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CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high-level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut-off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high-level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut-off for IHC to predict CCNE1 high-level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high-level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high-level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38-2.26, p < 0.0001). CCNE1 high-level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high-level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors., (© 2020 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons Ltd.)

Background/aim: Cyclins D1 and E play different roles in the cell cycle. Cyclin E promotes chromosome instability, whereas cyclin D1 regulates apoptosis of cells. This study evaluated the prognostic significance of G1 cyclins, p21 and pRb in tumor proliferation., Patients and Methods: A total of 102 patients with colon cancer were operated on and staged according to TNM. Follow-up was 2 to 68 months (mean 38.3±16.7 months). Expression of cyclin E and D1 were evaluated using immunohistochemistry., Results: Levels of cyclin E expression were correlated with cyclin D1 expression (p=0.038), p21 expression (p=0.047), and pRb expression (p=0.004). The 5-year survival rate along with prognosis of patients with advanced stage (III, IV) colon cancer and cyclin D1 positive tumors, were significantly worse (p=0.009). Statistically significant association was observed between tumor proliferative capacity Ki-67, cyclin D1 (p=0.009), pRb (p=0.031) and p21 (p=0.050)., Conclusion: Cyclin D1 is highly expressed in advanced stage colon cancer patients, implying a potential prognostic value., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Objective: To explore the mechanism by which lobaplatin, as monotherapy and in combination with paclitaxel, inhibits the proliferation of human gastric cancer SGC-7901 cells., Methods: After treatment, the MTT assay was used to assess cell viability; cell cycle distribution was evaluated flow-cytometrically. Western blot was used to quantitate cyclin D1, E1, B1, and Cdk2/4 protein levels., Results: Lobaplatin and paclitaxel inhibited SGC-7901 cell growth in a concentration and timedependent manner, with IC25 values at 48h of 1.97±0.17µg/ml and 1.98±0.19 ng/ml, respectively. Interestingly, both drugs synergistically inhibited SGC-7901 cells (combination index [CI]<0.95). Lobaplatin did not affect cyclin D1 and CDK4 protein expression, while cyclin E1 and CDK2 levels were significantly increased, with cyclin B1 amounts markedly decreased (p<0.05). More S phase cells were observed after lobaplatin treatment compared with controls (60.03±1.25 vs. 18.69±0.96%; p<0.05). After treatment with paclitaxel, cyclin D1 and CDK4 protein levels were similar to control values; meanwhile, cylinE1 and CDK2 protein amounts were reduced, with increased cyclin B1 levels, compared with control values (p<0.05). More G2/M cells were obtained after treatment with paclitaxel compared with control values (74.54±0.92 vs. 18.62±0.44% (p<0.05). Lobaplatin and paclitaxel combination did not affect cyclin D1 and CDK4 protein levels (p>0.05); meanwhile, cyclin E1 and CDK2 levels were increased, with reduced cyclin B1 amounts, compared with control values (p<0.05). Notably, more S (43.23±0.81 vs. 22.32±0.86%) and G2/M (31.22±0.96 vs. 25.81±2.08%) phase cells were obtained after combined treatment compared with control values., Conclusion: Lobaplatin and paclitaxel synergistically inhibit SGC-7901 cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background-Aim: To evaluate the prognostic role of elaborate molecular clusters encompassing cyclin D1, cyclin E1, p21, p27 and p53 in the context of various breast cancer subtypes., Methods: Cyclin E1, cyclin D1, p53, p21 and p27 were evaluated with immunohistochemistry in 1077 formalin-fixed paraffin-embedded tissues from breast cancer patients who had been treated within clinical trials. Jaccard distances were computed for the markers and the resulted matrix was used for conducting unsupervised hierarchical clustering, in order to identify distinct groups correlating with prognosis., Results: Luminal B and triple-negative (TNBC) tumors presented with the highest and lowest levels of cyclin D1 expression, respectively. By contrast, TNBC frequently expressed Cyclin E1, whereas ER-positive tumors did not. Absence of Cyclin D1 predicted for worse OS, while absence of Cyclin E1 for poorer DFS. The expression patterns of all examined proteins yielded 3 distinct clusters; (1) Cyclin D1 and/or E1 positive with moderate p21 expression; (2) Cyclin D1 and/or E1, and p27 positive, p53 protein negative; and, (3) Cyclin D1 or E1 positive, p53 positive, p21 and p27 negative or moderately positive. The 5-year DFS rates for clusters 1, 2 and 3 were 70.0%, 79.1%, 67.4% and OS 88.4%, 90.4%, 78.9%, respectively., Conclusions: It seems that the expression of cell cycle regulators in the absence of p53 protein is associated with favorable prognosis in operable breast cancer.

Purpose: Cell cycle is mainly mediated by cyclins, cyclin- dependent kinases (CDK), and CDK inhibitors. Cyclin E is the main regulator for transition from G1 to S phase, and is involved in cancer pathogenesis, progression and metastasis. Nevertheless, there is still a controversy of the prognostic value of cyclin E overexpression in ovarian cancer patients. This meta-analysis is the first study aimed at analyzing the effect of cyclin E overexpression on the prognosis of ovarian cancer., Methods: By systematically searching the PUBMED, EMBASE and MEDLINE databases for relevant articles with publication dates up to January 2016 and selection following inclusion and exclusion criteria, 8 studies with 1470 patients were enrolled in our meta-analysis. The overall survival (OS) of patients with cyclin E overexpression was calculated using hazard ratio (HR) with 95% confidence intervals (CIs). The studies were categorized according to the author and year, demographic data in each study, ovarian cancer related information, and cyclin E cut-off value., Results: Cyclin E overexpression in ovarian cancer was a poor prognostic factor with statistical significance for OS (HR=1.48, 95%CI: 1.12,1.85). Using confunnel, we found no publication bias in our analysis., Conclusion: Cyclin E might be considered as a prognostic factor for ovarian cancer, as supported by our meta-analysis. However, more high-quality studies should be conducted to find better clinical use of cyclin E in ovarian cancer.

Minichromosomal maintenance (MCM) proteins are participants of DNA replication and may represent more accurate markers in determining the proliferative fraction within a tumor than proliferative marker Ki-67. Our study investigated the correlation between MCM4 and MCM7 expression and Ki-67, Bmi1, and cyclin E expression in esophageal adenocarcinoma, squamous cell carcinoma, and precancerous lesions. MCM4 and MCM7 expression had similar distribution as Ki-67 and Bmi1 expression in esophageal carcinoma and precancerous lesions. The mean percentage of MCM4, MCM7, and Ki-67 expression increased from squamous epithelium (5.5%, 7.3%, and 5.9%, respectively), to columnar cell metaplasia (11.2, 13.5%, and 3.4%), Barrett's esophagus (27.7%, 35.3%, and 8.3%), low-grade dysplasia (42.6%, 52.2%, and 12.9%), high-grade dysplasia (63.2%, 77.7%, and 29.6%), adenocarcinoma (61.3%, 75.5%, and 24.5%), and squamous cell carcinoma (74.1, 85.4%, and 36.3%). The percentages of MCM4 and MCM7 expression were significantly higher than Ki-67 expression. Using univariate analysis we found a high percentage of MCM4 expression (>70%) to be significantly associated with lymph node metastasis and shorter survival in the adenocarcinoma group. We also demonstrated the percentage of MCM4 and MCM7 expression to be significantly correlated with Ki-67, Bmi1, and cyclin E expression in esophageal carcinoma and precancerous lesions. MCM4 and MCM7 may serve as more sensitive proliferative markers for the evaluation of esophageal lesions., Competing Interests: ✩ Competing interests: All authors have disclosed that they have no conflict of interest., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: Homologous recombination (HR) proficient ovarian cancers, including CCNE1 (cyclin E)-amplified tumors, are resistant to poly (ADP-ribose) polymerase inhibitors (PARPi). Histone deacetylase inhibitors (HDACi) are effective in overcoming tumor resistance to DNA damaging drugs. Our goal was to determine whether panobinostat, a newly FDA-approved HDACi, can sensitize cyclin E, HR-proficient ovarian cancer cells to the PARPi olaparib., Methods: Expression levels of CCNE1 (cyclin E), BRCA1, RAD51 and E2F1 in ovarian tumors and cell lines were extracted from The Cancer Genome Atlas (TCGA) and Broad-Novartis Cancer Cell Line Encyclopedia (CCLE). In HR-proficient ovarian cancer cell line models (OVCAR-3, OVCAR-4, SKOV-3, and UWB1.289+BRCA1 wild-type), cell growth and viability were assessed by sulforhodamine B and xenograft assays. DNA damage and repair (pH2AX and RAD51 co-localization and DRGFP reporter activity) and apoptosis (cleaved PARP and cleaved caspase-3) were assessed by immunofluorescence and Western blot assays., Results: TCGA and CCLE data revealed positive correlations (Spearman) between cyclin E E2F1, and E2F1 gene targets related to DNA repair (BRCA1 and RAD51). Panobinostat downregulated cyclin E and HR repair pathway genes, and reduced HR efficiency in cyclin E-amplified OVCAR-3 cells. Further, panobinostat synergized with olaparib in reducing cell growth and viability in HR-proficient cells. Similar co-operative effects were observed in xenografts, and on pharmacodynamic markers of HR repair, DNA damage and apoptosis., Conclusions: These results provide preclinical rationale for using HDACi to reduce HR in cyclin E-overexpressing and other types of HR-proficient ovarian cancer as a means of enhancing PARPi activity., Competing Interests: DK received olaparib from AstraZeneca and is a co-investigator for a clinical trial of olaparib. MAC is the local principal investigator for a clinical trial of olaparib., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives: Pulmonary arterial hypertension (PAH) is a fast progressing vascular disease characterized by uncontrolled cell proliferation of pulmonary artery smooth muscle cells (PASMCs). Some studies have suggested that PAH and cancers share an apoptosis-resistant state, featuring excessive cell proliferation. The miR-34 family consists of tumour-suppressive miRNAs, and its reduced expression has been reported in numerous cancers; however, its role in hypoxia-induced PAH has not been previously studied., Materials and Methods: miR-34 family expression was evaluated in a rat model with hypoxia and in cultured hypoxic PASMCs, using real-time quantitative PCR (RT-qPCR). Function of miR-34 family was assessed by transfecting miR-34 mimics and inhibitors. Dual luciferase reporter gene assays, RT-qPCR and Western blotting were performed to validate target genes of miR-34., Results: Significant down-regulation of miR-34a in hypoxic lung tissue, pulmonary artery and PASMCs was identified and then effects of miR-34a in modulating cell proliferation in human pulmonary artery smooth muscle cells (hPASMCs) was investigated in vitro. Reduction of miR-34a levels in hPASMCs caused increased proliferation and these effects were reversed by overexpression of miR-34a. miR-34a overexpression down-regulated platelet-derived growth factor receptor alpha (PDGFRA) expression, which is a key factor in PAH development. These results suggest that miR-34a is a potential regulator of proliferation in PASMCs, and that it could be used as a novel treatment strategy in PAH., (© 2016 John Wiley & Sons Ltd.)

Cyclin E and its co-activator, phospho-cyclin-dependent kinase 2 (p-CDK2), regulate G1 to S phase transition and their deregulation induces oncogenesis. Immunohistochemical assessments of these proteins in cancer have been reported but were based only on their nuclear expression. However, the oncogenic forms of cyclin E (low molecular weight cyclin E or LMW-E) in complex with CDK2 are preferentially mislocalized to the cytoplasm. Here, we used separate nuclear and cytoplasmic scoring systems for both cyclin E and p-CDK2 expression to demonstrate altered cellular accumulation of these proteins using immunohistochemical analysis. We examined the specificity of different cyclin E antibodies and evaluated their concordance between immunohistochemical and Western blot analyses in a panel of 14 breast cell lines. Nuclear versus cytoplasmic staining of cyclin E readily differentiated full-length from LMW-E, respectively. We also evaluated the expression of cyclin E and p-CDK2 in 1676 breast carcinoma patients by immunohistochemistry. Cytoplasmic cyclin E correlated strongly with cytoplasmic p-CDK2 (P < 0.0001), high tumor grade, negative estrogen/progesterone receptor status, and human epidermal growth factor receptor 2 positivity (all P < 0.0001). In multivariable analysis, cytoplasmic cyclin E plus phosphorylated CDK2 (as one variable) predicted breast cancer recurrence-free and overall survival. These results suggest that cytoplasmic cyclin E and p-CDK2 can be readily detected with immunohistochemistry and used as clinical biomarkers for aggressive breast cancer., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: The purpose of this study was to investigate the expression of A-kinase anchor protein 95 (AKAP95), cell cycle protein E1 (cyclinE1) and D1 (cyclinD1), and gap junction protein connexin 43 (Cx43) in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins., Methods: The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method., Results: The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30% (P<0.05). The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%, which was lower than that of ovarian pericarcinoma tissues expressing as 75%; respectively, and the difference was statistically significant between groups (P<0.05). The expression of cyclinD1 in ovarian cancer tissues was related to the histologic type (P<0.05) while it showed no correlation with the degree of differentiation (P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the histologic type (P>0.05). Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other (P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other (P<0.05). However, AKAP95 and cyclinD1 showed no correlation (P>0.05)., Conclusion: AKAP95, cyclinD1 and cyclinE1 play an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened. The expression of cyclinD1 in ovarian cancer tissues is related to the histologic type while it shows no correlation with the degree of differentiation. Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues shows no correlation with the degree of differentiation or the histologic type. AKAP95 expression is correlated with Cx43 and cyclinE1 expression; Cx43 expression is correlated with AKAP95, cyclinD1 and cyclinE1 expression; cyclinE1 expression is correlated with AKAP95, Cx43, cyclinD1 expression; cyclinD1 expression is correlated with Cx43 and cyclinE1 expression, while AKAP95 and cyclinD1 show no correlation.

Background: Luminescent semiconductor nanocrystals, also known as quantum dots (QD), possess highly desirable optical properties that account for development of a variety of exciting biomedical techniques. These properties include long-term stability, brightness, narrow emission spectra, size tunable properties and resistance to photobleaching. QD have many promising applications in biology and the list is constantly growing. These applications include DNA or protein tagging for in vitro assays, deep-tissue imaging, fluorescence resonance energy transfer (FRET), and studying dynamics of cell surface receptors, among others. Here we explored the potential of QD-mediated labeling for the purpose of tracking an intracellular protein inside live cells., Results: We manufactured dihydrolipoic acid (DHLA)-capped CdSe-ZnS core-shell QD, not available commercially, and coupled them to the cell cycle regulatory protein Cyclin E. We then utilized the QD fluorescence capabilities for visualization of Cyclin E trafficking within cells of Xenopus laevis embryos in real time., Conclusions: These studies provide "proof-of-concept" for this approach by tracking QD-tagged Cyclin E within cells of developing embryos, before and during an important developmental period, the midblastula transition. Importantly, we show that the attachment of QD to Cyclin E did not disrupt its proper intracellular distribution prior to and during the midblastula transition. The fate of the QD after cyclin E degradation following the midblastula transition remains unknown.

The limited effectiveness of therapy for patients with advanced stage head and neck squamous cell carcinoma (HNSCC) or recurrent disease is a reflection of an incomplete understanding of the molecular basis of HNSCC pathogenesis. MUC4, a high molecular weight glycoprotein, is differentially overexpressed in many human cancers and implicated in cancer progression and resistance to several chemotherapies. However, its clinical relevance and the molecular mechanisms through which it mediates HNSCC progression are not well understood. This study revealed a significant upregulation of MUC4 in 78% (68/87) of HNSCC tissues compared with 10% positivity (1/10) in benign samples (P=0.006, odds ratio (95% confidence interval)=10.74 (2.0-57.56). MUC4 knockdown (KD) in SCC1 and SCC10B HNSCC cell lines resulted in significant inhibition of growth in vitro and in vivo, increased senescence as indicated by an increase in the number of flat, enlarged and senescence-associated β-galactosidase (SA-β-Gal)-positive cells. Decreased cellular proliferation was associated with G0/G1 cell cycle arrest and decrease expression of cell cycle regulatory proteins like cyclin E, cyclin D1 and decrease in BrdU incorporation. Mechanistic studies revealed upregulation of p16, pRb dephosphorylation and its interaction with histone deacetylase 1/2. This resulted in decreased histone acetylation (H3K9) at cyclin E promoter leading to its downregulation. Orthotopic implantation of MUC4 KD SCC1 cells into the floor of the mouth in nude mice resulted in the formation of significantly smaller tumors (170±18.30 mg) compared to those (375±17.29 mg) formed by control cells (P=0.00007). In conclusion, our findings showed that MUC4 overexpression has a critical role by regulating proliferation and cellular senescence of HNSCC cells. Downregulation of MUC4 may be a promising therapeutic approach for treating HNSCC patients.

Objective: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships., Methods: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues., Results: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05)., Conclusion: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer.

Objective: Upregulation of cyclin E and cyclin D1-6 accelerates the transition from G1 to S phase. The objective of this study was to determine if cyclin D1 and E are prognostic indicators in endometrial cancer., Materials and Methods: Surgically-treated patients with endometrial carcinoma had their tumors stained for nuclear expression of cyclin D1 and E. Quantification of staining and measurement of growth phase fraction were performed using image analysis. FIGO stage, grade, and histology were also analyzed., Results: Cyclin D1 and E expression was unrelated to DNA index (p = 0.93). While cyclin D1 expression did not correlate with S+G2M phase fraction (p = 0.69), increased cyclin E expression was directly correlated with increased S+G2M phase fraction (p = 0.002). Cyclin E expression was highest in clear cell carcinomas (p = 0.042) while cyclin D1 expression was highest in adenosquamous carcinomas (p = 0.028). Patients dying from cancer had significantly higher expression of cyclin D1 (p = 0.042) and E (p = 0.02) as compared to patients surviving their disease. Multivariate logistic regression revealed FIGO stage, grade, and lack of cyclin E overexpression to be independent prognostic indicators of survival., Conclusion: Cyclin E expression is related to increased growth fraction, clear cell histology, and decreased survival in patients with endometrial cancer.

A number of studies have indicated that cyclin E plays an important role in a variety of neoplastic processes. In our study we evaluated cyclin E1 expression and the possible prognostic value of this protein in neuroblastic tumors in children. Cyclin E1 expression was investigated by means of immunohistochemical analysis of 25 neuroblastic tumor tissue samples. We found a significant correlation between high cyclin E1 expression and deaths due to neoplastic disease. The mean values of cyclin E1 indexes in fatal cases were twice as high as in other cases. The results indicate that high cyclin E1 expression may have prognostic importance in neuroblastic tumors in children.

Background: The role of estrogen in the growth and survival of ovarian cancer cells is controversial. In this study, we investigated the changes in cell-cycle regulatory proteins in ovarian cancer cell lines after estrogen treatment to explore the role of estrogen in ovarian cancers., Methods: Two ovarian adenocarcinoma cell lines were used for the study: the first, OC-117-VGH, was deficient in estrogen receptors (ER)α and ERβ, and the second, OVCAR3, was positive for ERα and ERβ. Serial concentrations of estrogen were used to evaluate the effects of estrogen on the survival of ovarian cancer cells. The cell-cycle regulatory proteins, including cyclin D1, cyclin E, p16/INK4a, and p27/KIP1, were used to check the possible mechanism of an estrogen effect on survival of the cancer cell line., Results: Estrogen 0.01-1.0 μM inhibited the growth of both cell lines. There were no differences in cyclin D1 and E expression between the two cell lines after estrogen treatment, but the expression of p16/INK4a and p27/KIP1 was significantly higher in the OC-1170-VGH cell line than in the OVCAR3 cell line., Conclusion: Although the ER-positive and ER-negative ovarian cancer cell lines were inhibited by estrogen, the influence of cell-cycle regulatory proteins was different between the two, suggesting that the inhibitory effect of estrogen on ovarian cancer cell lines might be mediated through different pathways., (Copyright © 2012. Published by Elsevier B.V.)

Embryonal tumors constitute the most common malignant brain tumor group in children. Experimental results indicate that genes involved in cell cycle and signal transduction are deregulated in medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT). The cell cycle is regulated by protein complexes composed of a regulatory subunit called Cyclin and a catalytic domain named Cyclin-dependent kinase (CDK). Cyclins and CDKs are in turn regulated by cyclin-dependent kinase inhibitors (CDKI) which inhibit cell-cycle progression. Cyclins D and Cyclin E are important for the passage of cells through G1 to S phase. P-27, a member of the universal CDKI family, is important in regulating the G1/S transition. Thus, the purpose of this study was to investigate the expression of p-27, Cyclin D3, and Cyclin E, and their possible prognostic significance in pediatric embryonal brain tumors. We retrospectively evaluated 51 children with embryonal tumors that were treated surgically in our institute. All patients had regular follow up examinations. The streptavidin-biotin HRP method was performed on paraffin sections for detection of p-27, Cyclin D3, and Cyclin E. There were 42 cases of MB and nine cases of AT/RT. Cyclin D3 expression was detected in 11/42 MB and 3/9 AT/RT patients. Cyclin E expression was detected in 28/42 MB and 8/9 AT/RT patients. High expression of Ki-67 (>50 %) and p-27 (>50 %) was observed in 23.8-73.8 % of MB patients. Combined high Ki-67 and p-27 expression was observed in 21.4 % cases of MB. In these cases there was expression of Cyclin E in 88.8 % and Cyclin D3 in 22.2 % of MB. No significant correlation was found between Ki-67 and p-27, Cyclin D3, and E. No correlation was found between Cyclin D3, Cyclin E, p-27, and overall survival. Increased p-27 and Cyclin E expression was detected in a substantial number of MB patients and in nearly all AT/RT patients. Further studies on a larger number of patients are needed to clarify a possible correlation of p-27 and Cyclin E with tumor behavior.

The Polycomb transcription repressor BMI1 is highly expressed in human neuroblastomas and is required for the clonogenic self-renewal and tumorigenicity of human neuroblastoma cell lines. The molecular basis of BMI1 action in neuroblastoma cells is not well understood. Here we report that BMI1 has a critical role in stabilizing cyclin E1 by repressing the expression of FBXW7, a substrate-recognition subunit of the SCF E3 ubiquitin ligase that targets cyclin E1 for degradation. BMI1 binds to the FBXW7 locus in vivo and represses its mRNA expression. Overexpression of cyclin E1 or abrogation of FBXW7 induction rescues the cell-death phenotype of BMI1 knockdown. Moreover, MYCN, an oncoprotein in the pathogenesis of high-risk neuroblastomas, is able to counteract the death-inducing effect of BMI1 knockdown by activating CCNE1 transcription. We further show that high cyclin E1 expression is associated with Stage 4 neuroblastomas and poor prognosis in patients. These findings suggest a molecular mechanism for the oncogenic activity of BMI1 and MYCN in neuroblastoma pathogenesis and progression by maintaining cyclin E1 levels.

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. The connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin's efficacy on the growth and viability of human HNSCC FaDU and Detroit cells. Our results show that metformin treatment (5-20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18-57% and 35-81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20-57% and 33-82% inhibition, respectively. Mechanistically, metformin caused G 1 arrest, which coincided with a decrease in the protein levels of CDKs (2, 4 and 6), cyclins (D1 and E) and CDK inhibitors (p15, p16, p18 and p27), but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and β-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites, but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.

Aims: To investigate the expression of cyclin E isoforms in rectal cancer and its relations to clinicopathological factors and survival., Materials and Methods: Cyclin E expression was assessed by Western blot in 360 resected rectal cancer patients of stage I to III. Multivariate analysis was applied to indicate the independent prognostic markers in this cohort., Results: Nineteen percent, 24% or 29% patients exhibited elevated levels of full-length (FL) cyclin E, low-molecular-weight (LMW) cyclin E or total cyclin E in their tumors respectively. Significant correlation was observed between cyclin E expression with blood vessel invasion, deeply invasive tumors, histology grade and lymph node metastasis. Moreover, patients with high levels of LMW-cyclin E or total cyclin E had a poorer 5-year overall survival than did patients with low levels of LMW-cyclin E or total cyclin E. In multivariate analysis, both the LMW-cyclin E and total cyclin E, but not FL-cyclin E, remained independent prognostic indicators in both patients with stage I to III and in those with early stage. Patients with elevated LMW- or total cyclin E levels had a hazard ratio for death from rectal cancer of 6.302 (95% CI, 1.903-17.81, p = 0.001) or 4.332 (95% CI, 1.298-16.362, p = 0.001)., Conclusion: Overexpression of the LMW-cyclin E or total cyclin E is a strong predictor for poorer survival in patients with rectal cancer. Therefore, evaluating cyclin E expression may provide useful prognostic information for resectable rectal cancer patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Cyclin E and p27 expression is easy to assess in human tissues by standard immunohistochemical techniques. Immunohistochemistry is cost effective, relatively easy to perform and will play more of a role in the future management of cancer. The aim of this study was to investigate the role of p27 and cyclin E immunoexpression as a prognostic factor in early breast carcinoma., Methods: Cyclin E and p27 immunohistochemistry was performed on sixty six cases of breast carcinoma submitted over a five year period to the Division of Anatomical Pathology, Groote Schuur hospital; Whittaker and Associates; and PathCare. All tumours included in this study were less than 5 cm in diameter (pT1 and pT2 stage) and all the patients had wide local excisions performed. Follow up information was obtained from patient folders in the Department of Radiation Oncology., Results: There was no significant association of cyclin E and p27 expression with distant metastasis free survival (MFS) for all invasive carcinomas in contrast to grade, lymph node spread and vascular invasion. However, there was a statistically significant direct association of cyclin E with distant metastases in all invasive carcinomas, in the subgroup of infiltrating duct carcinomas (IDC) and in the node negative group when cyclin E was stratified as negative and positive (low/high). In this study of early breast carcinoma, only 9/66 cases showed cyclin E expression. Of these, four patients had distant metastases, one patient had a local recurrence and four patients were alive at last follow-up. Furthermore, cyclin E expression was significantly associated with grade, lymph node spread, oestrogen receptor status and histological type. None of the lobular carcinomas showed cyclin E positivity and only one case of lobular carcinoma presented with distant metastases.59/66 cases were positive (low/high) for p27 while seven cases were negative, 22 cases showed low expression and 37 cases demonstrated high p27 expression.p27 was significantly associated with oestrogen receptor status only for all invasive carcinomas and in the IDC group. There was no statistical relationship between p27 and cyclin E, but 50 (76%) tumours with positive p27 expression were negative for cyclin E. There were similar results for the invasive ductal carcinoma subgroup., Conclusion: This study shows that p27 and cyclin E are not good independent prognostic markers for early breast carcinoma in contrast to grade, lymph node spread and vascular invasion for all invasive carcinomas. However, cyclin E provides some prognostic value as there is a direct statistical association with the development of distant metastases. Many previous studies have correlated overexpression of cyclin E with an aggressive course. The inverse relationship between p27 and cyclin E expression which has been reported in the literature has been highlighted, but this was not statistically significant. Most cases showed positive p27 expression and negative Cyclin E expression. This may be due to the early stage of the disease.

Background: Cyclins drive cell-cycle progression by associating with their kinase partners, cyclin-dependent kinases (CDK). We investigated cyclin D1/CDK6, cyclin E/CDK2 complexes, and the cell-cycle negative regulators p15 and p27 in an incisional skin wound model., Methods: Wounds were produced on the back of female diabetic mice and their normoglycemic littermates. Animals were treated with polydeoxyribonucleotide (PDRN, 8 mg/kg/i.p.), an agonist of adenosine A2(A) receptors, or its vehicle daily. Granulation tissue proliferation by Ki67 immunostaining, cyclin D/CDK6 and cyclin E/CDK2 complexes, and p21 and p16 proteins (Western blot analysis), and the histologic changes were assessed at different days (3, 6, and 12 days after injury)., Results: Numerous Ki67 positive cells were observed at day 3 and day 6 in the granulation tissue of normoglycemic mice. Ki67 positive cells were fewer in diabetic than in normoglycemic mice. PDRN increased Ki67 positive cells in diabetic mice. Normoglycemic mice showed the greatest upregulation of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. Diabetic mice had a markedly lower expression of cyclin D1, CDK6, cyclin E, and CDK2 at day 6. They also showed a greater expression of p15 and p27 at day 6. PDRN administration in diabetic mice increased cyclin D1/CDK6 and cyclin E/CDK2 expression and reduced p15 and p27 inhibitors at day 6 after injury; moreover, it improved the impaired wound healing at day 12., Conclusion: Our results suggest that adenosine A2(A) receptor activation by PDRN might represent a therapeutic strategy to overcome the diabetes-impaired cell-cycle machinery., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Malignant mesothelioma and ovarian/peritoneal serous carcinoma are two of the most common tumours affecting the serosal cavities. Unlike other malignant tumours diagnosed at this anatomical site, such as lung and breast carcinoma, malignant mesothelioma and serous carcinoma share a common histogenesis, may be difficult to differentiate morphologically, and co-express many of the diagnostic markers used by cytopathologists in effusion diagnosis. Selected markers have nevertheless shown sufficient sensitivity and specificity to differentiate serous carcinoma from malignant mesothelioma effectively. Recently, our group applied high throughput technology to the identification of new markers that may aid in differentiating these two cancer types and validated several of these markers in follow-up studies. This review will present current data regarding the diagnostic and biological aspects of malignant mesothelioma and ovarian/peritoneal serous carcinoma., (© 2010 Blackwell Publishing Ltd.)

Recent identification of the modular CLS motifs responsible for cyclins A and E localization on centrosomes has revealed a tight linkage between the nuclear and centrosomal cycles. These G1/S cyclins must localize on the centrosome in order for DNA replication to occur in the nucleus, whereas essential DNA replication factors also function on the centrosome to prevent centrosome overduplication. Both events are dependent on the presence of an intact CLS within each cyclin. Here we compare the cyclins A and E CLSs at the structural and functional levels and identify a new cyclin A CLS mutant that disrupts all CLS functions and reduces the affinity of cyclin A for Cdk2. Analysis of interactions of the CLS motif within the cyclin molecules highlights the importance of the cyclin CBOX1 region for Cdk2 binding.

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Ovarian epithelial cancer is the leading cause of death among gynecological malignancies. FSH may increase the risk of ovarian malignancy and play an important role in ovarian carcinogenesis. Our previous studies showed that FSH increases the expression of VEGF through survivin. In this study, the function and mechanism of FSH in ovarian cancer were further explored. We found that FSH promoted proliferation and prevented apoptosis of ovarian cancer cells by activating survivin through the SAPK/JNK and PI3K/AKT pathways. FSH also down-regulated the expression of programmed cell death gene 6 (PDCD6) and death receptor 5 (DR5), two molecules required for induction of apoptosis. RNA interference was applied to knock down survivin and PDCD6 expression, and we found that the blockage of survivin reversed the effects of FSH on apoptosis and proliferation, whereas knock down of PDCD6 enhanced these effects. The expression of DR5, cyclin D1, and cyclin E correlated with survivin expression, but PDCD6 did not. Using immunohistochemical staining, we further showed that ovarian serous cystadenocarcinoma samples had higher expression of survivin than did benign ovarian cystadenoma and borderline cystadenoma samples (P<0.01). Furthermore, survivin expression in the ovarian serous cystadenocarcinoma specimens was correlated with disease stage (P<0.05). Our results suggest that FSH promotes ovarian cancer development by regulating the expression of survivin, PDCD6, and DR5. Greater understanding of the molecular mechanisms of FSH in ovarian epithelial carcinogenesis and development will ultimately help in the development of a novel targeted therapy for ovarian cancer.

Cyclin E-associated CDK2 activity is required for the initiation of DNA synthesis in human cells. CDK2 activity is tightly regulated; CDK2 must be in the nucleus, bound to a cyclin, phosphorylated on T160, and dephosphorylated on T14/Y15 for complete kinase activation. Nuclear localization exposes CDK2 to activating enzymes (CAK, Cdc25A) in stimulated cells. Previous studies from our lab indicate CDK2 nuclear localization and cyclin E co-expression are insufficient to cause CDK2 activation or T160 phosphorylation in stimulated IIC9 cells; these activities still require serum stimulation and ERK kinase activity. Recent studies have implicated a role for origin of replication (ORC) licensing proteins in the activation of G1/S Cdks. In this study, we show that CDK2 associates with chromatin and Cdc6 in an ERK-dependent manner following stimulation of IIC9 CHEF cells. We show that nuclear-localized CDK2 (CDK2-NLS) ectopically expressed with cyclin E requires mitogenic stimulation and ERK activation for chromatin association, in addition to previously shown kinase activation and T160 phosphorylation in IIC9 cells. Additionally, we show that expression of Cdc6 in stimulated IIC9 cells treated with ERK inhibitor rescues CDK2-NLS chromatin association, kinase activation, and T160 phosphorylation. From the above data, we deduce ERK-dependent CDK2 activation is due in part to ERK-dependent Cdc6 expression. To examine the role of Cdc6 directly in stimulated primary human fibroblasts, we used RNA interference to attenuate the expression of Cdc6. We show that Cdc6 expression is required for CDK2 chromatin association and kinase activation in stimulated primary human fibroblasts. Additionally, we show that Cdc6 expression is required for the initiation of DNA synthesis and S phase entry in stimulated primary human fibroblasts. Ultimately, this data implicates Cdc6 expression as an important mitogen-induced mechanism in the activation of CDK2/cyclin E, the initiation of DNA synthesis, and the regulation of G1-S phase progression.

Cystic ovarian disease (COD) is an important cause of infertility that affects cattle. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, the objective in the present study was to evaluate apoptosis and proliferation in induced ovarian cystic follicles in cows to investigate the follicular persistence. Stage of estrous cycle was synchronized in 10 heifers and 5 were then subjected to the induction of COD by administration of ACTH. After the ovariectomy number of in situ apoptotic cells by TUNEL assay, active caspase-3, FAS/FASLG and members of the BCL2 family were compared by immunohistochemistry and multiplex PCR and cell proliferation by evaluation of Ki-67 protein and cyclin D1 and E mRNA. Significantly (p<0.05) lesser proliferative and apoptotic rates were found in cystic follicles from cows with COD compared with those with regular cycles. The relatively minimal proliferation found by immunohistochemistry with Ki-67 marker were confirmed by the gene expression of cyclin D1 and E. Lesser apoptotic rates were associated with decreased amounts of apoptotic-related proteins BAX, FASLG and caspase-3 as well as the in situ apoptosis detected by TUNEL assay, and increased amounts of the anti-apoptotic survival factor cellular BCL2 in the cystic follicles of the COD group. The BAX/BCL2 gene expression profile confirmed the immunohistochemical findings. Results from the present study indicate that cellular proliferation and apoptosis are altered in cystic follicles of cattle. The present study provides new insights into the molecular mechanisms underlying the aberrant persistence of follicular cysts and related diseases., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Serous tubal intraepithelial carcinoma (STIC) has been proposed as a precursor for many pelvic high-grade serous carcinomas. Our previous analysis of the ovarian cancer genome identified several genes with oncogenic potential that are amplified and/or overexpressed in the majority of high-grade serous carcinomas. Determining whether these genes are upregulated in STICs is important in further elucidating the relationship of STICs to high-grade serous carcinomas and is fundamental in understanding the molecular pathogenesis of high-grade serous carcinomas. In this study, 37 morphologically defined STICs were obtained from 23 patients with stage IIIC/IV high-grade serous carcinomas. Both STICs and the high-grade serous carcinomas were analyzed for expression of Rsf-1 (HBXAP), cyclin E, fatty acid synthase (FASN) and mucin-4. In addition, they were examined for expression of established markers including p53, Ki-67 and p16. We found that diffuse nuclear p53 and p16 immunoreactivity was observed in 27 (75%) of 36 and 18 (55%) of 33 STICs, respectively, whereas an elevated Ki-67 labeling index (>or=10%) was detected in 29 (78%) of 37 STICs. Cyclin E nuclear staining was seen in 24 (77%) of 35 STICs, whereas normal tubal epithelial cells were all negative. Increased Rsf-1 and FASN immunoreactivity occurred in 63%, and 62% of STICs, respectively, compared with adjacent normal-appearing tubal epithelium. Interestingly, only one STIC showed increased mucin-4 immunoreactivity. Carcinomas, when compared with STICs, overexpressed p16, Rsf-1, cyclin E and FASN in a higher proportion of cases. In conclusion, STICs express several markers including Rsf-1, cyclin E and FASN in high-grade serous carcinomas. In contrast, mucin-4 immunoreactivity either did not change or was reduced in most STICs. These results suggest that overexpression of Rsf-1, cyclin E and FASN occurs early in tumor progression.

Aims: To clarify which immunohistochemical markers could be helpful in distinguishing between classical Hodgkin's lymphoma (cHL) and primary mediastinal B-cell lymphoma (PMBCL) to more narrowly define 'B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and cHL'., Methods and Results: Two hundred and 83 cHLs and 51 PMBCLs were analysed on validated tissue microarray platforms with antibodies to BOB.1, CD15, CD20, CD23, CD30, CD79a, cyclin E, LMP-1, MUM1p, p63 and Oct2. The marker cut-off scores were calculated using receiver-operating characteristic curves. Markers with the highest positive predictive value for cHL were: CD15, cyclin E, LMP-1 (all 100%), MUM1p (93%) and CD30 (83%). High sensitivity was achieved only by CD30 (92%) and cyclin E (79%). Nineteen percent of PMBCLs were also positive for CD30, which led to a lower specificity of CD30 as regards cHL (81%) compared with cyclin E (100%). The antibodies with the highest positive predictive value for PMBCL were: CD23 (98%), p63 (96%), BOB.1 (94%) and CD79a (90%), with high sensitivity for BOB.1 (100%), CD79a (89%) and p63 (82%)., Conclusions: The use of at least three of the most accurate immunohistochemical markers, cyclin E, CD79a and BOB.1, may be helpful in the differential diagnosis of cHL and PMBCL.

Longan flower extract (LFE) has been shown to exhibit free radical scavenging ability and anti-inflammatory effects. However, the effect of LFE treatment on the growth of colorectal cancer cells has not been evaluated. This study investigated the effect of LFE on two colorectal cancer cell lines, SW-480 and Colo 320DM, and the possible mechanisms involved. LFE-treated cells were assessed for viability by trypan blue exclusion, for in vitro tumorigenesis by seeding cells in soft agar to allow anchorage independent growth, for cell cycle distribution by flow cytometry, for loss of mitochondrial membrane potential by rhodamine 123 staining, for increased apoptosis by DNA fragmentation assay, and for changes in the levels of proteins involved in cell cycle control and apoptosis by immunoblotting. LFE (25-400 microg/ml) could inhibit proliferation in a dose- and time-dependent manner. The cell cycle of both LFE-treated cell lines showed obvious S phase block. Western blotting further showed the S phase block in these two cell lines was mainly due to cyclin E accumulation and cyclin A decrease. LFE treatment increased rhodamine 123-negative cells and DNA fragmentation in Colo 320DM cells but not in SW480 cells. Increased levels of the apoptosis activation protein, caspase 3, were also found in Colo 320DM cells. The activation of caspase 3 in LFE-treated SW480 cells was not significant. The caspase 3 activation in Colo 320DM cells by LFE was mediated by the suppression of Bcl-2 protein levels. LFE treatment could inhibit the proliferation and malignancy of colorectal cancer cell lines and was associated with S phase block of the cell cycle. An apoptotic mechanism induced by LFE involving a loss of mitochondrial membrane potential and caspase 3 activation was found in Colo 320DM cells but not in SW480 cells. The results of this study indicate that LFE has potential to be developed as a novel functional food or chemopreventive agent for colorectal cancer.

The development of hepatocellular carcinomas (HCC) appears to be a multistep process that takes several decades in humans. However, the identities of specific gene alterations and their contribution to HCC pathogenesis remain poorly understood. We previously reported that Lkb1(+/-) mice spontaneously develop multiple hepatic nodular foci (NdFc) followed by HCC, and that the conditional activation of beta-catenin in Catnb(lox(ex3)) mouse livers alone does not cause tumor formation. We show here that the conditional activation of beta-catenin accelerates HCC development in Catnb(+/lox(ex3))Lkb1(+/-) compound mutant mice, affecting displastic hepatocytes in NdFc that suffered LOH at the Lkb1 locus. We further show that beta-catnin activation provides HCC with a growth advantage as well as transplantability. These results suggest that the loss of Lkb1 contributes to the formation of dysplastic NdFc, and that Wnt signaling activation is involved in ensuing progression toward HCC. A combination of these sequential changes can be a practical model for a subset of human HCC.

Background: Cyclin E is a protein that plays a key role in the G1 --> S transition of the normal cell cycle. The product of retinoblastoma gene (pRb) is the master regulator of entry into the cell cycle and p21 protein is a cyclin-dependent kinase inhibitor that disturbs the progression through the cell cycle. The expression of these proteins, among many others, is being deregulated in tumorogenesis. The aim of this study was to investigate whether cyclin E, pRb, and p21 can be used as prognostic indicators in gastric cancer., Material and Methods: Fifty-six patients with gastric adenocarcinoma, who underwent curative resection, constituted the group of our study. The immunohistochemical expression of cyclin E, pRb, and p21 proteins was examined and correlated with clinical-pathological parameters and survival., Results: Positive cyclin E immunostaining was observed in 23 tumors (41.1%). It was associated with intestinal Lauren classification (P=0.003), nodal infiltration (P=0.0025), size of the tumor >5 cm (P=0.032), and lymphatic (P=0.042) and vascular invasion (P= 0.0029). Nevertheless, the survival of patients with positive cyclin E tumors was not significantly shorter than that of negative patients. Positive pRb immunostaining was found in 24 (42.9%) cases and it was associated with the absence of Helicobacter pylori (P=0.044), whereas positive p21 immunostaining was found in 21 tumors (37.5%) and it was associated with less depth of gastric wall infiltration (P=0.001), the absence of lymphatic (P=0.019) and vascular infiltration (P=0.024), and the absence of liver metastasis (P=0.044). Cyclin E expression was associated with pRb expression (P=0.023), but was correlated inversely with p21 expression (P=0.009). The survival of patients with pRb-positive tumors and the survival of patients with p21-positive tumors were significantly longer than that of negative patients (P= 0.0044 and P<0.001, respectively)., Conclusion: The expression of cyclin E could not predict the survival in this series of patients with gastric cancer, whereas the expression of pRb and p21 was associated with a favorable prognosis.

Background: Cell cycle progression is regulated by cyclin dependent kinases (cdk) and cdk inhibitors. Recent immunohistological studies suggested that dysregulation of cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), and p27(kip1) are of prognostic value in patients with breast cancer. Our study represents the first comprehensive immunohistochemical cell cycle marker analysis for cdc25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb in tumor tissue and adjacent benign breast tissue from 69 primarily untreated breast cancer patients., Methods: Immunhistochemistry using primary monoclonal antibodies to detect cdc 25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb has been performed., Results: Sixty-nine patients with untreated, invasive breast cancer (n = 69) were divided into a low/ intermediate and a high risk group according to the St. Gallen 2005 consensus conference. High risk patients (n = 22) had a significantly (p = 0.003) shorter mean and median survival (282.85 weeks; 383.0 weeks, respectively) than low/intermediate risk patients (375.41 weeks; not reached yet, respectively). A subgroup of high risk breast cancer patients characterized in addition by overexpression of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) experienced a shortened mean survival of 222.03, 235.71, 257.25, 239.18, and 261.94 weeks, respectively. Regarding benign breast tissue adjacent to breast cancer tissue, 59.4% of the patients investigated overexpressed cdc25A, 23.2% overexpressed pRb, and 63.2% exerted dysregulation of p27(kip1) while they proved to be negative for immunohistochemical staining regarding all other markers tested., Conclusion: The immunohistological analyses of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) have the potential for further refining the risk assessment in patients with untreated breast cancer who belong to the high risk category defined according to the St. Gallen 2005 consensus conference. These cell cycle markers define a subgroup of high risk patients with even higher risk of metastazation and shortened survival. For confirmation a prospective study using standardized laboratory procedures in a larger population is needed.

The cell-cell adhesion system plays a pivotal role in the maintenance of tissue structure and cell-cell communication. E-cadherin is a major adhesion protein of the epithelial cells, and E-cadherin expression may be involved in the regulation of cell proliferation or differentiation. To address the relationship between cell-cell adhesion and cell proliferation, we focused on the alteration of p27Kip1 (p27), a cyclin-dependent kinase inhibitor, by E-cadherin-mediated adhesion. In an immunohistochemical study of 76 cases of renal cell carcinoma (RCC), the p27-labeling index (LI) was 67% in E-cadherin-reduced RCC, but only 28% in E-cadherin-preserved RCC. E-cadherin-expressing cells rarely expressed p27 in various cancers including those of the breast, colon, liver and prostate. In a subconfluent monolayer culture, the E-cadherin levels were increased steadily in the E-cadherin positive RCC cell line ACHN, whereas the p27 levels were decreased. Subsequent exposure of ACHN cells to the E-cadherin-specific function-blocking antibody reduced the growth associated with the increase in p27 and the decrease in phosphorylated epidermal growth factor receptor (EGFR). In the E-cadherin negative RCC cell line Caki-1, these effects were not observed. These results suggest that E-cadherin-mediated adhesion may be involved in the contact stimulation for cell proliferation in part through the downregulation of p27 and the activation of EGFR in human cancers.

In this study, the effect of high glucose (HG) on endothelial progenitor cell (EPC) proliferation and its relationship with cyclins and reactive oxygen species (ROS) were investigated. Mouse EPCs were isolated from bone marrow using a magnetic activated cell-sorting system and cultured in the presence or absence of HG (30 mmol/l). We found that in the early stage of incubation (3 days), HG promoted cell proliferation, and increased the expressions of cdk2 and cyclin E, while in the late stage of culture (7 days) it inhibited cell proliferation and decreased the expressions of cdk2, cyclin E, and proliferating cell nuclear antigen (PCNA). Moreover, on the third day after incubation, HG significantly inhibited the apoptosis of EPCs, while in the late stage it markedly activated caspase-3 and promoted apoptosis. ROS generation in cells and maleic dialdehyde level in medium were significantly increased in HG group on the seventh day, whereas the expressions of superoxide dismutase and glutathione levels decreased. Tempol, a membrane-permeable radical scavenger, significantly inhibited ROS production in EPCs and partially reversed the HG-mediated inhibition of EPCs proliferation on the seventh day. We hypothesize that in the HG environment, the biphasic response of EPC proliferation may be related to the generation of ROS, which causes modulation of cyclins and cell cycle effect.

Background: Post-translational cleavage of full-length cyclin E from the N-terminus can produce low molecular weight (LMW) isoforms of cyclin E containing the C-terminus only., Aim: To assess their presence in early-onset gastric cancer (EOGC), stump cancers and conventional gastric cancers and ascertain how they influence survival in EOGC., Methods: The expression of full-length and LMW isoforms of cyclin E in 330 gastric cancers, including early-onset gastric cancer (EOGC), stump cancer and conventional gastric cancer (>45 years old) was compared using antibodies targeted to the N- and C-terminals., Results: LMW isoforms were found in 35% of EOGCs, compared to 8% of conventional gastric cancers and 4% of stump cancers; their presence was visualised in cell lines using western blot analysis. In addition, C-terminal staining was a positive predictor of survival in EOGC. In contrast, no correlation with survival was found with the N-terminal antibody which detects only full-length cyclin E., Conclusion: EOGCs have a unique molecular phenotype and LMW isoforms of cyclin E may independently influence survival in EOGC.

Background: Tested was whether the assessment of 5 established bladder cancer biomarkers (p53, pRB, p21, p27, and cyclin E1) could improve the ability to predict disease recurrence and cancer-specific survival after radical cystectomy in patients with pTa-3N0M0 urothelial carcinoma of the bladder (UCB)., Methods: The study comprised 191 patients with pTa-3N0M0 UCB treated with radical cystectomy and bilateral lymphadenectomy (median follow-up, 3.1 years). Biomarker expression was assayed on serial tissue microarray slides using quantitative immunohistochemistry using advanced cell imaging and color detection software. Predictive accuracy was quantified using the concordance index and 200-bootstrap resamples were used to reduce overfit bias. Bootstrap-adjusted predictive accuracy estimates were compared using the Mantel-Haenszel test., Results: UCB recurred in 36 (18.8%) patients and 30 (15.7%) died of bladder cancer; 157 (82.2%) patients had altered expression of at least 1 biomarker. In univariate analyses the number of altered biomarkers had the highest predictive accuracy for both disease recurrence (76.8%, P< .001) and cancer-specific mortality (78.3%, P< .001). Addition of the number of altered biomarkers increased the predictive accuracy of nomograms based on the TNM staging system for disease recurrence and cancer-specific mortality by 10.9% (83.4% vs 72.5%, P< .001) and 8.6% (86.9% vs 78.3, P< .001), respectively., Conclusions: Assessment of the number of altered biomarkers in the cystectomy specimen improves the prediction of bladder cancer recurrence and survival in patients with pTa-3N0M0 disease. Prospective evaluation of alteration in these biomarkers can help identify patients who would benefit from adjuvant treatment after radical cystectomy.

Purpose: To update the recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer., Methods: For the 2007 update, an Update Committee composed of members from the full Panel was formed to complete the review and analysis of data published since 1999. Computerized literature searches of MEDLINE and the Cochrane Collaboration Library were performed. The Update Committee's literature review focused attention on available systematic reviews and meta-analyses of published tumor marker studies. In general, significant health outcomes (overall survival, disease-free survival, quality of life, lesser toxicity, and cost-effectiveness) were used for making recommendations. Recommendations and, Conclusions: Thirteen categories of breast tumor markers were considered, six of which were new for the guideline. The following categories showed evidence of clinical utility and were recommended for use in practice: CA 15-3, CA 27.29, carcinoembryonic antigen, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, urokinase plasminogen activator, plasminogen activator inhibitor 1, and certain multiparameter gene expression assays. Not all applications for these markers were supported, however. The following categories demonstrated insufficient evidence to support routine use in clinical practice: DNA/ploidy by flow cytometry, p53, cathepsin D, cyclin E, proteomics, certain multiparameter assays, detection of bone marrow micrometastases, and circulating tumor cells.

The Cul-1 protein is the scaffold element of SCF complexes that are involved in the proteasomal degradation of numerous proteins regulating cell cycle progression. Owing to this central role in cell growth control, aberrant expression of the components of SCF is thought to play a role during tumourigenesis. Nothing is known about Cul-1 expression in human tumours. In this study, we have analysed its status in a series of 128 human lung carcinomas, comprising 50 non-small cell lung cancers (NSCLCs; 29 squamous cell carcinomas and 21 adenocarcinomas) and 78 neuroendocrine (NE) lung tumours (24 typical and atypical carcinoids, 19 large cell NE carcinomas and 35 small cell lung carcinomas), using immunohistochemistry. We report for the first time an altered pattern of Cul-1 expression in human tumours; indeed, we show that Cul-1 expression is up-regulated in 40% (51/128) of all lung tumours as compared to normal lung tissues, including 34% (17/50), 75% (18/24) and 30% (16/54) of NSCLCs, carcinoids and high grade neuroendocrine lung carcinomas, respectively. Furthermore, we demonstrate that high levels of Cul-1 protein are associated with a low KI67 proliferative index (p = 0.005) and with a decrease in the cyclin E oncoprotein (p = 0.0003), one of the major targets of SCF complexes. These data suggest that up-regulation of Cul-1 could protect cells from hyperproliferative signals through cyclin E down-regulation. Cul-1 is modified by neddylation, a post-translational modification that grafts ubiquitin-like Nedd8/Rub1 residues and controls Cul-1 activity. We also provide evidence that neddylated forms of Cul-1 are specifically expressed in high-grade NE lung tumours and are associated with down-regulation of the Cul-1 inhibitor CAND1 (p = 0.03) and a high level of cyclin E (p = 0.0002). These data support the notion that alterations in the Cul-1 neddylation/deneddylation pathway could contribute to the development of these highly aggressive lung tumours., (Copyright 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: The authors recently reported on the role of cyclin E in differentiating ovarian/primary peritoneal carcinoma from malignant peritoneal mesothelioma using gene expression arrays. In the current study, they analyzed the expression of low-molecular weight (LMW) forms of cyclin E in ovarian carcinoma, malignant mesothelioma, and benign reactive effusions., Methods: Cyclin E protein expression was analyzed in 98 effusions (72 ovarian carcinomas, 14 malignant mesotheliomas, and 12 reactive specimens) using immunoblotting. Sixty-two ovarian carcinoma effusions were studied further for cyclin E expression using immunohistochemistry. The correlations between cyclin E expression in ovarian carcinoma and clinical parameters, including chemotherapy response, were analyzed., Results: LMW forms of cyclin E were identified in 54 of 72 ovarian carcinoma effusions (75%) compared with 1 of 14 malignant mesothelioma effusions (7%) and 1 of 12 reactive effusions (8%) (P < .001). Their presence in ovarian carcinoma was associated with a higher percentage of cyclin E-positive cells (P = .001) and increased staining intensity (P < .001) using immunohistochemistry. The presence of LMW forms of cyclin E was correlated with shorter overall survival (P = .021) and progression-free survival (P = .020). The presence of a higher percentage of cyclin E-positive cells using immunohistochemistry was correlated with shorter progression-free survival (P = .026). No association with chemotherapy response was observed., Conclusions: LMW forms of cyclin E differentiated ovarian carcinoma from benign and malignant mesothelial cells and were associated with increased protein expression using immunohistochemistry. The expression of LMW cyclin E forms was not associated with chemotherapy response, although it may be a marker of aggressive disease in patients with metastatic ovarian carcinoma., ((c) 2007 American Cancer Society.)

Objectives: To identify the relationships between key components of the proliferative and apoptotic pathways in bladder tumors., Methods: A tissue array of 88 bladder tumors was assembled. Immunohistochemical analyses were used to investigate the relationship between nine different parameters: stage, proliferation (Ki67), apoptosis (in situ DNA nick end labeling), the anti-apoptotic protein Bcl-2, tumor suppressors p53 and retinoblastoma protein (Rb), the Rb-related protein p130, cyclin E, and the cyclin-dependent kinase inhibitor p27. The protein expression in each tumor was reported as the percentage of positively staining cells., Results: The analysis focused on Stage 1 to 3 tumors. Analysis found that p53 expression increased progressively with stage, and Rb and p27 decreased with increasing stage. Overall, the cyclin E levels correlated with the proliferative index. Cyclin E levels were low in Stage 1 tumors and elevated in Stage 2 tumors, but were decreased in Stage 3 tumors. Multivariate analysis uncovered a correlation between cyclin E and proliferation (Ki67) and a weak correlation between p53 and Bcl-2 and between p27 and Rb. A strong correlation was found between the expression of p53 and p130, which was apparent in Stages 1 and 3, but not in Stage 2. Furthermore, high levels of p130 protein were detected primarily in the cytoplasm., Conclusions: These results suggest a novel p53/p130 axis in bladder tumors.

Purpose: The role of cyclin E as a predictive marker of response to chemotherapy remains unknown. We have previously shown that deregulation of cyclin E in an ovarian tumor cell line model enhances cyclin E-associated kinase activity and sensitizes tumor cells to cisplatinum. We hypothesized that cyclin E deregulation would predict for responsiveness to platinum-based regimens in ovarian cancer patients., Experimental Design: Patients who met the following criteria were retrospectively identified from the institutional tumor bank records: (a) high-grade ovarian epithelial malignancy, (b) stage III/stage IV disease, (c) optimally debulked, (d) completed platinum-based therapy. Tumor samples were analyzed for cyclin E, p21, and p27 by Western blot analysis and assessed for cyclin E-associated kinase activity., Results: Seventy-five patients, who met the study criteria, were identified. Cyclin E protein levels did not correlate with cyclin E-cdk2 kinase activity (Spearman's rho, 0.07; P = 0.58). Cyclin E-associated kinase activity was the only significant predictive marker for response to platinum-based therapy, with higher response rates seen in patients with higher levels of activity (P = 0.045). Cyclin E protein levels did not predict for platinum sensitivity (P = 0.20). In contrast, cyclin E protein levels, but not cyclin E-associated kinase activity, was a significant predictor for freedom from recurrence (P = 0.01 and P = 0.25, respectively)., Conclusions: Cyclin E overexpression and cyclin E-associated kinase activity have distinct roles in predicting for response to chemotherapy and outcome in ovarian cancer patients. These results suggest a compartmentalization of cyclin E functions in the oncogenic process.

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.

Amplification of 8p11-12 has been recurrently reported in sporadic breast cancer. These studies define a complex molecular structure with a set of minimal amplified regions, and different putative oncogenes that show a strong correlation between amplification and over-expression such as ZNF703/FLJ14299, SPFH2/C8orf2, BRF2 and RAB11FIP. However, none of these studies were carried out on familial breast malignancies. We have studied the incidence, molecular features and clinical value of this amplification in familial breast tumors associated with BRCA1, BRCA2 and non-BRCA1/2 gene mutations. We detected 9 out of 80 familial tumors with this amplicon by chromosomal comparative genomic hybridization. Next, we used a high-resolution comparative genomic hybridization array covering the 8p11-12 region to characterize this chromosomal region. This approach allowed us to define 2 cores of common amplification that largely overlap with those reported in sporadic tumors. Our findings confirm the molecular complexity of this chromosomal region and indicate that this genomic event is a common alteration in breast cancer, present not only in sporadic but also in familial tumors. Finally, we found correlation between the 8p11-12 amplification and proliferation (Ki-67) and cyclin E expression, which further proves in familial tumors the poor prognosis association previously reported in sporadic breast cancer.

Deregulation of the G1 cyclin, cyclin E, has been shown to be both the most powerful predictor of prognosis in early stage breast cancer as well as a significant determinant of tumor aggressiveness. It may also contribute to treatment failure due to chemoresistance. Because some form of cell cycle deregulation is present in all malignant cells,3 increasing understanding of these processes is starting to provide new opportunities to overcome the cells' resistance mechanisms. One particular form of cyclin E deregulation, the generation of hyperactive low molecular weight isoforms, is especially intriguing. Because only tumor cells contain the machinery necessary to generate these isoforms, they not only provide a mechanism of targeting critical cell cycle events, but their presence may also provide both a means of increased specificity for targeting malignant cells, as well as an objective measure of response. This review describes the mechanisms of resistance to commonly used systemic therapies for the treatment of breast cancer, with particular respect to the role of the cell cycle. The mechanisms and effects of the deregulation of cyclin E in breast cancer are reviewed and novel approaches to circumventing chemoresistance through abrogation of the malignant cell cycle are proposed.

The tubal fimbria is a common site of origin for early (tubal intraepithelial carcinoma or TIC) serous carcinomas in women with familial BRCA1 or 2 mutations (BRCA+). Somatic p53 tumour suppressor gene mutations in these tumours suggest a pathogenesis involving DNA damage, p53 mutation, and progressive loss of cell cycle control. We recently identified foci of strong p53 immunostaining-termed 'p53 signatures'-in benign tubal mucosa from BRCA+ women. To examine the relationship between p53 signatures and TIC, we compared location (fimbria vs ampulla), cell type (ciliated vs secretory), evidence of DNA damage, and p53 mutation status between the two entities. p53 signatures were equally common in non-neoplastic tubes from BRCA+ women and controls, but more frequently present (53%) and multifocal (67%) in fallopian tubes also containing TIC. Like prior studies of TIC, p53 signatures predominated in the fimbriae (80-100%) and targeted secretory cells (HMFG2 + /p73-), with evidence of DNA damage by co-localization of gamma-H2AX. Laser-capture microdissected and polymerase chain reaction-amplified DNA revealed reproducible p53 mutations in eight of 14 fully-analysed p53 signatures and all of the 12 TICs; TICs and their associated ovarian carcinomas shared identical mutations. In one case, a contiguous p53 signature and TIC shared the same mutation. Morphological intermediates between the two, with p53 mutations and moderate proliferative activity, were also seen. This is the first report of an early and distinct alteration in non-neoplastic upper genital tract mucosa that fulfils many requirements for a precursor to pelvic serous cancer. The p53 signature and its malignant counterpart (TIC) underline the significance of the fimbria, both as a candidate site for serous carcinogenesis and as a target for future research on the early detection and prevention of this disease., (Copyright (c) 2006 Pathological Society of Great Britain and Ireland.)

Although the human and the nonhuman primate fetal adrenal glands undergo a highly unique pattern of cortical zone-specific intrauterine growth and development, studies of the regulatory components of the cell cycle responsible for this growth have not been conducted. Therefore, the present study determined expression of the cell cycle regulators, cyclin D1 and cyclin E, and their cyclin-dependent kinases, Cdk2, Cdk4, and Cdk6, and Ki67 a marker of cell proliferation within the baboon fetal adrenal cortex during advancing stages of gestation. Fetal adrenal glands were obtained on days 60 (early), 100 (mid), and 160-170 (late) of gestation (term = 184 days). Mean (+/- s.e.) cyclin D1 mRNA levels, determined by RT-PCR and expressed relative to 18S rRNA, were similar at early (0.85 +/- 0.09) and mid (1.04 +/- 0.08) gestation, then decreased (P < 0.001, ANOVA) approximately 50% by late gestation (0.57 +/- 0.04). Cyclin E mRNA levels were also similar at early (2.03 +/- 0.07) and mid (1.63 +/- 0.31) gestation, and decreased by 70% (P < 0.001) in late gestation (0.53 +/- 0.09). Coinciding with the decrease in cyclin D1 and cyclin E, the percentage of Ki67 positive cells in the definitive zone decreased twofold (P < 0.01) between mid (28.2 +/- 3.6) and late (13.8 +/- 1.7) gestation. The cyclin D1 and cyclin E proteins, determined by immunocytochemistry, were expressed at high levels in the definitive zone of baboon fetal adrenal gland, where they decreased between mid- and late gestation. In contrast, immunocytochemical expression of the functionally important steroidogenic enzyme Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) became abundant in the definitive and transitional zones with advancing pregnancy. However, fetal adrenal Cdk2, Cdk4, and Cdk6 mRNA levels and protein immunoexpression were similar in the baboon fetal adrenal at early-, mid-, and late gestation. In summary, expression of cyclin D1, cyclin E, and Ki67 decreased, while 3beta-HSD expression increased, in the fetal adrenal cortex, particularly in the definitive zone, between mid- and late-baboon gestation. We propose that a developmental decline in cellular proliferation permits functional differentiation of fetal adrenal cortical cells, leading to increased production of steroid hormones important for placental estrogen synthesis and maturation of organ systems within the developing fetus.

Background: Cell cycle proteins and HIF-1alpha with downstream factors are often abberrantly expressed in (pre)neoplastic tissue., Methods: Paraffin-embedded specimens of inactive endometrium with TM (n=15), ovarian inclusion cysts (n=6), cervix with TM (tubal metaplasia) (n=3), Fallopian tubes (n=7), cycling endometrium (n=9) and a ciliated cell tumor of the ovary were stained for p16 and LhS28. 39 Endometrioid endometrial carcinomas and 5 serous endometrial carcinomas were stained for p16. Additionally, inactive endometrium (n=15) was immunohistochemically stained for p21, p27, p53, cyclin A, cyclin D1, cyclin E, HIF-1alpha, CAIX, Glut-1 and MIB-1., Results: A mosaic pattern of expression of p16 was seen throughout in all cases of endometrial TM (15/15), in 2/6 of the ovarian inclusion cysts with TM, in all (3/3) cervical TM and focal in 5/7 of Fallopian tube cases. Mosaic expression was also seen in a ciliated cell tumor of the ovary and in 18/39 of endometrioid endometrial carcinomas, and diffuse p16 expression was seen in 5/5 serous carcinomas. In comparison with normal endometrium, TM areas in the endometrium showed significantly increased expression of HIF-1alpha, cyclin E, p21 and cyclin A, and decreased expression of p27. Membranous expression of CAIX and Glut-1 was only seen in TM areas, pointing to functional HIF-1alpha., Conclusion: As p16 is consistently expressed in TM, less and only patchy expressed in the normal Fallopian tube, is paralleled by aberrant expression of cell cycle proteins, HIF-1alpha, CAIX and Glut-1 and resembles the pattern of p16 expression frequently seen in endometrial carcinomas, we propose endometrial TM to be a potential premalignant endometrial lesion.