Loss of Both USP10 and p14ARF Protein Expression Is an Independent Prognostic Biomarker for Poor Prognosis in Patients With Epithelial Ovarian Cancer.

BACKGROUND/AIM: The prognostic role of USP10 in epithelial ovarian cancer has been studied in various human cancers. Our aim was to evaluate the clinical and pathological significance of USP10 in epithelial ovarian cancer. MATERIALS AND METHODS: Immunohistochemical analyses of the expression of USP10 and p14ARF by using tissue microarrays were performed in 336 ovarian tumours and the data were compared with clinicopathological variables. We examined their level of DNA methylation around the putative transcriptional start site in 5' CpG islands in fresh frozen tissues and ovarian cancer cells. RESULTS: Expression of USP10 and p14ARF was significantly lower in cancer tissues than in normal epithelium. Low USP10 expression and a combined USP10/p14ARF low expression were revealed to be independent prognostic factors. A high degree of methylation in USP10 and p14ARF CpG islands was found by methylation specific PCR analysis in cancer than in normal tissues and cells. CONCLUSION: Decreased expression of USP10 or combined USP10/p14ARF decreased expression is a strong indicator of poor prognosis in patients with ovarian cancer.

Increased expression of senescence markers p14(ARF) and p16(INK4a) in breast cancer is associated with an increased risk of disease recurrence and poor survival outcome.

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 p14(ARF) , p16(INK4a) and p21(WAF1/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 p14(ARF) and p16(INK4a) but lower expression of p21(WAF1/Cip1) than non-malignant tissues. There were significant correlations of normal, benign, preinvasive and malignant tissues with p14(ARF) , p16(INK4a) and p21(WAF1/Cip1) expression (P < 0.05). Univariate comparison showed a correlation between high p16(INK4a) expression and poor survival (P = 0.000) and an increased risk of relapse (P = 0.000), whereas high p14(ARF) expression correlated only with an increased risk of relapse (P = 0.038). Multivariate analysis showed p16(INK4a) to be an important prognostic factor for overall survival (P = 0.011) and disease-free survival (P = 0.004), with p14(ARF) also being a significant prognostic factor for disease-free survival (P = 0.043). Moreover, patients showing both high p16(INK4a) expression and and high p14(ARF) 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 p16(INK4a) expression and p14(ARF) expression may play an important role in the progression of proliferative breast tissue to invasive cancer, and may be useful as prognostic factors.

Hepatitis C virus core protein overcomes H2O2-induced apoptosis by downregulating p14 expression via DNA methylation.

Infection with hepatitis C virus (HCV) is characterized by systemic oxidative stress that is caused by either viral core protein or chronic inflammation. It is well recognized that reactive oxygen species (ROS) such as H2O2 can induce apoptotic cell death and can therefore function as anti-tumorigenic species. However, the detailed mechanisms by which ROS induce apoptotic cell death and HCV copes with the oxidative conditions are largely unknown. In the present study, we found that H2O2 induced apoptotic cell death in p53-positive human hepatocytes, but not in p53-negative human hepatocytes. For this effect, H2O2 upregulated levels of p14, increased ubiquitin-dependent degradation of mouse double minute 2 (MDM2), and reduced the interaction between MDM2 and p53 to prevent p53 degradation, resulting in accumulation of p53 and subsequent activation of p53-dependent apoptotic pathways. Interestingly, HCV core repressed p14 expression via promoter hypermethylation to abolish the potential of H2O2 to activate the p14-MDM2-p53 pathway. As a consequence, HCV core-expressing cells could overcome p53-mediated apoptosis provoked by H2O2. Taken together, HCV core could contribute to hepatocellular carcinoma formation by removing deleterious roles of ROS inducing cell death.

Epigenetic regulation of p14ARF and p16INK4A expression in cutaneous and uveal melanoma.

Inactivation of p14ARF and p16INK4A by epigenetic changes in cutaneous and uveal melanoma has been here investigated. Compared with melanocytes, p14ARF mRNA reduction and p16INK4A inactivation were frequently noticed. No association between p14ARF promoter methylation and mRNA levels was found, whereas aberrant p16INK4A methylation was associated with gene silencing (p<0.001). Comparative analysis within melanomas of different Breslow's thicknesses showed that drastic reductions in p14ARF and p16INK4A expression appeared at the level of thin/intermediate and intermediate/thick transitions. The effects of 5-aza-2'-deoxycytidine (5-aza-dC) and suberanilohydroxamic acid (SAHA) on in vivo binding of DNA methyltransferases (DNMTs) and acetyl histone H3/H4 to p14ARF and p16INK4A promoters were tested together with the impact of ectopic expression of p14ARF and p16INK4A on cell proliferation, migration, and invasion. SAHA treatment induced H3 and H4 hyperacetylation at the p14ARF promoter followed by increased p14ARF expression, whereas exposure to 5-aza-dC decreased the recruitment of DNMT1 and DNMT3b at the p16INK4A promoter and reactivated p16INK4A. Studies on promoter-associated di-methyl histone H3 (Lys4) levels ruled out an involvement of this epigenetic trait on p14ARF and p16INK4A expression. The enforced expression of p14ARF or p16INK4A and, even more so, their co-expression, significantly reduced cell proliferation, migration and invasion. Our data pinpoint: i) a frequent impairment of p14ARF and p16INK4A gene expression by epigenetic modifications in melanoma; ii) histone hypoacetylation as the dominant mechanism of p14ARF silencing; and iii) 5' CpG promoter methylation as the major mechanism of p16INK4A gene inactivation. Collectively, our data suggest that selected epi-drugs may be useful in melanoma treatment.

Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC.

Knockdown of Bmi1 inhibits the stemness properties and tumorigenicity of human bladder cancer stem cell-like side population cells.

B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) is directly involved in cell growth, proliferation and self-renewal of cancer stem cells (CSCs). The aim of the present study was to assess the role of Bmi1 in the maintenance of stemness properties and tumorigenicity of human bladder CSC-like side population (SP) cells. SP cells were sorted by flow cytometry using Hoechst 33342 staining. Bmi1 mRNA and protein expression in SP and non-SP (NSP) cells was analyzed by quantitative PCR, immunofluorescence and western blotting. The stemness properties of SP cells included cell proliferation, migration, self-renewal, chemotherapy resistance and cell cycle progression were assessed. Tumor formation was also assessed in human bladder cancer xenografts after Bmi1 silencing. The mRNA expression of Bmi1 was upregulated in SP cells when compared with that in the NSP cells. Knockdown of Bmi1 in SP cells resulted in inhibition of cell proliferation, migration and tumor sphere formation, enhanced sensitivity to cisplatin, and cell cycle arrest in the G0/G1 phase. Bmi1 knockdown inhibited cell cycle progression through derepression of the p16INK4a/p14ARF locus. Bmi1-siRNA SP cells failed to produce tumors in recipient mice, while typical urothelial carcinoma formed from subcutaneously injected scramble-siRNA SP cells. Bmi1 is crucial for the maintenance of stemness properties and tumorigenicity of human bladder CSC-like cells. Bmi1 may be a potential therapeutic target for the eradication of CSCs in bladder cancer.

ARF induction in response to DNA strand breaks is regulated by PARP1.

The ARF tumour suppressor protein, the gene of which is frequently mutated in many human cancers, plays an important role in the cellular stress response by orchestrating up-regulation of p53 protein and consequently promoting cell-cycle delay. Although p53 protein function has been clearly linked to the cellular DNA damage response, the role of ARF protein in this process is unclear. Here, we report that arf gene transcription is induced by DNA strand breaks (SBs) and that ARF protein accumulates in response to persistent DNA damage. We discovered that poly(ADP-ribose) synthesis catalysed by PARP1 at the sites of unrepaired SBs activates ARF transcription through a protein signalling cascade, including the NAD(+)-dependent deacetylase SIRT1 and the transcription factor E2F1. Our data suggest that poly(ADP-ribose) synthesis at the sites of SBs initiates DNA damage signal transduction by reducing the cellular concentration of NAD(+), thus down-regulating SIRT1 activity and consequently activating E2F1-dependent ARF transcription. Our findings suggest a vital role for ARF in DNA damage signalling, and furthermore explain the critical requirement for ARF inactivation in cancer cells, which are frequently deficient in DNA repair and accumulate DNA damage.

Restoration of INK4a/ARF gene inhibits cell growth and cooperates with imatinib mesylate in Philadelphia chromosome-positive leukemias.

VSV-G-pseudotyped lentiviral vectors expressing p16(INK4a) or p14(ARF) were used to infect at high-efficiency Philadelphia chromosome (Ph)-positive leukemia cell lines lacking endogenous transcripts. Restoration of p16(INK4a) accumulated cells in the G0/G1 phase of cell cycle and restoration of p14(ARF) induced their apoptosis, followed by significant growth inhibition. Transduction of primary blast cells from chronic myeloid leukemia in blast crisis (CML-BC) and Ph-positive acute lymphoblastic leukemia (ALL) with p16(INK4a) or p14(ARF) virus also resulted in cell growth inhibition and/or apoptosis with a patient-to-patient variation, whereas clonal growth and differentiation of cord blood progenitor cells were not affected by enforced expression of INK4a/ARF. Furthermore, upon viral transduction at low multiplicity of infection, INK4a/ARF potentiated the effect of imatinib mesylate on Ph-positive leukemia cell lines in an additive but not synergistic manner. These results suggest that INK4a/ARF protein-mimetic agents may be promising options for Ph-positive leukemias in combination with imatinib mesylate.

Regulation of p14ARF expression by HPV-18 E6 variants.

A common causative agent for uterine cervical cancer is the human papillomavirus type 18 (HPV-18) which has three phylogenic variants: Asian-Amerindian, European, and African. Each variant shows significant molecular differences in the E6 gene. E6 oncoprotein is a negative regulator of tumor suppressor protein p53, hence, this oncoprotein indirectly regulates the expression of tumor-suppressor p14(ARF) . p14(ARF) and p16(INK4A) genes are overexpressed in--and have been proposed as markers for--HPV-related cervical cancer. In order to dissect the role of E6 on the regulation of p14(ARF) expression, separating it from that of other intervening factors, transfection of E6 variants to MCF-7 cells was performed, assessing cDNA transcript levels by RT-PCR, whereas p14(ARF) and p53 expression were evaluated by immunocytochemistry and Western blot. E6 transfected cells differentially expressed transcripts of two molecular forms: E6 and E6*. The ratio of these two forms varied with the transfected E6 variant. With the Asian-Amerindian variant, the ratio was E6 > E6*, whereas with the European and the African the ratio was E6* > E6. As expected with the E6* construct, E6* transcripts were solely observed. In addition, when E6 > E6* and p53 expression was low, p14(ARF) was high and when E6* > E6 and p53 expression was high, p14(ARF) was low. In conclusion, each E6 variant distinctively affects p53 levels and consequently p14(ARF) expression, finding that could be related with the differences in oncogenic effect of infection with the diverse high-risk HPV variants.

Different expression pattern and significance of p14ARF-Mdm2-p53 pathway and Bmi-1 exist between gastric cardia and distal gastric adenocarcinoma.

Recent studies have suggested that adenocarcinoma of gastric cardia (GCA) is distinct from distal stomach, with different risk factors, tumor characteristics, and biological behavior. The aim of this study is to evaluate the possible difference in the expressions of p14ARF, Mdm2, p53, and Bmi-1 by immunohistochemical staining on paraffin-embedded tissues of gastric cardia adenocarcinoma (GCA; n = 74) and distal gastric adenocarcinoma (DGA; n = 41). The results showed that the percentage of p14ARF-negative expression, Mdm2 overexpression, p53-positive expression, and p53 pathway abnormality (p14ARF(-)/Mdm2(+)/p53(+)) were all significantly higher in GCA than those in DGA (P < .05). Further analysis showed that in GCA, the negative expression of p14ARF was significantly associated with poor differentiation, Mdm2 overexpression with tumor stage and lymph node metastasis, and positive p53 expression with tumor stage (P < .05), whereas in DGA, only Mdm2 overexpression was related with well/moderate differentiation (P < .05). Abnormality of the p53 pathway was significantly correlated with poorer differentiation only in GCA (P < .05). The positive expression of Bmi-1 in all cases of GCA and DGA was significantly higher than normal gastric mucosa epithelium, but no difference was found between GCA and DGA (P > .05). Thus, the results in this study confirmed that different expression pattern and clinicopathologic significance of the p14ARF-Mdm2-p53 pathway did exist between GCA and DGA. The results further support the hypothesis that different mechanisms may be involved in the development and progression of adenocarcinoma from cardia and distal portion of stomach.

p14(ARF) inhibits the growth of lung adenocarcinoma cells harbouring an EGFR L858R mutation by activating a STAT3-dependent pro-apoptotic signalling pathway.

Epidermal growth factor receptor (EGFR) stimulates proliferative and survival signals. Activating mutations of EGFR are involved in the aetiology and maintenance of the malignant phenotype of lung tumours. We previously described the frequent association of these mutations with the decreased expression of the p14(ARF) tumour suppressor, another common feature of lung cancer. Based on these data, we postulated that p14(ARF) could protect cells against untimely or excessive mitotic signals induced by mutant EGFR. In this study, we demonstrate that p14(ARF) promotes apoptosis in lung tumour cells harbouring the EGFR L858R mutation through the accumulation of phosphorylated signal transducer and activator of transcription 3 (STAT3) on Tyr 705 residue, which leads to Bcl-2 downregulation. Using siRNA against PTP-RT, the phosphatase that specifically targets Tyr 705 residue, we show that accumulation of pSTAT3-Tyr705 promotes EGFR L858R mutant cell death, thereby confirming the existence of a STAT3-dependent pro-apoptotic pathway in these cells. Finally, we show that the expression of the EGFR L858R mutant represses p14(ARF) expression and inhibits STAT3/Bcl-2 signalling. These results identify a novel link between the p14(ARF) and EGFR pathways and suggest that EGFR L858R counteracts the pro-apoptotic function of p14(ARF) by downregulating its expression to promote carcinogenesis.

Stress-regulated transcription factor ATF4 promotes neoplastic transformation by suppressing expression of the INK4a/ARF cell senescence factors.

Many cancers overexpress ATF4, a stress-induced transcription factor that promotes cell survival under hypoxic conditions and other stresses of the tumor microenvironment, but the potential contributions of ATF4 to oncogenesis itself have been little explored. Here, we report that ATF4 promotes oncogene-induced neoplastic transformation by suppressing the expression of cellular senescence-associated genes. Strikingly, primary embryo fibroblasts from ATF4-deficient mice were resistant to transformation by coexpression of H-ras(V12) and SV40 large T antigen. In wild-type cells these oncogenes induced expression of the murine Atf4 gene along with the cyclin-dependent kinase inhibitor Cdkn2a, which encodes the cell senescence-associated proteins p16INK4 and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, genetic ablation of ATF4 led to constitutive expression of p16INK4a and p19ARF, triggering cellular senescence. Our findings define a central function for ATF4 in promoting oncogenic transformation by suppressing a central pathway of cellular senescence.

p53 binds to and is required for the repression of Arf tumor suppressor by HDAC and polycomb.

The expression of tumor suppressor Arf is tightly repressed during normal cell growth at a young age and is activated by oncogenic insults, and during aging, results in p53 activation and cell-cycle arrest to prevent hyperproliferation. The mechanisms of both transcriptional repression and activation of Arf are not understood. We show that p53 binds to and represses Arf expression and that this repression requires the function of both histone deacetylases (HDAC) and polycomb group (PcG) proteins. Inactivation of p53 leads to increased Arf transcription in both mouse embryonic fibroblasts (MEF) cultured in vitro and in tissues and organs of p53 null mice. Activation of endogenous p53 enhances Arf repression, and reintroduction of p53 back into p53 null MEFs restores Arf repression. Both DNA binding and transactivation activities of p53 are required for Arf repression. We show that p53 is required for both HDAC and PcG to repress Arf expression. Bindings of both HDAC and PcG to Arf are disrupted by inactivation of p53 and can be restored in p53 null MEFs by the reintroduction of wild-type, but not mutant, p53. These results indicate that p53 recruits both HDAC and PcG to Arf locus to repress its expression, and this repression constitutes a second feedback loop in p53 regulation.

The RB (pRb2/p16) and p53 (p14/p53/p21) tumor-suppressor pathways in endemic Burkitt lymphoma.

Burkitt lymphoma accounts for approximately 50% of pediatric cancers in equatorial Africa and a majority of NHL in Uganda. The aim of the study was to examine the expression profile of the RB (pRb2 or p16) and p53 (p53, p14, or p21) pathways in biopsies of endemic BL, and compare it to the pattern found in reactive lymphoid hyperplasia (RLH). A total of 51 BL and 10 RLH biopsy specimens were included in the study. p16 expression was found in 8 (16.3%) BL and 2 (20%) RLH cases. p27 was revealed in 29 (65.9%)BL and 9(90%) RLH cases, whereas 29(59.2%) BL and only 1 RLH expressed p53. Positivity for pRb2 was found in 42 (84.0%) of the BL and 8(80%)of the RLH cases. p21 and p14 were negative in all BL and RLH cases. In conclusion, our data indicate that heterogeneous RB (pRb2 or p16) and p53 (p53, p14, or p21) pathway alterations occur frequently in BL. Except for a much higher frequency of p53 protein expression in BL, close similarities were found between BL and RLH.

E2F1 localizes predominantly to neuronal cytoplasm and fails to induce expression of its transcriptional targets in human immunodeficiency virus-induced neuronal damage.

As human immunodeficiency virus (HIV) does not induce neuronal damage by direct infection, the mechanisms of neuronal damage or loss in HIV-associated dementia (HAD) remain unclear. We have shown previously that immunoreactivity of transcription factor, E2F1, increases in neurons, localizing predominantly to the cytoplasm, in HIV-associated pathologies. Here we confirm that E2F1 localization is predominantly cytoplasmic in primary postmitotic neurons in vitro and cortical neurons in vivo. To determine whether E2F1 contributes to neuronal death in HAD via transactivation of target promoters, we assessed the mRNA and protein levels of several classical E2F1 transcriptional targets implicated in cell cycle progression and apoptosis in an in vitro model of HIV-induced neurotoxicity and in cortical autopsy tissue from patients infected with HIV. By Q-PCR, we show that mRNA levels of E2F1 transcriptional targets implicated in cell cycle progression (E2F1, Cyclin A, proliferating cell nuclear antigen (PCNA), and dyhydrofolate reductase (DHFR)) and apoptosis (caspases 3, 8, 9 and p19(ARF)) remain unchanged in an in vitro model of HIV-induced neurotoxicity. Further, we show that protein levels of p19(ARF), Cyclin A, and PCNA are not altered in vitro or in the cortex of patients with HAD. We propose that the predominantly cytoplasmic localization of E2F1 in neurons may account for the lack of E2F1 target transactivation in neurons responding to HIV-induced neurotoxicity.

Intensive expression of Bmi-1 is a new independent predictor of poor outcome in patients with ovarian carcinoma.

BACKGROUND: It has been suggested that the B-cell specific moloney leukemia virus insertion site 1 (Bmi-1) gene plays an oncogenic role in several types of human cancer, but the status of Bmi-1 amplification and expression in ovarian cancer and its clinical/prognostic significance are unclear. METHODS: The methods of immunohistochemistry and fluorescence in situ hybridization were utilized to examine protein expression and amplification of Bmi-1 in 30 normal ovaries, 30 ovarian cystadenomas, 40 borderline ovarian tumors and 179 ovarian carcinomas. RESULTS: Intensive expression of Bmi-1 was detected in none of the normal ovaries, 3% cystadenomas, 10% borderline tumors, and 37% ovarian carcinomas, respectively. Amplification of Bmi-1 was detected in 8% of ovarian carcinomas. In ovarian carcinomas, significant positive associations were found between intensive expression of Bmi-1 and the tumors ascending histological grade, later pT/pN/pM and FIGO stages (P < 0.05). In univariate survival analysis of the ovarian carcinoma cohorts, a significant association of intensive expression of Bmi-1 with shortened patient survival (mean 49.3 months versus 100.3 months, p < 0.001) was demonstrated. Importantly, Bmi-1 expression provided significant independent prognostic parameters in multivariate analysis (p = 0.005). CONCLUSIONS: These findings provide evidence that intensive expression of Bmi-1 might be important in the acquisition of an invasive and/or aggressive phenotype of ovarian carcinoma, and serve as a independent biomarker for shortened survival time of patients.

Endogenous LKB1 knockdown accelerates G(1)/S transition through p53 and p16 pathways.

The tumor suppressor LKB1 is inactivated in 90% of Peutz-Jeghers cancer syndrome, 30-40% of non-small cell lung carcinoma, and a variety of other cancers, indicating the loss of LKB1 activity is a critical step in oncogenesis. However, current understanding of LKB1 function is largely limited to the results from cancer cells, and how LKB1 inactivation initiates malignant transformation in normal cells remains unclear. Here we ablated endogenous expression of LKB1 in two normal cell lines: human embryonic kidney 293T cells (HEK-293T cells) and human umbilical vein endothelial cells (HUVECs) by LKB1-specific short hairpin RNAs. Downregulation of endogenous LKB1 lead to a facilitated G(1)/S transition, accompanied by a concomitant increase in Rb phosphorylation (Ser(807/811)). Furthermore, reduced expression of p53 and p16 was observed in LKB1 ablated cells, while no differences were detected for cyclin D1 and cyclin E. These results jointly suggest that endogenous LKB1 knockdown accelerates cell cycle progression through G(1)/S checkpoint in HEK-293T cells and HUVECs, which is at least in part, mediated by decline of p53 and p16 pathways. Our findings provided a plausible mechanism by which loss of LKB1 expression in normal cells contributes to the formation of malignancies.

P21 Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis.

Human papillomavirus is involved in the carcinogenesis of tonsillar squamous cell carcinomas. Here, we investigated the expression and the prognostic value of key cell cycle proteins in the pRb and p53 pathways in both human papillomavirus type 16-positive and -negative tonsillar squamous cell carcinomas. Using immunohistochemistry, 77 tonsillar squamous cell carcinomas with known human papillomavirus type 16 status and clinical outcome were analyzed for expression of Ki67, p16(INK4A,) cyclin D1, pRb, p14(ARF), MDM2, p53, p21(Cip1/WAF1), and p27(KIP1). Results were correlated with each other and with clinical and demographic patient data. A total of 35% of tonsillar carcinomas harbored integrated human papillomavirus type 16 DNA and p16(INK4A) overexpression, both being considered essential features for human papillomavirus association. These tumors also showed the overexpression of p14(ARF) (P<0.0001) and p21(Cip1/WAF1) (P=0.001), and downregulation of pRb (P<0.0001) and cyclin D1 (P=0.027) compared with the human papillomavirus-negative cases. Univariate Cox regression analyses revealed a favorable survival rate for non-smokers (P=0.006), as well as for patients with T1-2 tumors (P<0.0001) or tumors showing low expression of cyclin D1 (P=0.028), presence of human papillomavirus and overexpression of p16(INK4A) (P=0.01), p14(ARF) (P=0.02) or p21(Cip1/WAF1) (P=0.004). In multivariate regression analyses, smoking and tumor size, as well as expression of cyclin D1 and p21(Cip1/WAF1), were found to be independent prognostic markers. We conclude that human papillomavirus positivity in tonsillar squamous cell carcinomas strongly correlates with p21(Cip1/WAF1) and p14(ARF) overexpression and downregulation of pRb and cyclin D1. In particular p21(Cip1/WAF1) overexpression is an excellent favorable prognosticator in tonsillar squamous cell carcinomas.

Expression of p14ARF, MDM2, and MDM4 in human retinoblastoma.

It is still not clear whether the p53 pathway is altered in retinoblastoma development. We assessed the expression of the p53 pathway genes p14(ARF), 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 p14(ARF) and MDM4 mRNA by quantitative RT-PCR. p14(ARF), MDM2, and MDM4 protein were measured by immunoblot and immunohistochemistry. Compared to retina, p14(ARF) mRNA expression was notably increased in retinoblastoma but p14(ARF) 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 p14(ARF) protein and resultant lack of functional inactivation of these p53 inhibitors may contribute to retinoblastoma development by constitutive inhibition of p53.

Overexpression of Pokemon in non-small cell lung cancer and foreshowing tumor biological behavior as well as clinical results.

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 p14(ARF) 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 p14(ARF) 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.