Cyclin-dependent Protein Research Articles

Screening out molecular pathways and prognostic biomarkers of ultraviolet-mediated melanoma through computational techniques.

Ultraviolet radiation causes skin cancer, but the exact mechanism by which it occurs and the most effective methods of intervention to prevent it are yet unknown. For this purpose, our study will use bioinformatics and systems biology approaches to discover potential biomarkers of skin cancer for early diagnosis and prevention of disease with applicable clinical treatments. This study compared gene expression and protein levels in ultraviolet-mediated cultured keratinocytes and adjacent normal skin tissue using RNA sequencing data from the National Center for Biotechnology Information-Gene Expression Omnibus (NCBI-GEO) database. Then, pathway analysis was employed with a selection of hub genes from the protein-protein interaction (PPI) network and the survival and expression profiles. Finally, potential clinical biomarkers were validated by receiver operating characteristic (ROC) curve analysis. We identified 32 shared differentially expressed genes (DEGs) by analyzing three different subsets of the GSE85443 dataset. Skin cancer development is related to the control of several DEGs through cyclin-dependent protein serine/threonine kinase activity, cell cycle regulation, and activation of the NIMA kinase pathways. The cytoHubba plugin in Cytoscape identified 12 hub genes from PPI; among these 3 DEGs, namely, AURKA, CDK4, and PLK1 were significantly associated with survival (P < 0.05) and highly expressed in skin cancer tissues. For validation purposes, ROC curve analysis indicated two biomarkers: AURKA (area under the curve (AUC) value = 0.8) and PLK1 (AUC value = 0.7), which were in an acceptable range. Further translational research, including clinical experiments, teratogenicity tests, and in-vitro or in-vivo studies, will be performed to evaluate the expression of these identified biomarkers regarding the prognosis of skin cancer patients.

ACTL6A suppresses p21Cip1 tumor suppressor expression to maintain an aggressive mesothelioma cancer cell phenotype

Mesothelioma is a poor prognosis cancer of the mesothelial lining that develops in response to exposure to various agents including asbestos. Actin-Like Protein 6A (ACTL6A, BAF53a) is a SWI/SNF regulatory complex protein that is elevated in cancer cells and has been implicated as a driver of cancer cell survival and tumor formation. In the present study, we show that ACTL6A drives mesothelioma cancer cell proliferation, spheroid formation, invasion, and migration, and that these activities are markedly attenuated by ACTL6A knockdown. ACTL6A expression reduces the levels of the p21Cip1 cyclin-dependent kinase inhibitor and tumor suppressor protein. DNA binding studies show that ACTL6A interacts with Sp1 and p53 binding DNA response elements in the p21Cip1 gene promoter and that this is associated with reduced p21Cip1 promoter activity and p21Cip1 mRNA and protein levels. Moreover, ACTL6A suppression of p21Cip1 expression is required for maintenance of the aggressive mesothelioma cancer cell phenotype suggesting that p21Cip1 is a mediator of ACTL6A action. p53, a known inducer of p21Cip1 expression, is involved ACTL6A in regulation of p21Cip1 in some but not all mesothelioma cells. In addition, ACTL6A knockout markedly reduces tumor formation and this is associated with elevated tumor levels of p21Cip1. These findings suggest that ACTL6A suppresses p21Cip1 promoter activity to reduce p21Cip1 protein as a mechanism to maintain the aggressive mesothelioma cell phenotype.

TYLER, a fast method that accurately predicts cyclin-dependent proteins by using computation-based motifs and sequence-derived features

Cyclins and related cyclin-dependent kinases play vital roles in regulating the progression in the cell cycle. Understanding the intrinsic mechanisms of cyclins promises knowledge about cell uncontrolled proliferation and prevention of cancer cells. Therefore, accurate recognition of cyclins is important for the investigation of tumor cells and biomedical engineering. This study proposes a novel sequence-based predictor named TYLER (predicT cYcLin-dEpendent pRoteins) for addressing the long challenge problem of predicting cyclin-dependent proteins (CDPs). We use information theory to compute selectively enriched CDP-related motifs and build the motif-based model. For those proteins without sharing enriched motifs, we compute sequence-derived features and construct machine learning-based models. We optimize the weights of two different models to build a more accurate predictor. We estimate these two types of models by using 5-fold cross-validations on the TRAINING dataset. We prove that the combination of two models and optimization of the corresponding weights promises decent and robust results on both TRAINING and independent TEST dataset. The empirical test demonstrates that TYLER is robust predictor and statistically significantly better than current methods. The runtime assessment reveals TYLER is a high-throughput effective method. We use TYLER to make predictions on the human proteome, and use the results to hypothesize CDPs. The latest experimental verified CDPs and GO analysis proves that some of our novel predictions shall be potential CDPs. TYLER is implemented as a public user-friendly web server at http://www.inforstation.com/webservers/TYLER/. We share all data and source code that used in this research at https://github.com/biocomputinglab/TYLER.git.

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

ARHGEF10L Promotes Cervical Tumorigenesis via RhoA-Mediated Signaling.

Background Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, thus contributing to tumorigenesis. Our previous study demonstrated a strong association between the ARHGEF10L gene and the risk of cervical carcinoma. This study investigated the pathogenic role and mechanism of ARHGEF10L in cervical tumors. Methods The HeLa cell line, which was derived from cervical carcinoma, was transfected with ARHGEF10L-overexpressing plasmids or anti-ARHGEF10L siRNA. Cell counting kit-8 assays, wound-healing assays, and cell apoptosis assays were performed to investigate the effects of ARHGEF10L on cell activities. A Rho pull-down assay and RNA-sequencing analysis were performed to investigate the pathogenic pathway of ARHGEF10L involvement in cervical tumors. Results ARHGEF10L overexpression promoted cell proliferation and migration, reduced cell apoptosis, and induced epithelial-to-mesenchymal transition (EMT) via downregulation of E-cadherin and upregulation of N-cadherin and Slug in transfected HeLa cells. The overexpression of ARHGEF10L also upregulated GTP-RhoA, ROCK1, and phospho-ezrin/radixin/moesin (ERM) expression in HeLa cells. RNA-sequencing analysis detected altered transcription of 31 genes in HeLa cells with ARHGEF10L overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors.

CKS protein overexpression renders tumors susceptible to a chemotherapeutic strategy that protects normal tissues.

The cyclin-dependent kinase-interacting proteins Cyclin-dependent Kinase Subunit 1 and 2 (CKS1 and 2) are frequently overexpressed in cancer and linked to increased aggressiveness and poor prognoses. We previously showed that CKS protein overexpression overrides the replication stress checkpoint activated by oncoproteins. Since CKS overexpression and oncoprotein activation/overexpression are often observed in the same tumors, we have hypothesized that CKS-mediated checkpoint override could enhance the ability of premalignant cells experiencing oncoprotein-induced replication stress to expand. This tumor advantage, however, could represent a vulnerability to exploit therapeutically. Here, we first show in vitro that CKS protein overexpression selectively sensitizes tumor-derived cell lines to nucleoside analog-mediated toxicity under replication stress conditions. A treatment combination of the nucleoside analog gemcitabine and an agent that induces replication stress (thymidine or methotrexate) resulted in selective targeting of CKS protein-overexpressing tumor-derived cells while protecting proliferative cells with low CKS protein levels from gemcitabine toxicity. We validated this strategy in vivo and observed that Cks2-overexpressing mammary tumors in nude mice were selectively sensitized to gemcitabine under conditions of methotrexate-induced replication stress. These results suggest that high CKS expression might be useful as a biomarker to identify subgroups of cancer patients who might benefit from the described therapeutic approach.

The features of the expression of sex steroids hormone receptors, pro-inflammatory markers and cyclin-dependent kinase inhibitor protein p16ink4a in endometrium at external genital endometriosis

Background. A local inflammatory reaction in the endometrium caused by viral infection and associated with external genital endometriosis (EGE) can affect the implantation properties of endometrium and be one of the causes of infertility. The aim of the study was to investigate the expression of estrogen and progesterone receptors, pro-inflammatory markers and inhibitor of cyclin-dependent kinases p16ink4a in the endometrium of women with infertility associated with EGE. Materials and methods. The object of the study was the material taken from 133 patients aged 19-35 years. Inclusion criteria were the presence of external genital endometriosis confirmed by laparoscopy and the absence of hormone treatment for 6 months prior to this study. Endometrial biopsies were obtained on 19-24 day of the menstrual cycle. Histological and immunohistochemical studies of endometrial biopsies were performed using standard methods. The expression of estrogen (ER) and progesterone receptors (PgR), inflammatory markers (CD8+, CD20+, CD4+, CD138+) and inhibitor of cyclin-dependent kinase p16ink4a were examined by immunohistochemistry. The evaluation of the expression of studied markers was performed in the epithelium of the glands and endometrial stromal component separately, using semiquantitative method H-Score, as well as qualitative and quantitative methods in image analysis system “Morphology 5.0” (VideoTest, Russia). Statistical analysis was performed using statistical software (STATGRAPHICS v.6.0). Results. A significant decrease in the expression of ER and PgR in endometrial stromal component was noted on the background of the high incidence of chronic endometritis in patients with EGE. All patients of the main group had a positive expression of p16ink4a. Conclusions. The obtained results indicate a high incidence of disorders of eutopic endometrial secretory transformation in patients with infertility associated with external genital endometriosis. The high incidence of the virus associated with chronic endometritis and sensitivity violation of endometrial tissue to sex steroid hormones, such as progesterone, may be a major determinant of infertility of patients in this group.

Abstract C202: CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models

Abstract The group of transcriptional regulatory proteins known collectively as the super enhancer complex (SEC) coordinate the expression of entire genetic programs directing cell fate. The SEC is also important in driving cancer progression mediated by transcription of key oncogenes such as c-Myc and Bcl-2. The SEC requires the interaction and coordination of many proteins, including cyclin-dependent kinases (CDK), bromodomain proteins (BRD), histone deacetylases (HDAC), and histone methyltransferases (HMT). Each of these proteins are the focus of significant development efforts for the treatment of cancer. SEC-regulated transcription requires recruitment of CDK9/cyclin T1 from the 7SK RNA/Hexim1 inhibitory complex by BRD4 to transcriptional start sites. CDK9 then phosphorylates RNA polymerase II, releasing it from the start site leading to productive transcriptional elongation and gene expression. Considering the close association of CDK9 and BRD4, we hypothesized that the combination of CDK9 and BRD4 inhibitors would have synergistic effects in cancer cells. Alvocidib is a potent CDK9 inhibitor with validated clinical activity in AML from multiple Phase II studies in over 400 patients. Additionally, BRD4 inhibitors have demonstrated early promise in clinical studies with a focus on hematologic malignancies. However, we have found that CDK9 inhibitors, combined with bromodomain inhibitors, produced a synergistic effect by inhibiting the SEC more effectively than either of these compounds alone. Cell viability studies with various combinations resulted in an increase in potency. This was observed with alvocidib combined with JQ-1 (BRD4 inhibitor) in A549 lung cancer cells. Furthermore, the combination of alvocidib with JQ-1 completely abrogated SEC function, as measured by c-Myc or Mcl-1 expression through RT-qPCR. Similar results were achieved with other combinations of CDK9 and BRD4 inhibitors. These data, primarily focused on alvocidib and JQ-1, suggest a strong rationale for combining CDK9 and BRD4 inhibitors as a treatment strategy for multiple tumor types, including lung cancer. Furthermore, these findings may be more broadly applied to additional therapeutic targets in the SEC. These strategies yield synergistic effects at inhibiting SEC function and are highly active in tumor growth studies of cancer, in vivo. Clinical studies utilizing these combination strategies will explore this therapeutic approach. Citation Format: Ye Sol Lee, Wontak Kim, Katherine K. Soh, Peter Peterson, Clifford J. Whatcott, Adam Siddiqui-Jain, David J. Bearss, Steven L. Warner. CDK9 inhibition synergizes with BRD4 inhibitor-mediated super enhancer transcriptional repression in multiple preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C202.

Overexpression of transforming growth factor typeIII receptor restores TGF-β1 sensitivity in human tongue squamous cell carcinoma cells.

The transforming growth factor typeIII receptor (TβRIII), also known as β-glycan, is a multi-functional sensor that regulates growth, migration and apoptosis in most cancer cells. We hereby investigated the expression of TβRIII in clinical specimens of tongue squamous cell carcinoma (TSCC) and the underlying mechanism that TβRIII inhibits the growth of CAL-27 human oral squamous cells. The TSCC tissues showed a significant decrease in TβRIII protein expression as detected by immunohistochemistry (IHC) and western blot analysis. Transfection of TβRIII-containing plasmid DNA dramatically promoted TGF-β1 (10ng/ml)-induced decrease in cell viability, apoptosis and cell arrest at the G0-/G1-phase. Moreover, transient overexpression of TβRIII enhanced the TGF-β1-induced cyclin-dependent kinase inhibitor 2b (CDKN2b) and p38 protein activity, but did not affect the activities of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2 (JNK1/2) in CAL-27 cells. These results suggest overexpression of TβRIII receptor restored TGF-β1 sensitivity in CAL-27 cells, which may provide some new insights on exploiting this molecule therapeutically.

P58 * BRAIN TUMOUR CELLS CAN SURVIVE AND THRIVE IN THE HYPOXIC SOLID TUMOUR MICROENVIRONMENT

INTRODUCTION: Normal brain tissue is exposed to 5-8% oxygen whilst the average oxygen level within brain tumours is just 1% (hypoxia). Hypoxia is associated with the increased malignancy, invasiveness, and treatment resistance of brain tumours. Paradoxically, hypoxia has also been shown to induce cell cycle arrest in certain cells; however the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this project was to investigate the consequences of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an aggressive brain tumour with an extremely poor prognosis. METHOD: GBM cell lines were exposed to a range of oxygen tensions and their cell cycle distribution and survival was assessed using flow cytometry. Changes in the expression level of genes involved in regulation of the cell cycle were quantified by qRT-PCR. RESULTS: Results indicate that GBM proliferation and survival is unaffected by long-term exposure to pathophysiological levels of oxygen (1% O2). Cell cycle arrest and/or cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). Sensitivity to severe hypoxia was associated with increased expression of the cyclin-dependent kinase inhibitor protein p21. CONCLUSION: These findings demonstrate that the pro-tumorigenic effects of pathophysiological hypoxia are not balanced out by reduced cellular proliferation, strengthening the association between hypoxia and tumour malignancy. This helps to explain how a hypoxic tumour such as GBM can be incredibly fast growing and aggressive.

Abstract 2121: Resveratrol regulates phospho-serine 81 androgen receptor and its stability to inhibit growth of prostate cancer cells

Abstract Resveratrol, a polyphenol, has been reported as a cell growth inhibitor of various types of cancer. The molecular mechanisms of resveratrol are involved in different cellular signal transductions such as anti-inflammation and anti-oxidation. Resveratrol inhibits growth of prostate cancer cells through regulating cyclin-dependent kinases, anti-apoptotic proteins, and pro-apoptotic proteins. Notably, resveratrol may down-regulate androgen receptor (AR) and its downstream gene to inhibit prostate cancer cell growth. In previous study, serine 81 phosphorylation of AR increases its protein stability and transcriptional activity, which promotes prostate cancer cell growth. The aim of this study is to investigate whether AR serine 81 phosphorylation would be a target of resveratrol in prostate cancer cells. Our data indicate that resveratrol inhibited the growth of prostate cancer cell line, LNCaP, with the decrease of AR phospho-serine 81 levels. Moreover, AR protein stability was declined by resveratrol without affecting AR mRNA expression. Indeed, resveratrol inhibited AR transcriptional activity in results of AR reporter assay and PSA level in LNCaP cells. In summary, the results suggest that resveratrol might inhibit growth of LNCaP cells, at least, via decreasing phospho-serine 81 of AR, AR protein stability, and AR transcriptional activity. Citation Format: Yu-Ting Peng, Tzu-Yin Chen, Mei-Chih Chen, Eugene Lin, Ho Lin. Resveratrol regulates phospho-serine 81 androgen receptor and its stability to inhibit growth of prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2121. doi:10.1158/1538-7445.AM2014-2121

Effects of melittin on the cell cycle of bladder cancer cell line T24 and its mechanism

Objective To investigate the effects of melittin on the cell cycle of bladder cancer cell line T24 and the related regulatory mechanisms.Methods In vitro cultured bladder cancer T24 cells were treated with melittin (0,7.5,10.0,and 12.5 mg/L).Flow cytometry was applied to examine the cell cycle of T24 cells.Western blotting method was applied to detect the protein expression of Cyclin A,cyclindependent kinase 2 (CDK2),CDK inhibitor p21 cip1 and cyclin dependent kinase inhibitor protein 27 (p27kip1).Results Corresponding to different concentration groups of melittin,the proportion of T24 cells in S phase was (25.73 ±2.11)％,(31.57 ±1.91)％,(45.37±0.74)％,and (53.67±1.12)％,showing significant difference between melittin-treated groups and control group (P ＜ 0.01).The protein expression of p21cip1 and p27kip1 in melittin-treated T24 cells was increased (p21cip1:0.156 1 ±0.011 6,0.209 2 ±0.010 8,0.287 9 ±0.012 9,0.351 8 ±0.013 0;p27kip1:0.250 3 ±0.012 3,0.342 1 ±0.012 3,0.400 3 ±0.009 9,0.469 2 ±0.013 0),while that of Cyclin A and CDK2 significantly decreased as compared with control group (Cyclin A:0.950 7 ± 0.017 8,0.870 6 ± 0.011 9,0.806 5 ± 0.012 0,0.726 7 ± 0.018 4;CDK2:0.965 4 ±0.014 5,0.868 3 ±0.015 3,0.755 4 ±0.008 5,0.652 6 ±0.018 3,P ＜0.01).Conclusion Melittin can cause S phase arrest of T24 cells,which may be associated with the down-regulation of Cyclin A and CDK2 expression while up-regulation of p21cip1 and p27kip1. Key words: Bladder neoplasm; Melittin; Cell cycle

Knockdown of CDK2AP1 by RNA interference inhibits cell growth and tumorigenesis of human glioma

Previous reports support the role of cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) as a tumor suppressor that functions as a key player in cell cycle regulation. Although the misadjustment of CDK2AP1 has been revealed in several types of human malignancies, the functional role of CDK2AP1 in human glioma remains unknown. The present study was undertaken to investigate the effects of CDK2AP1 knockdown by RNA interference (RNAi) on glioma cell growth and tumorigenesis. We employed lentivirus-mediated RNAi to down-regulate CDK2AP1 expression in U251 and U373 cells. Knockdown of CDK2AP1 resulted in a significant reduction in U251 and U373 cell proliferation, as determined by MTT and colony formation assays. Cell cycle analysis showed CDK2AP1 silencing caused U251 cells arrest in G0/G1 phase, especially in the sub-G1 phase representing apoptotic cells. In vivo tumorigenesis was assessed using xenograft formation and CDK2AP1 depletion remarkably inhibited glioma growth and tumorigenesis. Taken together, these results suggest that CDK2AP1 siRNA may have an anti-tumorigenic effect on human glioma.

Dynamic cyclin profiles as a tool to segregate the cell cycle

Background and novelty Mammalian cells growth, productivity and cell death are highly regulated and coordinated processes. The cell cycle is at the centre of cellular control and should play a key role in determining optimization strategies towards improving productivity [1]. Specifically, cell productivity is cell cycle, cell-line and promoter dependant [2]. The cyclins are key regulators that activate their partner cyclin-dependent kinases (CDKs) and target specific proteins driving the cell cycle. To our knowledge, there is no information on cyclin phase-dependent expression profiles of industrial relevant mammalian cell lines. We use the cyclin profiles as a tool to identify and quantify the landmarks of the cell cycle and implement a modelling approach to describe the bioprocess. Hereby, we introduce two possible experimental approaches to obtain such dynamic cyclin profiles. Experimental approach Cyclin expression (cyclin E - G1 class and cyclin B - G2 class) was studied in GS-NS0 batch cultures by flow cytometry. Two set of experiments were performed: a) culture of cells under perturbed (cell arrest) and unperturbed growth (control run) and b) culture of cells for DNA labelling to perform a proliferation assay as well as a non-exposed cells (control run). The static profiles were obtained by direct cyclin staining and the dynamic profiles were reconstructed by either a) tracking a partially synchronized population or b) combining the timings from proliferation assays with the static profiles. Result discussion Both cyclins showed a clear cell cycle phase-specific pattern (cyclin E was 10% higher at G1 and cyclin B was 40% higher at G2). These results were consistent among all the different culture conditions and were inferred from the static cyclin profiles. After the arrest release the dynamic cyclin profiles can be directly reconstructed by plotting the relevant cyclin content from the partially synchronized moving population traversing the cycle. An advantage of this approach is a clear view of the cyclin accumulation and transition threshold levels. However, this approach requires testing using different arrest agents, exposure levels and timings, which could have an effect on the cell behaviour. A second approach included an indirect dynamic cyclin profile reconstruction by combining the acquired proliferation times for different cell cycle phases (e.g. G1/G0, G2/M) with the static cyclin profiles. If the static cyclin profiles are considered as the most representative cyclin values (and near to the transition threshold level), it is possible to reconstruct the dynamic profile by linking the threshold values with the cycling times (from the proliferation assay). The advantage of such approach is the ability to formulate different dynamic cyclin profiles such as constant functions, piece-wise linear functions or more elaborated profiles. However, implementation of such an approach requires the tuning of the proliferation assay and the frequency of sampling since it will affect the quality of the assay. The two approaches showed comparable results both for the static cyclin profiles (also when compared to the control runs) and the dynamic cyclin profiles.

Abstract 080: Eya4 Induces Hypertrophy Via Regulation Of p27kip1

Introduction: E193, a truncating mutation in the transcription cofactor Eyes absent 4 (Eya4) causes hearing impairment followed by heart failure. Here we identified the Eya4 dependent molecular mechanisms leading to the cardiac phenotype in the E193 mutation. Methods and Results: First we showed in vitro that the cyclin-dependent kinase inhibitor protein p27kip1 is a direct target of Eya4/Six1 and is suppressed upon Eya4 overexpression, whereas E193 has a dominant negative effect, releasing Eya4 mediated suppression of p27. We next generated transgenic mice with cardiac specific constitutive overexpression of full-length Eya4 or the mutant form E193. While E193 transgenic mice developed age-dependent DCM, Eya4 mice displayed cardiac hypertrophy already under basal conditions as judged by increases in heart weight and cardiomyocyte cross-sectional areas along with increases in myocardial dimension and mass. These two distinct cardiac phenotypes were even more aggravated upon pressure overload suggesting Eya4 is a regulator of cardiac hypertrophy. We also observed that the activity of Casein Kinase 2-α and the phosphorylation status of HDAC2 were significantly upregulated in the Eya4 transgenic mice, while they were significantly reduced in E193 mice, under baseline conditions and pressure overload. We were also able to identify a new human mutation (E215) with a phenotype comparable to the one seen in E193 patients. Conclusion: Our results implicate that Eya4/Six1 regulates cardiac hypertrophic reactions via p27/CK2-α/HDAC2 and indicate that truncating mutations in Eya4 interfere with this newly established signalling pathway.

Expression,mutation and promoter methylation of cyclin dependent kinase inhibitor protein 27 gene in gastric carcinoma

Objective To explore the relationship between the expression,mutation and promoter methylation of cyclin dependent kinase inhibitor protein 27 (P27KiP1) gene in gastric carcinoma.Methods Expression of P27KiP1 was investigated by using SP immunohistochemical staining,mutation was assayed by single-strand conformation polymorphism analysis (PCR-SSCP) and Promoter Methylation was assayed by methylation-specific PCR (MSP) in 97 cases of carcinoma,25 cases normal gastric tissues.Results The expression of P27KiP1 in normal gastric cases was higher than its in gastric carcinoma tissues (x2 =31.02,P ＜ 0.01).The expression of P27KiP1 in early gastric cases was also higher than its in advanced gastric carcinoma tissues (x2 =13.55,P ＜0.01).mutation and promoter methylation chould not be assayed in normal gastric tissues aud they were chould be assayed in carcinoma tissues,but there were no difference between early and advanced carcinoma tissues (x2 =0.13,0.32. P ＞ 0.05).There was a inverse association between expression of P27KiP1 and Promoter Methylation,mution (x2 =17.89,P ＜ 0.01).Conclusion The expression of P27KiP1 was associated with the occurrence and development of gastric carcinoma.P27KiP1 gene mutation and promoter methylation maybe occurred in early stage of this carcinoma and they might lead to the down or loss exprssion of P27KiP1 of gastric carcinoma. Key words: Gastric carcinoma; Cyclin dependent kinase inhibitor protein 27; Mutation; Promoter Methylation

Expression of S-phase kinase-associated protein-2 in rat injured aorta and its significance in neointimal hyperplasia

Objective To investigate the change of S-phase kinase-associated protein-2 (Skp2) in the aorta of rats after balloon injury and its effect on neointimal hyperplasia.Methods Fisty-six SD male rats were randomly divided into two groups:sham operated group (n =8),and balloon injured group (including:2-,4-,7-,10-,14-and 28-day subgroups,n =8 each).The model of rat aorta injury was established.The vascular tissues were harvested on the postoperative day (POD) 2,4,7,10,14 and 28 respectively and examined histomorphologically.Inmunohistochemistry streptavid-in-peroxidase (SP) method was used to detect the differential expression level of proliferating cell nuclear antigen (PCNA),Skp2 and cyclin dependent kinase inhibitor protein 27 (p27KiP1) at each time point.The expression levels of Skp2 and p27Kip1 mRNA were detected by using real-time quantitative polymerase chain reaction (Real-time PCR).Results (1) The normal artery wall didn' t express PCNA,hut PCNA was detected on POD 2.The amount of positive cells was increased and reached a peak on POD 7 [(38.46 ± 2.01),P ＜ 0.05],then gradually decreased; (2) The expression of Skp2 was very low in uninjured aorta,and increased on POD 2 [(27.90 ±2.37)] and peaked on POD 10 [(47.36 ±2.37),P ＜ 0.05].It was declined on POD 14 and further decreased on POD 28; (3) p27Kip1 was expressed in normal aorta (27.82 ±3.26) and significantly reduced on POD 2 [(14.71 ± 1.37)],significantly increased on POD 10 [(32.06 ± 2.57),P ＜ 0.05] and remained high until POD 28; (4) The expression of PCNA was temporally associated with expression of Skp2 (r =0.892,P ＜ 0.01) ; (5) The Real-time PCR results showed that the expression of Skp2 mRNA was extremely low in normal aorta [(1.28 ± 1.11)],increased on POD 2 and reached the peak on POD 10 [(10.49 ±3.17),P＜ 0.05],then decreased turther on POD 28.The expression of p27Kip1 mRNA could be detected in normal aorta (1.14 ±0.64),decreased on POD 4 (P ＜ 0.05),and remained low from POD 7 to 10,then significantly increased on POD 14 [(1.64 ±0.45),P ＜ 0.05] and returned to normal level on POD 28 (P ＞ 0.05).Conclusion Skp2 was one of the important promoting factors that induced VSMC proliferation and subsequent neointimal hyperplasia. Key words: S-phase kinase-associated protein 2; Artery injury; Vascular smooth muscle cell; Neointimal hyperplasia

Abstract 1655: Synthetic lethal interaction of CDK inhibition and autophagy inhibition in human solid cancer cell lines.

Abstract Autophagy is a stress-induced cellular reaction in cancer cells. Recent studies showed that cyclin dependent kinase inhibitor proteins, such as p27 or p16, also induced cytoprotective autophagy in cancer cells. However, it is not clear whether small molecule CDK inhibitors also induce autophagy in solid cancer cells. In this study, we revealed that CDK4 inhibitor and a broad-range CDK inhibitor (flavopiridol) induced autophagy in some solid cancer cell lines, but not all. The autophagy induced by CDK4 inhibitors was observed in BT474, MDA-MB435S, SKBr3 (derived from breast cancer), A431 (derived from lung cancer) and SW480 (derived from colorectal cancer), While it was not observed in MCF7, MDA-MB231 (derived from breast cancer), NCI-N87 (derived from gastric cancer) and KMST-6 (derived from fibroblast). In the cell lines which were induced autophagy by CDK inhibitors, the combination of a CDK4 inhibitor and an autophagy inhibition, by either chloroquine or knockdown of ATG5 or BECN1, induced apoptosis. However it did not induce apoptosis in the cell lines which were not induced autophagy by CDK4 inhibitor. From these results, the autophagy induced by CDK4 inhibitor mimic the stress-induced autophagy in some solid cancer cell lines. The combination of a small-molecule CDK inhibitor and an autophagy inhibitor is a synthetic lethal interaction and could become a new antitumor strategy in solid tumors which are induced cytoprotective autophagy by CDK inhibitors. Citation Format: Yoshinari Okada, Shunsuke Kato, Yasuhiro Sakamoto, Takayuki Oishi, Chikashi Ishioka. Synthetic lethal interaction of CDK inhibition and autophagy inhibition in human solid cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1655. doi:10.1158/1538-7445.AM2013-1655

The Diagnostic Value of p27 in Comparison to p57 in Differentiation Between Different Gestational Trophoblastic Diseases

The histologic features that permit the identification of complete mole (CM) and partial mole (PM) as well as hydropic abortion (HA) may be overlapping. The cyclin-dependent kinase inhibitor p57Kip2 protein (p57) has been included among the paternally imprinted genes in humans that commonly used for diagnosis of CM. P27 is one of the cell-cycle controlling molecules that may be involved in the proliferation, differentiation and oncogenesis of trophoblastic cells. The current study tried to test the diagnostic validity of several histopathological parameters together with p57 and p27 immunostaining in differentiation between different gestational trophoblastic diseases. The current study was carried out on 13 cases product of conception, 13 cases PM, 25 cases CM and 8 cases choriocarcinoma. Maximal villous diameter at 1.5 mm cut-off point was found to be the most reliable factor in discrimination between PM and product of conception followed by presence of villous cistern, p57 expression by extravillous cytotrophoblasts at 1.5% cut-off point and p27 expression by villous cytotrophoblasts at 25% cut-off point. P27 expression by stromal cells and total trophoblastic population at 7.5% cut-off point could discriminate between PM and CM. P57 and p27 are co-parallely expressed in non-molar as well as partial molar gestations, but they did not show this coordination in choriocarcinoma. This study demonstrated diagnostic values for several cut-off points for p57 and p27 in discrimination between different categories of gestational trophoblastic diseases. The co-expression of p27 and p57 by extravillous cytotrophoblasts and their positive correlation in non-molar gestations may indicate its suppressive role on the proliferation of these cells to provide them the capacity for differentiation and invasion.

Solution NMR Structure of Cyclin-dependent kinase 2-associated protein 2 (CDK2AP2, DOC-1R) from Homo sapiens, Northeast Structural Genomics Consortium (NESG) Target HR8910C

Solution NMR Structure of Cyclin-dependent kinase 2-associated protein 2 (CDK2AP2, DOC-1R) from Homo sapiens, Northeast Structural Genomics Consortium (NESG) Target HR8910C