An F-box protein, beta-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by beta-TrCP has been widely studied, the regulation of beta-TrCP itself is not well understood yet. In this study, we found that the level of beta-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of beta-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of beta-TrCP1 prior to its degradation. In addition, knockdown of beta-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the beta-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of beta-TrCP1 in TNBC cells and targeting beta-TrCP1 is a potential approach to treat human TNBC.
Cell Line, Tumor ; Cell Proliferation ; Cell Survival/drug effects ; Cyclin E/genetics/metabolism ; Dose-Response Relationship, Drug ; Female ; Furans/pharmacology ; Gene Knockdown Techniques ; Humans ; Models, Biological ; Multiprotein Complexes/antagonists & inhibitors ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors ; Phosphorylation/drug effects ; Protein Kinase Inhibitors/*pharmacology ; Proteolysis/drug effects ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Pyridines/pharmacology ; Pyrimidines/pharmacology ; TOR Serine-Threonine Kinases/*antagonists & inhibitors ; Triple Negative Breast Neoplasms/genetics/*metabolism ; beta-Transducin Repeat-Containing Proteins/genetics/*metabolism

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.
Animals ; Apoptosis ; genetics ; Carcinogenesis ; genetics ; metabolism ; pathology ; Carcinoma ; genetics ; metabolism ; pathology ; therapy ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cyclin D1 ; antagonists & inhibitors ; genetics ; metabolism ; Cyclin E ; antagonists & inhibitors ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Injections, Intralesional ; Ki-67 Antigen ; genetics ; metabolism ; Mice ; Mice, Nude ; Polycomb Repressive Complex 1 ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Small Interfering ; administration & dosage ; genetics ; metabolism ; Signal Transduction ; Tumor Burden ; Urinary Bladder ; metabolism ; pathology ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; agonists ; genetics ; metabolism

Animals ; Carcinogenesis ; Cell Cycle Proteins ; genetics ; metabolism ; Cyclin E ; metabolism ; F-Box Proteins ; genetics ; metabolism ; F-Box-WD Repeat-Containing Protein 7 ; Gene Silencing ; Genes, Tumor Suppressor ; Humans ; Mutation ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Neoplasms ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Receptors, Notch ; metabolism ; Signal Transduction ; TOR Serine-Threonine Kinases ; metabolism ; Ubiquitin-Protein Ligases ; genetics ; metabolism

OBJECTIVETo detect the expression of prostate cancer antigen-1 (PCA-1) in prostate cancer, and to analyze the effects of downregulation of PCA-1 expression on malignant biological behavior of prostate cancer LNCaP cells, and to explore their possible molecular mechanisms.

METHODSPCA-1-siRNA and control siRNA were transfected into LNCaP cells with lipofectamine 2000. The cell cycle, proliferation and migration were determined by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry and Transwell chambers, respectively. Western blotting was used to detect the expression of cyclin E, matrix metallopeptidase 9 (MMP-9) and p21. Immunohistochemistry was used to detect the expression of PCA-1 protein in 126 cases of prostate cancer and 88 cases of benign prostatic hyperplasia (BPH).

RESULTSThe positive rate of PCA-1 expression was 77.8% (98/126) in prostate cancer, and 10.2% (9/88) in BPH, and its expression was not significantly related to age, prostate specific antigen (PSA), Eastern Cooperative Oncology Group (ECOG) score (P > 0.05), and was associated with Gleason score, TNM staging and bone metastasis (P < 0.05). Downregulation of PCA-1 expression inhibited cell proliferation, arrested cell cycle at S phase and decreased cell migration of LNCaP cells. The downregulation of PCA-1 expression decreased the expression of Bcl-xl, cyclin E and MMP-9 proteins, but increased the expression of p21 proteins.

CONCLUSIONSPCA-1 may play an important role in the development of prostate cancer. The downregulation of PCA-1 expression can lead to changes in the proliferation, cell cycle and migration of prostate cancer LNCaP cells, and these effects may be associated with the decrease of Bcl-xl, cyclin E and MMP-9 proteins and increase of p21 protein.

Aged ; Aged, 80 and over ; Antigens, Neoplasm ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin E ; metabolism ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Staging ; Oncogene Proteins ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; bcl-X Protein ; metabolism

OBJECTIVETo evaluate the value of FOXP1 and Cyclin E gene in primary central nervous system lymphoma(PCNSL) of immunocompetent patients on prognostic significance.

METHODSClinical data of 71 patients with newly diagnosed PCNSL from 2002 to 2007 was analyzed retrospectively. Immunohistochemistry method (HRP-EnVision(TM)) was performed to observe the expression of FOXP1 and Cyclin E gene in tumor tissue samples. The survival was analyzed by Kaplan-Meier survival curve, survival factors analysis by the Log-rank test and COX proportional hazards regression model.

RESULTSFOXP1 positive was observed in 35 of 51 patients (68.63%) and Cyclin E staining was present in 29 of 50 cases (58.00%). FOXP1(+) patients had a shorter overall survival (OS) than FOXP1(-) ones. 2-year OS rate in FOXP1(+) and FOXP1(-) patients were 23.33% and 73.56%, respectively(P = 0.0015). Cyclin E(+) patients had a shorter overall survival(OS) than cyclinE(-) ones. 2-year OS rate in Cyclin E(+) and Cyclin E(-) patients were 17.56% and 69.76%, respectively (P = 0.0017). Multivariate analysis showed that Cyclin E expression was an independent prognostic factor for shorter OS (P = 0.048). FOXP1 expression might be an important prognostic factor for shorter OS (P = 0.065).

CONCLUSIONCyclin E expression is an independent prognostic factor and FOXP1 expression is a possible prognostic factor for poor clinical outcome in patients with PCNSL.

Aged ; Aged, 80 and over ; Central Nervous System Neoplasms ; genetics ; metabolism ; Cyclin E ; genetics ; metabolism ; Female ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Lymphoma, Non-Hodgkin ; genetics ; metabolism ; Male ; Middle Aged ; Prognosis ; Repressor Proteins ; genetics ; metabolism ; Retrospective Studies ; Survival Rate

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration

OBJECTIVETo study the effect of down-regulation of histone deacetylase 2 (HDAC2) expression on cell proliferation and cell cycle in cervical carcinoma cell lines HeLa.

METHODSHDAC2 siRNA and control siRNA were transfected to HeLa cells. CCK-8 and flow cytometry were used to analyze the changes of cell proliferation and cell cycle, respectively. Western blot was employed to detect the changes of cell proliferation and cell cycle-related proteins.

RESULTSHDAC2 siRNA significantly down-regulated the expression of HDAC2 protein in HeLa cells, resulting in marked inhibition of cell proliferation. In addition, the percentage of cells in G(0)/G(1) phase in HDAC2 siRNA group (63.3% ± 2.0%) was significantly higher than that in untreated group (29.3% ± 1.7%) or control siRNA group (29.4% ± 1.7%), F = 354.181, P = 0.000. Furthermore, Western blot demonstrated that down-regulation of HDAC2 expression decreased the expression of cyclin D1, cyclin E and CDK2 proteins but increased the expression of p21 protein.

CONCLUSIONSDown-regulation of HDAC2 expression mediates proliferation inhibition and cell cycle arrest. It is associated with decrease in cyclin D1, cyclin E and CDK2 protein expression and increase in p21 protein expression.

Cell Cycle ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Down-Regulation ; HeLa Cells ; Histone Deacetylase 2 ; genetics ; metabolism ; Humans ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo detect the expression of histone deacetylase 6 (HDAC6) in laryngeal squamous cell carcinoma, and to analyze the effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration of laryngeal squamous cell carcinoma cell line Hep-2 cells, and to explore their possible molecular mechanisms.

METHODSImmunohistochemistry was used to detect the expression of HDAC6 protein in 55 cases of laryngeal squamous cell carcinoma and 20 cases of normal laryngeal mucosa. HDAC6 siRNA and control siRNA were transfected into Hep-2 cells via lipofectamine 2000, and the interfering effect was analyzed using Western blotting. The effects of downregulation of HDAC6 expression on cell cycle, proliferation and migration were determined by cell counting kit-8 (CCK-8), flow cytometry and Boyden chamber, respectively. Finally, Western blotting was used to detect the expressions of cell cycle, proliferation and migration related proteins.

RESULTSThere was a high level expression of HDAC6 protein in laryngeal squamous cell carcinoma, and its expression was not related to age and sex of the patients (P > 0.05), but closely associated with the degree of histological differentiation, TNM staging and lymph node metastasis (P < 0.05). HDAC6 siRNA effectively down-regulated the expression of HDAC6 protein in laryngeal squamous cell carcinoma cell line Hep-2 cells, and downregulation of its expression obviously inhibited cell proliferation, arrested cell cycle at G(0)/G(1) phase and decreased cell migration ability in Hep-2 cells. Additionally, the downregulation of HDAC6 protein expression markedly decreased the expressions of cyclin D1, cyclin E, cdk2 and MMP-9 proteins, but increased the expressions of p21 and E-cadherin proteins.

CONCLUSIONSHDAC6 may play a pivotal role in the carcinogenesis and development of laryngeal squamous cell carcinoma. The downregulation of HDAC6 expression-mediated cell proliferation inhibition, cell cycle arrest and decreased cell migration ability may be closely associated with the decrease of cyclin D1, cyclin E, cdk2 and MMP-9 proteins and increase of p21 and E-cadherin proteins.

Adult ; Aged ; Cadherins ; metabolism ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; Down-Regulation ; Female ; Histone Deacetylase 6 ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Matrix Metalloproteinase 9 ; metabolism ; Middle Aged ; Neoplasm Staging ; Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo study the roles of DNA dependent protein kinase (DNA-PK)in silica-induced cell cycle changes and expressions of CyclinE and CDK2 in human embryo lung fibroblasts (HELF).

METHODSThe expressions of Ku80 and DNA-PKcs proteins were inhibited by siRNA plasmids, respectively. Flow cytometry was used to detect the distributions of cell cycle and western blot assay was used to determine the expression levels of CyclinE and CDK2 after cells were exposed to 200 microg/ml silica for 0, 3, 6, 12, 24 h.

RESULTSThe proportion of G1 phases in negative control cells decreased from 83.53% +/- 2.24% to 69.11% +/- 3.12% after exposure to silica; the proportion of G1 phases in H-Ku80 and H-PKcs cells exposed to silica decreased from 85.16% +/- 3.73% to 59.92% +/- 3.31% and from 75.06% +/- 2.23% to 58.32% +/- 1.35%, respectively (P < 0.05). The exposure to silica resulted in the increasing protein expression levels of CyclinE and CDK2 in negative control cells, and the expression levels of CyclinE were obviously suppressed in H-Ku80 and H-PKcs as compared with control cells. However, the expression level of CDK2 protein did not change significantly.

CONCLUSIONDNA-PK might play a role in silica-induced alternations of cell cycle and regulate silica-induced overexpression of CyclinE in human embryo lung fibroblasts.

Cell Cycle ; drug effects ; Cells, Cultured ; Cyclin E ; metabolism ; Cyclin-Dependent Kinase 2 ; metabolism ; DNA-Activated Protein Kinase ; genetics ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Lung ; cytology ; Nuclear Proteins ; genetics ; metabolism ; Oncogene Proteins ; metabolism ; Silicon Dioxide ; pharmacology

OBJECTIVE: To investigate the role of histone deacetylase 7 (HDAC7) in the occurrence and development of hepatocellular carcinoma. METHODS: HepG2 cells and human microvascular endothelial cells (HMEC-1) were divided into 3 groups after transfection pSUPER-HDAC7 retroviral interference plasmid: a pSUPER(HDAC7RNAi+) group, a pSUPER(HDAC7RNAi-) group, and a pSUPER group as the control group. The expression of HDAC7, p21, cyclin E, matrix metalloproteinases 10 (MMP10), and hypoxiainducible factor-1alpha (HIF-1alpha) were detected by Western blot. The expression of HDAC7 in cell lines was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry, the nude mice modle, and vascular endothelial cells 2-dimensional tubulogenesis in vitro and in vivo. RESULTS: Compared with the control group and the pSUPER(HDAC7RNAi-) group, the expression of HDAC7 was downregulated, the rate of cell growth inhibition and apoptosis in the pSUPER(HDAC7RNAi+) group increased more significantly; the expression of p21 and HIF-1alpha was increased significantly, while the expression of cyclin E and MMP10 in the pSUPER(HDAC7RNAi+) group was downregulated (P<0.05). CONCLUSION The expression of HDAC7 protein plays an important role in the apoptosis and vascular tubulogenesis of hepatocellular carcinoma by the upregulation of p21 and HIF-1alpha and the downregulation of cyclin E and MMP10.
Animals ; Apoptosis ; genetics ; Cells, Cultured ; Cyclin E ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Hep G2 Cells ; Histone Deacetylases ; genetics ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Matrix Metalloproteinase 10 ; genetics ; metabolism ; Mice ; Mice, Nude ; Neovascularization, Pathologic ; genetics ; RNA Interference ; Transfection