indicate that cellular senescence is an important event in p53-dependent cell death in ATL cells and is inducible without p16(INK4a) and p14(ARF). Furthermore, knockdown of Tp53-induced glycolysis and apoptosis regulator (TIGAR), a novel target gene of p53, by small interfering RNA(siRNA) indicated its important role in the induction of cellular senescence. As many patients with ATL carry wild-type p53, our study suggests that p53 activation by Nutlin-3a is a promising strategy in ATL. We also found synergism with a combination of Nutlin-3a and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), suggesting the application of Nutlin-3a-based therapy to be broader than expected. [ABSTRACT FROM AUTHOR]

Background: 8-Chloro-adenosine (8-Cl-Ado) inhibits tumor cell proliferation by inducing cell-cycle arrest and apoptosis. We speculate that upregulation of p14ARF by E2F1 might contribute to 8-Cl-Ado-induced late apoptosis. Methods: Hoechst staining, cell proliferation and TUNEL assays, real-time quantitative PCR, Western blotting, chromatin immunoprecipitation and RNA interference were employed in investigating the role of induction of p14ARF by E2F1 in 8-Cl-Ado-induced apoptosis in human lung cancer H1299 cells. Results: Exposure of H1299 to 8-Cl-Ado led to apoptosis after long exposure (48 h), revealed by the appearance of nucleus fragmentation and apoptotic bodies and the activation of procaspase-3 pathway. Western blotting and RT-PCR showed that the upregulation of p14ARF was in parallel with E2F1 expression during exposure. Furthermore, induction of p14ARF was attributed to increased E2F1 expression, evidenced by E2F1 transfection and chromatin immunoprecipitation/real-time quantitative PCR. Knockdown of p14ARF expression in H1299 decreased TUNEL-positive cell numbers and relatively increased survival cell numbers during 8-Cl-Ado exposure, indicating insensitivity of p14ARF-knocked down cells to 8-Cl-Ado. Conclusions: Induction of p14ARF by E2F1 contributes to 8-Cl-Ado-induced late apoptosis. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population. Objectives To determine the involvement of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in BCC. Methods We investigated the integrity of the CDKN2A locus in 15 BCC samples by analysing the presence of allelic imbalance/loss of heterozygosity (LOH). Moreover, we studied the mRNA expression levels of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in the BCC samples and compared them with mRNA levels in the corresponding normal tissue. The presence of mutations was examined by sequencing for exons 1a and 2 of p16INK4a. Results We found LOH in one BCC sample for the marker D9S1748. A polymorphism (G442A) of exon 2 was detected in three cases. p14ARF, p15INK4b and p53 presented high expression levels, whereas p16INK4a exhibited low mRNA levels compared with the corresponding normal tissue. Significant correlations were detected among the genes studied. Conclusions Our results demonstrate a different expression profile between p16INK4a and p14ARF, p15INK4b and p53 in BCC. Moreover, we found a low percentage of LOH and of a polymorphic sequence variant (Ala148Thr) for the CDKN2A locus. [ABSTRACT FROM AUTHOR]

Abstract: The Alternative Reading Frame (ARF) protein suppresses tumorigenesis through p53-dependent and p53-independent pathways. Most of ARF''s anti-proliferative activity is conferred by sequences in its first exon. Previous work showed specific amino acid changes occurred in that region during primate evolution, so we programmed those changes into human p14ARF to assay their functional impact. Two human p14ARF residues (Ala14 and Thr31) were found to destabilize the protein while two others (Val24 and Ala41) promoted more efficient p53 stabilization and activation. Despite those effects, all modified p14ARF forms displayed robust p53-dependent anti-proliferative activity demonstrating there are no significant biological differences in p53-mediated growth suppression associated with simian versus human p14ARF residues. In contrast, p53-independent p14ARF function was considerably altered by several residue changes. Val24 was required for p53-independent growth suppression whereas multiple residues (Val24, Thr31, Ala41 and His60) enabled p14ARF to block or reverse the inherent chromosomal instability of p53-null MEFs. Together, these data pinpoint specific residues outside of established p14ARF functional domains that influence its expression and signaling activities. Most intriguingly, this work reveals a novel and direct role for p14ARF in the p53-independent maintenance of genomic stability. [Copyright &y& Elsevier]

In acute myeloid leukaemia (AML), nucleophosmin-1 (NPM1) mutations create a nuclear export signal (NES) motif and disrupt tryptophans at NPM1 C-terminus, leading to nucleophosmin accumulation in leukaemic cell cytoplasm. We investigated how nucleophosmin NES motifs (two physiological and one created by the mutation) regulate traffic and interaction of mutated NPM1, NPM1wt and p14ARF. Nucleophosmin export into cytoplasm was maximum when the protein contained all three NES motifs, as naturally occurs in NPM1-mutated AML. The two physiological NES motifs mediated NPM1 homo/heterodimerization, influencing subcellular distribution of NPM1wt, mutated NPM1 and p14ARF in a ‘dose-dependent tug of war’ fashion. In transfected cells, excess doses of mutant NPM1 relocated completely NPM1wt (and p14ARF) from the nucleoli to the cytoplasm. This distribution pattern was also observed in a proportion of NPM1-mutated AML patients. In transfected cells, excess of NPM1wt (and p14ARF) relocated NPM1 mutant from the cytoplasm to the nucleoli. Notably, this distribution pattern was not observed in AML patients where the mutant was consistently cytoplasmic restricted. These findings reinforce the concept that NPM1 mutants are naturally selected for most efficient cytoplasmic export, pointing to this event as critical for leukaemogenesis. Moreover, they provide a rationale basis for designing small molecules acting at the interface between mutated NPM1 and other interacting proteins.Leukemia (2009) 23, 501–509; doi:10.1038/leu.2008.326; published online 13 November 2008 [ABSTRACT FROM AUTHOR]

Summary: Chemotherapy and/or radiotherapy are established measures in treatment protocols of head and neck squamous cell carcinoma (HNSCC). However, we still lack reliable predictive markers for the response to radio- and chemotherapy. The p53 pathway is involved in stress response and thus might influence chemo-/radiosensitivity. Using 29 HNSCC cell lines previously characterized for p53 mutations, we simultaneously analyzed several key players in the p53 pathway by RT-PCR, transcript sequencing and immunohistochemistry, and investigated their association with chemosensitivity and radiosensitivity. Cell lines with p53 mutations were slightly more sensitive to cisplatin than those with wild-type p53. The type of mutation did not influence radio- or chemosensitivity. p14ARF, an activator of p53, was lost or mutated in all cell lines. Three cell lines showed overexpression of HDM-2, a major negative regulator of p53; however, HDM-2 levels did not correlate with radio- or chemosensitivity. HPV-16 oncoproteins were detected in one highly chemoresistant cell line. Our findings suggest that molecular events resulting in the inactivation of the p53 pathway occur in all HNSCC cell lines. However, single alterations in the p53 pathway are not reliable predictors for the response to radio- or chemotherapy in HNSCC. [Copyright &y& Elsevier]

Abstract: We report here that the polycomb group protein Bmi1 promotes prostate tumorigenesis. Bmi1 is detected at higher levels in androgen-independent PC3 and DU145 than in androgen-dependent LNCaP prostate cancer (CaP) cells. Ectopic Bmi1 enhanced the expression of human telomerase reverse transcriptase (hTERT) and suppressed the exression of p16INK4A and p14ARF in CaP cells. Consistent with these observations, immunohistochemical staining of 51 cases of primary CaP specimens revealed 1.4 fold (p =0.014) and 1.3 fold (p =0.051) higher levels of Bmi1-positive cells in carcinoma compared to normal prostatic epithelial cells and PIN, respectively. In primary CaPs, Bmi1 expression was associated with a reduction in p16INK4A and p14ARF. Furthermore, in comparison to empty vector-transfected cells, Bmi1-expressing DU145 cells formed significantly larger tumors in NOD/SCID mice. Taken together, we demonstrate that Bmi1 promotes prostate tumorigenesis. [Copyright &y& Elsevier]

Abstract: It is still not clear whether the p53 pathway is altered in retinoblastoma development. We assessed the expression of the p53 pathway genes p14ARF , mouse double minute 2 (MDM2), and mouse double minute 4 (MDM4) in human retinoblastoma compared to normal retina. Primary human retinoblastomas, retinoblastoma cell lines and normal retinas were assessed for p14ARF and MDM4 mRNA by quantitative RT-PCR. p14ARF, MDM2, and MDM4 protein were measured by immunoblot and immunohistochemistry. Compared to retina, p14ARF mRNA expression was notably increased in retinoblastoma but p14ARF protein was undetectable. MDM2 and MDM4 proteins were expressed in 22/22 retinoblastomas. MDM2 was expressed in 3/10 retinas tested, and MDM4 in 10/10 retinas. The expression level of MDM2 protein in retinoblastomas and retina was comparable, while MDM4 protein was overexpressed in one retinoblastoma cell line Y79 and two primary retinoblastomas. We observe that overexpression of MDM2 and MDM4 is not a necessary step in retinoblastoma development. However, loss of detectable p14ARF protein and resultant lack of functional inactivation of these p53 inhibitors may contribute to retinoblastoma development by constitutive inhibition of p53. [Copyright &y& Elsevier]

Summary: Background: Transcription factor Pokemon, a central regulation gene of the important tumor suppressor alternative reading frame (ARF), exerted its activity by acting upstream of many tumor-suppressing genes and proto-oncogenes. Its expression in non-small cell lung cancer (NSCLC) and its clinical significance remains unclear. The aim of this study was to investigate the expression of Pokemon in non-small cell lung cancer and to explore its correlation with the clinical pathological characteristics and its influence on patients’ prognosis. Aim: Observe the expression of Pokemon in NSCLC and investigate its mechanism and clinical significance. Methods: Determine the expression of Pokemon in human NSCLC cell lines as well as 55 cases of NSCLC tumor tissues, tumor adjacent tissues and surrounding tissues by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and analyze the relationship between Pokemon expression in NSCLC tumor tissues and clinicopathological features. Determine 62 NSCLC tumor tissues (5 years ago) and p14ARF expression with immunohistochemical technique, discuss the correlation between them and assess the effect of Pokemon on prognosis of patients with lung cancer. Results: Pokemon mRNA and protein took on high expression in lung cancer cell lines, and the expression difference between cancer tissues, tumor adjacent tissues and surrounding tissues had statistical significance (P <0.05). Pokemon expression and p14ARF expression were negatively correlated (r =−0.287). The expression of Pokemon was determined not to be associated with the patient''s sex, age, smoking condition, tumor differentiation degree, histology and lymph node metastasis condition. However, its relationship with TNM staging was established (P <0.05). Furthermore, it was shown that the survival rate of patients with negative Pokemon expression was significantly higher than that of those with positive Pokemon expression (P =0.004), therefore, the expression of Pokemon is believed to be an independent factor affecting prognosis (P =0.034). Conclusion: There was high expression of Pokemon in NSCLC, Pokemon exerted carcinogenesis by inhibiting ARF and had some clinical significance for prognostic evaluation of the patients with NSCLC. [Copyright &y& Elsevier]

Abstract: We report an association between p14ARF and Brca1 in which both proteins co-immunoprecipitate (co-IP) in DU145 cells. The N-terminal 64 residues of p14ARF encoded by exon 1β are sufficient for this association. Inside the cell, ectopic p14ARF co-localizes with ectopic and endogenous Brca1 in A375 cells. Endogenous p14ARF co-localizes with endogenous Brca1 in DU145 cells but not in H1299 cells. Since p14ARF interacts with B23 in the nucleolus, Brca1 co-localizes with B23 in DU145 but not in H1299 cells. While ectopic ARF potently inhibited DU145 cell proliferation, it had no effect on the proliferation of H1299 cells, suggesting that the interaction between ARF and Brca1 contributes to ARF-mediated tumor suppression. Consistent with this notion, ectopic p14ARF modulates endogenous Brca1 expression in MCF7 breast cancer cells and p14ARF co-localizes with Brca1 in normal breast epithelial cells. This co-localization is enhanced in primary breast cancer. Taken together, the results show that p14ARF associates with Brca1, which may play a major role in tumor suppression. [Copyright &y& Elsevier]

The article discusses a study on the association of interindividual differences in p14ARF promoter methylation with single nucleotide polymorphism (SNP) in primary colorectal cancer. In the study, p14ARF methylation was detected in 61 of 188 colorectal cancers. Key findings suggested that SNP around the p14ARF promoter region may be responsible for the individual susceptability to p14ARF promoter methylation among colorectal cancer patients.