Computational analysis of Cyclin D1 gene SNPs and association with breast cancer.

CCND1 encodes for Cyclin D1 protein and single-nucleotide polymorphisms (SNPs) can modulate its activity. In the present study, the impact of CCND1 SNPs on structure and/or function of Cyclin D1 protein using in silico tools was investigated. Our analysis revealed only one splice site SNP (c.1988+5G

RNA-Binding RING E3-Ligase DZIP3/hRUL138 Stabilizes Cyclin D1 to Drive Cell-Cycle and Cancer Progression.

DZIP3/hRUL138 is a poorly characterized RNA-binding RING E3-ubiquitin ligase with functions in embryonic development. Here we demonstrate that DZIP3 is a crucial driver of cancer cell growth, migration, and invasion. In mice and zebrafish cancer models, DZIP3 promoted tumor growth and metastasis. In line with these results, DZIP3 was frequently overexpressed in several cancer types. Depletion of DZIP3 from cells resulted in reduced expression of Cyclin D1 and a subsequent G1 arrest and defect in cell growth. Mechanistically, DZIP3 utilized its two different domains to interact and stabilize Cyclin D1 both at mRNA and protein levels. Using an RNA-binding lysine-rich region, DZIP3 interacted with the AU-rich region in 3' untranslated region of Cyclin D1 mRNA and stabilized it. Using a RING E3-ligase domain, DZIP3 interacted and increased K63-linked ubiquitination of Cyclin D1 protein to stabilize it. Remarkably, DZIP3 interacted with, ubiquitinated, and stabilized Cyclin D1 predominantly in the G1 phase of the cell cycle, where it is needed for cell-cycle progression. In agreement with this, a strong positive correlation of mRNA expression between DZIP3 and Cyclin D1 in different cancer types was observed. Additionally, DZIP3 regulated several cell cycle proteins by modulating the Cyclin D1-E2F axes. Taken together, this study demonstrates for the first time that DZIP3 uses a unique two-pronged mechanism in its stabilization of Cyclin D1 to drive cell-cycle and cancer progression. SIGNIFICANCE: These findings show that DZIP3 is a novel driver of cell-cycle and cancer progression via its control of Cyclin D1 mRNA and protein stability in a cell-cycle phase-dependent manner. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/2/315/F1.large.jpg.

Integrated bioinformatics analysis to decipher molecular mechanism of compound Kushen injection for esophageal cancer by combining WGCNA with network pharmacology.

Compound Kushen injection (CKI), a medicine in widespread clinical use in China, has proven therapeutic effects on cancer. However, few molecular mechanism analyses have been carried out. To address this problem, bioinformatics approaches combining weighted gene co-expression network analysis with network pharmacology methods were undertaken to elucidate the underlying molecular mechanisms of CKI in the treatment of esophageal cancer (ESCA). First, the key gene modules related to the clinical traits of ESCA were analysed by WCGNA. Based on the results, the hub genes related to CKI treatment for ESCA were explored through network pharmacology. Molecular docking simulation was performed to recognize the binding activity of hub genes with CKI compounds. The results showed that the potential hub targets, including EGFR, ErbB2, CCND1 and IGF1R, are therapeutic targets of CKI for the treatment of ESCA. Moreover, these targets were significantly enriched in many pathways related to cancer and signalling pathways, such as the PI3K-Akt signalling pathway and ErbB signalling pathway. In conclusion, this research partially highlighted the molecular mechanism of CKI in the treatment of ESCA, offering great potential in the identification of the effective compounds in CKI and biomarkers for ESCA treatment.

The double dealing of cyclin D1.

The cell cycle is tightly regulated by cyclins and their catalytic moieties, the cyclin-dependent kinases (CDKs). Cyclin D1, in association with CDK4/6, acts as a mitogenic sensor and integrates extracellular mitogenic signals and cell cycle progression. When deregulated (overexpressed, accumulated, inappropriately located), cyclin D1 becomes an oncogene and is recognized as a driver of solid tumors and hemopathies. Recent studies on the oncogenic roles of cyclin D1 reported non-canonical functions dependent on the partners of cyclin D1 and its location within tumor cells or tissues. Support for these new functions was provided by various mouse models of oncogenesis. Finally, proteomic and transcriptomic data identified complex cyclin D1 networks. This review focuses on these aspects of cyclin D1 pathophysiology, which may be crucial for targeted therapy.Abbreviations: aa, amino acid; AR, androgen receptor; ATM, ataxia telangectasia mutant; ATR, ATM and Rad3-related; CDK, cyclin-dependent kinase; ChREBP, carbohydrate response element binding protein; CIP, CDK-interacting protein; CHK1/2, checkpoint kinase 1/2; CKI, CDK inhibitor; DDR, DNA damage response; DMP1, cyclin D-binding myb-like protein; DSB, double-strand DNA break; DNA-PK, DNA-dependent protein kinase; ER, estrogen receptor; FASN, fatty acid synthase; GSK3ß, glycogen synthase-3ß; HAT, histone acetyltransferase; HDAC, histone deacetylase; HK2, hexokinase 2; HNF4α, and hepatocyte nuclear factor 4α; HR, homologous recombination; IR, ionizing radiation; KIP, kinase inhibitory protein; MCL, mantle cell lymphoma; NHEJ, non-homologous end-joining; PCAF, p300/CREB binding-associated protein; PGC1α, PPARγ co-activator 1α; PEST, proline-glutamic acid-serine-threonine, PK, pyruvate kinase; PPAR, peroxisome proliferator-activated receptor; RB1, retinoblastoma protein; ROS, reactive oxygen species; SRC, steroid receptor coactivator; STAT, signal transducer and activator of transcription; TGFß, transforming growth factor ß; UPS, ubiquitin-proteasome system; USP22, ubiquitin-specific peptidase 22; XPO1 (or CRM1) exportin 1.

p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition.

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors.

Downregulation of TCEAL7 expression induces CCND1 expression in non-small cell lung cancer.

Transcription Elongation Factor A-like 7 (TCEAL7) was first reported as a candidate tumor suppressor gene because of its inactivation in ovarian cancer as a result of promoter methylation. Down-regulation of the TCEAL7 gene expression was also associated with other cancers such as endometrial, breast, brain, prostate, gastric cancers, glioblastoma and linked to tumor phenotypes and clinical outcomes. However, there is no report in the literature investigating the role of TCEAL7 in non-small cell lung cancer. Cyclin D1 is an important molecule in the transition from G1 to S phase of the cell cycle, and is frequently deregulated in cancers. Cylin D1 (CCND1) gene is amplified or overexpressed in a variety of tumors. In our previous study we reported that CCND1 over-expression was not associated with amplification in non-small cell lung cancer. Recently, it has been reported that TCEAL7 regulates CCND1 expression through myc-binding E-box sequences. The aim of this study was to investigate the expression of TCEAL7 gene in non-small cell lung cancer and to determine its effect on the CCND1 expression level. For this purpose, expression levels of TCEAL7 and CCND1 genes were investigated in 50 patients with non-small cell lung cancer by quantitative real time polymerase chain reaction (qRT-PCR). TCEAL7 was under-expressed (68%) in non-small cell lung cancer tumor tissues while CCND1 was over-expressed (42%). The TCEAL7 levels negatively correlated with increased CCND1 expression (p = 0.002).

Biodegradable Anisotropic Microparticles for Stepwise Cell Adhesion and Preparation of Janus Cell Microparticles.

The biomimetic anisotropic particles have different physicochemical properties on the opposite two sides, enabling diverse applications in emulsion, photonic display, and diagnosis. However, the traditional anisotropic particles have a very small size, ranging from submicrons to a few microns. The design and fabrication of anisotropic macron-sized particles with new structures and properties is still challenging. In this study, anisotropic polycaprolactone (PCL) microparticles well separated with each other were prepared by crystallization from the dilute PCL solution in a porous 3D gelatin template. They had fuzzy and smooth surfaces on each side, and a size as large as 70 µm. The fuzzy surface of the particle adsorbed significantly larger amount of proteins, and was more cell-attractive regardless of the cell types. The particles showed stronger affinity toward fibroblasts over hepatocytes, which paved a new way for cell isolation merely based on the surface morphology. After a successive seeding process, Janus cell microparticles with fibroblasts and endothelial cells (ECs) on each side were designed and obtained by making use of the anisotropic surface morphology, which showed significant difference in EC functions in terms of prostacyclin (PGl2) secretion, demonstrating the unique and appealing functions of this type of anisotropic microspheres.

Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana.

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants.

Structural basis of the phosphorylation-independent recognition of cyclin D1 by the SCFFBXO31 ubiquitin ligase.

Ubiquitin-dependent proteolysis of cyclin D1 is associated with normal and tumor cell proliferation and survival. The SCFFBXO31 (Skp1-Cul1-Rbx1-FBXO31) ubiquitin ligase complex mediates genotoxic stress-induced cyclin D1 degradation. Previous studies have suggested that cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and subsequent proteolysis in the cytoplasm. Here we present the crystal structures of the Skp1-FBXO31 complex alone and bound to a phosphorylated cyclin D1 C-terminal peptide. FBXO31 possesses a unique substrate-binding domain consisting of two ß-barrel motifs, whereas cyclin D1 binds to FBXO31 by tucking its free C-terminal carboxylate tail into an open cavity of the C-terminal FBXO31 ß-barrel. Biophysical and functional studies demonstrate that SCFFBXO31 is capable of recruiting and ubiquitinating cyclin D1 in a phosphorylation-independent manner. Our findings provide a conceptual framework for understanding the substrate specificity of the F-box protein FBXO31 and the mechanism of FBXO31-regulated cyclin D1 protein turnover.

Homeobox Transcription Factor NKX2-1 Promotes Cyclin D1 Transcription in Lung Adenocarcinomas.

The known oncogene cyclin D1 (CCND1) participates in progression of the cell cycle from G1 to S-phase. Expression of cyclin D1 is frequently promoted in multiple human cancers including non-small cell lung cancer (NSCLC). However, a relationship between cyclin D1 expression and the prognosis of NSCLC has not been confirmed. NKX2-1 is a homeobox transcription factor involved in pulmonary development as a differentiation-promoting factor. In NSCLC, it acts as a metastasis suppressor and correlates with a good prognosis. Here, NKX2-1-binding motifs were identified in the cyclin D1 promoter, but it has not been clarified whether NKX2-1 is involved in cyclin D1 expression in NSCLC. To shed light on this issue, endogenous NKX2-1 was depleted in NSCLC cell lines, which resulted in decreased cyclin D1 mRNA and protein. In contrast, forced overexpression of NKX2-1 increased cyclin D1 levels. Moreover, NKX2-1 directly bound to the cyclin D1 promoter and enhanced its activity. Finally, using human NSCLC clinical specimens, it was determined that both NKX2-1 protein and mRNA were significantly correlated with cyclin D1 expression status in adenocarcinomas. These results indicate that NKX2-1 directly and positively regulates transcription of cyclin D1 Finally, expression of NKX2-1, but not cyclin D1, was significantly associated with metastatic incidence as an independent good prognostic factor of adenocarcinoma.Implications: NKX2-1-expressing adenocarcinomas, whereas NKX2-1 promoted cyclin D1 expression, may show good prognosis features by the metastasis inhibition potency of NKX2-1 regardless cyclin D1 expression. Mol Cancer Res; 15(10); 1388-97. ©2017 AACR.

CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells.

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.

Conformational Equilibrium of CDK/Cyclin Complexes by Molecular Dynamics with Excited Normal Modes.

Cyclin-dependent kinases (CDKs) and their associated regulatory cyclins are central for timely regulation of cell-cycle progression. They constitute attractive pharmacological targets for development of anticancer therapeutics, since they are frequently deregulated in human cancers and contribute to sustained, uncontrolled tumor proliferation. Characterization of their structural/dynamic features is essential to gain in-depth insight into structure-activity relationships. In addition, the identification of druggable pockets or key intermediate conformations yields potential targets for the development of novel classes of inhibitors. Structural studies of CDK2/cyclin A have provided a wealth of information concerning monomeric/heterodimeric forms of this kinase. There is, however, much less structural information for other CDK/cyclin complexes, including CDK4/cyclin D1, which displays an alternative (open) position of the cyclin partner relative to CDK, contrasting with the closed CDK2/cyclin A conformation. In this study, we carried out normal-mode analysis and enhanced sampling simulations with our recently developed method, molecular dynamics with excited normal modes, to understand the conformational equilibrium on these complexes. Interestingly, the lowest-frequency normal mode computed for each complex described the transition between the open and closed conformations. Exploration of these motions with an explicit-solvent representation using molecular dynamics with excited normal modes confirmed that the closed conformation is the most stable for the CDK2/cyclin A complex, in agreement with their experimentally available structures. On the other hand, we clearly show that an open↔closed equilibrium may exist in CDK4/cyclin D1, with closed conformations resembling that captured for CDK2/cyclin A. Such conformational preferences may result from the distinct distributions of frustrated contacts in each complex. Using the same approach, the putative roles of the Thr(160) phosphoryl group and the T-loop conformation were investigated. These results provide a dynamic view of CDKs revealing intermediate conformations not yet characterized for CDK members other than CDK2, which will be useful for the design of inhibitors targeting critical conformational transitions.

Marine steroids as potential anticancer drug candidates: In silico investigation in search of inhibitors of Bcl-2 and CDK-4/Cyclin D1.

Star fishes (Asteroidea) are rich in polar steroids with diverse structural characteristics. The structural modifications of star fish steroids occur at 3ß, 4ß, 5α, 6α (or ß), 7α (or ß), 8, 15α (or ß) and 16ß positions of the steroidal nucleus and in the side chain. Widely found polar steroids in starfishes include polyhydroxysteroids, steroidal sulfates, glycosides, steroid oligoglycosides etc. Bioactivity of these steroids is less studied; only a few reports like antibacterial, cytotoxic activity etc. are available. In continuation of our search for bioactive molecules from natural sources, we undertook in silico screening of steroids from star fishes against Bcl-2 and CDK-4/Cyclin D1 - two important targets of progression and proliferation of cancer cells. We have screened 182 natural steroids from star fishes occurring in different parts of the world and their 282 soft-derivatives by in silico methods. Their physico-chemical properties, drug-likeliness, binding potential with the selected targets, ADMET (absorption, distribution, metabolism, toxicity) were predicted. Further, the results were compared with those of existing steroidal and non steroidal drugs and inhibitors of Bcl-2 and CDK-4/Cyclin D1. The results are promising and unveil that some of these steroids can be potent leads for cancer treatments.

Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide (CA224), a nonplanar analogue of fascaplysin, inhibits Cdk4 and tubulin polymerization: evaluation of in vitro and in vivo anticancer activity.

Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a nonplanar analogue of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in vitro. Compound 1 blocks the growth of cancer cells at G0/G1 phase of the cell cycle. It also blocks the cell cycle at G2/M phase, which is explained by the fact that it inhibits tubulin polymerization. Additionally, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21, and p27 proteins together with downregulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis of SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner, of lung cancer cells. It is efficacious at 1/10th of the MTD against human tumors derived from HCT-116 and NCI-H460 cells in SCID mouse models. The promising efficacy of compound 1 in human xenograft models as well as its excellent therapeutic window indicates its potential for clinical development.

A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3ß to phosphorylate cyclin D1 at Thr(286), not Thr(288).

DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G1-S-phase transition, is phosphorylated on Thr(286) by GSK3ß (glycogen synthase kinase 3ß) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr(288) rather than Thr(286). Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr(286), not Thr(288), in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr(286) phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3ß inhibitors or GSK3ß RNAi. DYRK1B expression causes a G1-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21CIP1 (21 kDa CDK-interacting protein 1) and p27KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr(286)-CCND1 kinase that acts independently of GSK3ß to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.

Assembly, activation, and substrate specificity of cyclin D1/Cdk2 complexes.

Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.

Comparative modeling and docking studies of p16ink4/cyclin D1/Rb pathway genes in lung cancer revealed functionally interactive residue of RB1 and its functional partner E2F1.

BACKGROUND: Lung cancer is the major cause of mortality worldwide. Major signalling pathways that could play significant role in lung cancer therapy include (1) Growth promoting pathways (Epidermal Growth Factor Receptor/Ras/ PhosphatidylInositol 3-Kinase) (2) Growth inhibitory pathways (p53/Rb/P14ARF, STK11) (3) Apoptotic pathways (Bcl-2/Bax/Fas/FasL). Insilico strategy was implemented to solve the mystery behind selected lung cancer pathway by applying comparative modeling and molecular docking studies. RESULTS: YASARA [v 12.4.1] was utilized to predict structural models of P16-INK4 and RB1 genes using template 4ELJ-A and 1MX6-B respectively. WHAT CHECK evaluation tool demonstrated overall quality of predicted P16-INK4 and RB1 with Z-score of -0.132 and -0.007 respectively which showed a strong indication of reliable structure prediction. Protein-protein interactions were explored by utilizing STRING server, illustrated that CDK4 and E2F1 showed strong interaction with P16-INK4 and RB1 based on confidence score of 0.999 and 0.999 respectively. In order to facilitate a comprehensive understanding of the complex interactions between candidate genes with their functional interactors, GRAMM-X server was used. Protein-protein docking investigation of P16-INK4 revealed four ionic bonds illustrating Arg47, Arg80,Cys72 and Met1 residues as actively participating in interactions with CDK4 while docking results of RB1 showed four hydrogen bonds involving Glu864, Ser567, Asp36 and Arg861 residues which interact strongly with its respective functional interactor E2F1. CONCLUSION: This research may provide a basis for understanding biological insights of P16-INK4 and RB1 proteins which will be helpful in future to design a suitable drug to inhibit the disease pathogenesis as we have determined the interacting amino acids which can be targeted in order to design a ligand in-vitro to propose a drug for clinical trials. Protein -protein docking of candidate genes and their important interacting residues likely to be provide a gateway for developing computer aided drug designing.

Immunohistochemical features of 3,3',4,4'-tetrachloroazobenzene-induced rat gingival lesions.

Gingival lesions of squamous hyperplasia, cystic keratinizing hyperplasia (CKH), and squamous cell carcinoma (SCC) can be induced in rats treated by chronic gavage with 10-100 mg/kg 3,3',4,4'-tetrachloroazobenzene. We evaluated gingival squamous hyperplasia (GSH), CKH, and SCC for the immunohistochemical pattern of expression of carcinogenesis-associated markers. The 3 types of lesions and controls were stained with proliferation markers (proliferating cell nuclear antigen [PCNA] and cyclin-D1), tumor-suppressor markers (ß-catenin and mammary serine protease inhibitor [maspin]) and stroma-related markers (α-smooth muscle actin [SMA] and osteonectin/SPARC). The lesions had common immunohistochemical characteristics that differed in their expression patterns among the various diagnoses. PCNA and cyclin-D1 expression was higher in GSH, CKH, and SCC than in controls. The normal membranous expression of ß-catenin was lower in GSH, and almost absent in CKH and SCC. Maspin expression was similar in GSH and controls, whereas both CKH and SCC showed decreased expression. SMA and/or osteonectin/SPARC were seen in stromal cells in CKH and SCC. Collectively, there appears to be a progression from hyperplastic and cystic lesions toward malignancy based on the morphological changes, supported by the expression of carcinogenesis-associated proteins. The exact sequence of events leading to SCC remains to be defined in a time-dependent manner.

Serum and glucocorticoid kinase 3 at 8q13.1 promotes cell proliferation and survival in hepatocellular carcinoma.

Amplification of broad regions of 8q is one of the most frequent genetic alterations in hepatocellular carcinoma (HCC), suggesting the existence of oncogenes in addition to MYC at 8q24.21. In this report we examine the potential role of the candidate amplified oncogene serum and glucocorticoid kinase 3 (SGK3) at 8q13.1 in HCC pathogenesis. We found amplification and overexpression of SGK3 was frequently detected in clinical HCC specimens and that SGK3 genomic activation was significantly associated with poor outcome of patients (P = 0.028). Functionally, we found that overexpression of SGK3 in HCC cells increased cell cycle progression through G(1), cell survival, clonogenicity, anchorage-independent growth, and tumor formation in nude mice. In contrast, RNA interference (RNAi) silencing of SGK3 inhibited its oncogenic effects. We provide evidence that SGK3 promotes HCC growth and survival through inactivating glycogen synthase kinase 3 beta and Bcl-2-associated death promoter, respectively. We also found that expression of SGK3, which like AKT is activated by PI3K/PDK1 signaling, has more significance than overexpression of AKT in predicting poor outcome in HCC patients. Taken together, our findings in the present study suggests that the SGK3 pathway may function in parallel with the AKT pathway and undergoes an AKT-independent signaling pathway in the pathogenesis of HCC. Further characterization of SGK3 may provide a prognostic biomarker for HCC outcome prediction and a novel therapeutic target in HCC treatment.