Over-expression of CD151 was found to be associated with metastasis and poor prognosis of prostatic carcinoma. This study was designed to examine the mechanism by which CD151 promotes the proliferation and migration of prostatic cancer cells. The pAAV-CD151, pAAV-GFP and pAAV-CD151-AAA mutant plasmids were constructed and used to transiently transfect PC3 cells (a prostatic carcinoma 3 cell line) by the mediation of Fugene HD. Then, the cells were assigned to control group, pAAV-GFP group, pAAV-CD151 group, and pAAV-CD151-AAA group respectively. Cell proliferation was evaluated by using the 3-[4,5-dimet-hylthiazol-2-yl]-2,5, diphenyltetrazolium bromide (MTT) method. Cell migration assay was performed by using Boyden chambers. The formation of CD151-integrin α3/α6 complex was determined by the method of co-immunoprecipitation. The protein expression levels of CD151 and extracellular signal-regulated kinase (ERK) were measured by Western blotting. The results showed that transfection of pAAV-CD151 or pAAV-CD151-AAA mutant increased the expression of CD151 protein in PC3 cells. Co-immunoprecipitation showed that more CD151-integrin α3/α6 complex was formed in the pAAV-CD151 group than in the control group, the pAAV-GFP group and the pAAV-CD151-AAA mutant group. Furthermore, the proliferative and migrating capacity of PC3 cells was substantially increased in the pAAV-CD151 group but inhibited in the pAAV-CD151-AAA mutant group. CD151 transfection increased the expression of phospho-ERK. Taken together, it was concluded that CD151 promotes the proliferation and migration of PC3 cells through the formation of CD151-integrin complex and the activation of phosphorylated ERK.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Integrin alpha3 ; metabolism ; Integrin alpha6 ; metabolism ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Tetraspanin 24 ; metabolism

OBJECTIVETo observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action.

METHODSAttachment assay was used to assess the adhesion of wwox-transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells. Integrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane.

RESULTSAttachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours (P<0.01). The level of membranous integrins alpha2 and alpha3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in vector-transfected controls (P<0.05), but there was no association with the level of functioning integrins betal or beta2 in clone cells (P>0.05). The attachment assays were repeated after pre-incubating the cells with integrin alpha2 or alpha3 function-blocking antibodies. These results showed that blocking integrin alpha3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody (P<0.05). In contrast, preincubation with alpha2 blocking antibody had very little effect on fibronectin binding in these cells (P>0.05). FACS analysis showed that membranous integrin alpha3 expression revealed a marked reduction in WWOX-transfected cells than that in vector-transfected cells.

CONCLUSIONWWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.

CD18 Antigens ; metabolism ; Cell Adhesion ; Female ; Fibronectins ; metabolism ; Humans ; Integrin alpha2 ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Oxidoreductases ; genetics ; metabolism ; Protein Binding ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

This study was aimed to investigate whether 2-methoxyestradiol (2ME2) could exert effect of inducing differentiation on myeloma cells. A myeloma cell line CZ-1 secreting lambda light chain protein was used as an object of study. The CZ-1 cell morphology was observed by Wright's staining, the CD49e expression on cell surface after treatment with 2ME2 was detected by flow cytometry, the concentration of lambda light chain protein in the supernatant was assayed by immuno-scattering turbidity method. The results showed that treatments with 0.1-0.5 micromol/L 2ME2 for 72 hours resulted in some mature morphological changes of CZ-1 cells, such as the ratio of karyoplasms going down, nucleolus reducing or disappearing, chromatin getting rougher and more compacted; the CD49e positive CZ-1 cells increased by 2ME2 with concentrations of 0.1 micromol/L to 0.5 micromol/L in a concentration-dependent manner. The statistical difference from the control group was significant; the concentration of lambda light chain protein increased from control group 29.3 +/- 2.77 microg/ml to 35.97 +/- 2.6 microg/ml (P < 0.05) after exposure to 0.1 micromol/L 2ME2 for 72 hours, and the treatment of 0.5 micromol/L 2ME2 up-regulated lambda light chain protein to 79.67 +/- 1.88 microg/ml (P < 0.01) continuously. It is concluded that 2ME2 at low-concentration can induce differentiation of the CZ-1 cells to mature, which provides a new, and safe strategy for myeloma therapy.
Cell Line, Tumor ; Cell Transformation, Neoplastic ; drug effects ; Estradiol ; analogs & derivatives ; pharmacology ; Humans ; Immunoglobulin lambda-Chains ; biosynthesis ; Integrin alpha3 ; biosynthesis ; Multiple Myeloma ; metabolism ; pathology

OBJECTIVETo explore the association between renal transforming growth factor-β1 (TGF-β1) and the expressions of α3 and β1 integrins and observe the effect of irbesartan on their expressions in diabetic rats.

METHODSThirty 8-week-old male Wistar rats were randomly divided into normal control group (n=7), diabetic control group (n=14) and irbesartan group (n=9). Rat models of diabetes were established by a single peritoneal injection of streptozotocin (STZ), and 4 weeks later the rats received irbesartan treatment for 8 weeks. Enzyme-linked immunosorbent assay (ELISA) was used to measure the urinary albumin excretion rate, and PAS staining was utilized to observe the renal pathologies. Immunohistochemistry was performed for semi-quantitative determination of podocyte density, and real-time RT-PCR was used to detect the renal TGF-β1 and α3/β1 integrin mRNA expressions.

RESULTSIn diabetic rats, the expression of renal TGF-β1 mRNA was significantly increased, while α3 and β1 integrin mRNA expressions and podocyte density significantly decreased with increased proteinuria. Irbesartan obviously improved such changes.

CONCLUSIONIn diabetic rats renal TGF-β1 can regulate α3 and β1 integrin mRNA expressions to reduce the number of podocytes, and inhibition of this pathway may be one of the mechanisms of the renal protective effect of irbesartan.

Animals ; Biphenyl Compounds ; pharmacology ; Diabetes Mellitus ; metabolism ; Diabetic Nephropathies ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Kidney ; drug effects ; metabolism ; Male ; Rats ; Rats, Wistar ; Tetrazoles ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.
Cell Movement ; physiology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblast Growth Factor 2 ; physiology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrin alpha3 ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; metabolism ; Umbilical Veins ; cytology

PURPOSE: To investigate the overall expression of extracellular matrix (ECM) and adhesion molecule genes using a gene array technique in the joint capsule of a frozen shoulder. MATERIALS AND METHODS: Tissues from 20 human shoulder joint capsules were harvested intraoperatively from patients (15 primary frozen shoulders, 5 controls) in our hospital. The RNA was isolated from the capsule tissue and the gene expression of ECM and adhesion molecules was analyzed using an oligo-array technique. RESULTS: The expression of several genes of the ECM and cell adhesion molecules was significantly higher in the capsule tissue from patients with a frozen shoulder than the controls. The gene expression of the collagen V alpha1/alpha3, VI alpha2/alpha3, VIII alpha1/alpha2, XV alpha1, XVIII alpha1 and ECM proteins including CD 44, connective tissue growth factor (CTGF), matrix metalloproteinase (MMP)-9/14, osteonectin, veriscan, hyaluronan synthase (HAS)-1, extra-cellular matrix (ECM)-1, secreted phosphoprotein (SSP)-1, tenascin C (TNC), thrombospondin 2/4 was two times higher in the frozen shoulder than the control. Several cell adhesion molecules genes including catenin alpha1, seletin p, integrin alpha3, beta2, beta4, beta5 and laminin alpha4, alpha5 were also two times higher in the in the patients with a frozen shoulder than the control. CONCLUSION: The gene expression of several ECM proteins and adhesion molecules is significantly higher in patients with frozen shoulder.
Bursitis ; Capsules ; Cell Adhesion Molecules ; Collagen ; Connective Tissue Growth Factor ; Extracellular Matrix ; Gene Expression ; Genes, vif ; Glucuronosyltransferase ; Humans ; Hyaluronic Acid ; Integrin alpha3 ; Joint Capsule ; Laminin ; Osteonectin ; Proteins ; RNA ; Shoulder ; Shoulder Joint ; Tenascin ; Thrombospondins