OBJECTIVETo investigate the significance of Rac subfamily members in the gastrointestinal carcinogenesis and progression.

METHODSThe mRNA expression of Rac1, Rac2 and Rac3 in 12 kinds of gastrointestinal cancer cell lines was examined by semi-quantitative RT-PCR. The activities of Rac1 protein in 5 kinds of gastric cancer cell lines were tested by pull-down assay.

RESULTSCompared with the normal gastric mucosa and intestinal epithelial cell line, the mRNA expression of Rac1 and Rac3 was up-regulated in most of gastrointestinal cancer cell lines. The activities of Rac1 protein increased markedly in gastric cancer cell lines.

CONCLUSIONThe increased mRNA expression of Rac1 and Rac3 in gastrointestinal cancer cell lines and the abnormal activation of Rac1 protein in gastric cancer cell lines might be correlated with the carcinogenesis of gastrointestinal cancer.

Cell Line, Tumor ; Gastrointestinal Neoplasms ; metabolism ; Humans ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; rac GTP-Binding Proteins ; genetics ; rac1 GTP-Binding Protein ; analysis ; genetics

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.
Actins ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; immunology ; metabolism ; Blood Platelets ; metabolism ; Cells, Cultured ; Humans ; Phosphatidylinositol 3-Kinases ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism ; pharmacology

Brain ; Cholesterol ; chemistry ; Cytoskeleton ; drug effects ; Endothelial Cells ; cytology ; metabolism ; Hemolysin Proteins ; chemistry ; pharmacology ; Humans ; Phalloidine ; pharmacology ; Pseudopodia ; drug effects ; Stress Fibers ; drug effects ; rac1 GTP-Binding Protein ; metabolism ; rhoA GTP-Binding Protein ; metabolism

OBJECTIVETo investigate modulatory role of Rac1 protein in epidermal stem cell (ESC) migration during wound healing, in order to provide a reference for enriching basic theory of wound healing and guiding clinical application.

METHODSConstitutively active mutant of Rac1 protein (Rac1Q61L) or dominant negative isoform of Rac1 protein (Rac1T17N) was transfected into ESC using a retroviral vector FUGW, and retroviral vector FUGW transfected into ESC in singles was used as blank control. The cells were divided into 3 parts according to the random number table and treated as follows. First, equal numbers of cells were inoculated into 24-well plates coated with collagen I (20 µg/mL), collagen IV (20 µg/mL) or fibronectin (10 µg/mL). Cells adhered to above matrices were quantitated using CytoTox 96 colorimetric kit. Second, 1000 cells adhered to collagen IV, after being stained with tetramethyl rhodamine isothiocyanate-phalloidin, were collected for observation of cell morphology and comparison of spreading area under confocal laser scanning microscope. Third, ESC with density of 2 × 10(5) cells per well were placed in upper compartment of Transwell chamber, DK-SFM culture medium alone or that containing stromal cell derived factor 1 (SDF-1) was added into lower compartment of Transwell chamber. Migration of ESC was observed using inverted phase contrast microscope, and the result was denoted as migration rate. Lastly, ESC with density of 7.5 × 10(5) cells per well was inoculated into 6-well plates for 12 hours, and treated with 4 µg/mL mitomycin C for 2 hours. The remaining scratch width of monolayer was respectively measured 6 hours or 12 hours after scratching to calculate the percentage of remaining scratch width. Data were processed with t test.

RESULTSCompared with that of blank control, the number of Rac1Q61L-transfected cells adhered to collagen I was significantly increased (t = 5.302,P < 0.05), while the number of Rac1T17N-transfected cells adhered to collagen I, IV, and fibronectin were all obviously decreased (with t value respectively 13.741, 15.676, 8.256, P values all below 0.05). Confocal laser scanning microscope showed that spreading area of Rac1Q61L-transfected ESC (with laminate pseudopodia on edge) and Rac1T17N-transfected ESC was respectively larger and smaller as compared with that of blank control. With SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 78.0% and Rac1Q61L-transfected ESC was increased by 43.4% as compared with that of blank control. Without SDF-1 effect, the migration rate of Rac1T17N-transfected ESC was decreased by 55.2%, while the migration rate of Rac1Q61L-transfected ESC was close to that of blank control. Six or 12 hours after scratching, the percentage of remaining scratch width in Rac1Q61L-transfected ESC was lower as compared with that in blank control [(39 ± 9)% vs. (43 ± 5)%, (6 ± 5)% vs. (18 ± 7)%, with t value respectively 1.027, 4.389, with P value respectively above and below 0.05], while that in Rac1T17N-transfected ESC [(81 ± 9)%, (71 ± 11)%, respectively] was obviously higher as compared with that in blank control (with t value respectively 11.386, 11.726, P values all below 0.05).

CONCLUSIONSRac1 protein may control the migration of ESC by regulating its adhesion, spreading, and chemotaxis, and it plays an active role in wound healing accelerated by ESC.

Cell Movement ; Cell Proliferation ; Epidermis ; cytology ; Epithelial Cells ; Humans ; Mutation ; Stem Cells ; cytology ; Transfection ; Wound Healing ; rac1 GTP-Binding Protein ; genetics ; metabolism

BACKGROUNDRecent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

METHODSA series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133 - U87 cells. The migration and invasive ability of CD133+ and CD133 - U87 cells were determined by cell migration and invasion assays in vitro. Student's t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

RESULTSIn the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14 ± 4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378 ± 0.007), compared to CD133- cells (0.195 ± 0.004) (t = 27.81; P < 0.05). CD133+ cells had stronger ability to migrate (74.34 ± 2.40 vs. 38.72 ± 2.60, t = 42.71, P < 0.005) and invade (52.00 ± 2.28 vs. 31.26 ± 1.82, t = 30.76, P < 0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

CONCLUSIONSThese data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

AC133 Antigen ; Antigens, CD ; metabolism ; Cell Line, Tumor ; Glioblastoma ; metabolism ; pathology ; Glioma ; metabolism ; pathology ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Peptides ; metabolism ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo investigate the role and signalling of microRNA(miR)-182 on regulating high glucose-induced cardiomyocyte hypertrophy.

METHODSThe candidates of miR which might potentially be involved on targeting Rac1 were predicted by applying bioinformatics analysis. The expression of all related candidates miRs was verified by real-time reverse transcription-PCR (RT-PCR) in cardiac tissues of db/db mice and db/m mice. Then the relationship between candidates miR and Rac1 was investigated with Pearson relevant analysis. Neonatal mice cardiomyocytes were cultured and divided into 2 groups: normal glucose group and high glucose group. The level of selected miR and Rac1 in two groups was detected by RT-PCR. Neonatal mice cardiomyocytes were then randomly divided into 4 groups: normal glucose group, selected microRNA mimics control group, high glucose group, high glucose plus selected miR mimics control group. The morphology of cardiomyocyte in each group was detected under light microscope. Furthermore, Rac1, β-MHC and α-SMA expressions were detected in cultured cardiomyocyte treated by high glucose for 48 h after transfecting selected miR mimics by RT-PCR and Western blot.

RESULTSA total of 6 miR candidates potentially targeting Rac1 were screened by bioinformatics, which were miR-182, miR-142-3p, miR-140, miR-101a, miR-429 and miR-200b. Among these candidates, miR-182 and miR-142-3p expression was significantly downregulated in cardiac tissues of db/db mice compared with db/m controls (P < 0.05). MiR-182 was negatively correlated with Rac1 by person analysis (r = -0.891 02). Downregulation of miR-182 and upregulation of Rac1, β-MHC, α-SMA were found in high glucose-induced cardiomyocyte. After transfection of miR-182 mimics, hypertrophic changes were significantly reduced and Rac1 as well β-MHC expression was significantly downregulated in cardiomyocyte incubated with high glucose.

CONCLUSIONMiR-182 might be involved in the regulation of high glucose-induced myocardial hypertrophy process via targeting Rac1.

Animals ; Cardiomyopathy, Hypertrophic ; metabolism ; Down-Regulation ; Glucose ; physiology ; Mice ; MicroRNAs ; physiology ; Myocytes, Cardiac ; metabolism ; Neuropeptides ; metabolism ; Rats, Sprague-Dawley ; Transfection ; Up-Regulation ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo construct a vector expressing small interfering RNA (siRNA) against Rac1 gene and observe its effect on soft agar colony formation of SW480 cells in vitro.

METHODSOligos of 64 base pairs for hairpin RNA targeting Rac1 were chemically synthesized and annealed. The siRNA constructs for Rac1, produced by inserting the annealed oligos into the downstream of H1 promoter of linearized pSUPER, were confirmed by restriction digestion and DNA sequencing. The constructed Rac1-siRNA was transfected into SW480 cells and Western blotting was performed to assess the expression and interference efficiency of siRNAs against Rac1.The soft agar colony formation assay was used to study the effect of Rac1 gene silencing on SW480 cells.

RESULTSRestriction digestion and DNA sequencing showed that the siRNA targeting Rac1 gene was successfully constructed. The siRNA could effectively down-regulate the expression of Rac1 in SW480 cells. Soft agar colony formation assay showed that the colony number and diameter of SW480 cells was reduced after siRNA transfection.

CONCLUSIONA vector expressing hairpin RNA against Rac1 gene are successfully produced, which significantly reduces the colony numbers and size of SW480 cells in vitro, suggesting that Rac1 plays an important role in the growth of colorectal cancer in vitro.

Base Sequence ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; pathology ; Down-Regulation ; Humans ; Molecular Sequence Data ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; rac1 GTP-Binding Protein ; genetics

This study sought to elucidate whether Rac1 mediates the migration of endothelial cell induced by IL-8. The Transwell chamber motility assay was conducted to disclose the effect of different matrigel dilution and different time of IL-8 treatment on the migration of endothelial cells. The mRNA of Rac1 was detected by RT-PCR. The results demonstrated that when the concentration of Matrigel was 1:2, there is significant difference on the amounts of migration cells than that of the concentration of 1:3 or 1:8; When the dilution of Matrigel was 1:4, 1:5 or 1:6, there is no significant difference on the amounts of migration cells than that of other dilution groups. So we choose the Matrigel concentration as 1:4. With the increase of IL-8 stimulation time, the cells which migrated from upper reservoirs to lower reservoirs progressively increased. After six hours stimulation by IL-8, the expression of Rac1 mRNA in migrated cells was increased, compared with that of other groups. The results suggest that Rac1 may mediate the migration of endothelial cells induced by IL-8. It can also be the foundation for further investigation on the role of Rac1 in the migration of endothelial cells induced by IL-8.
Cell Movement ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; metabolism ; Humans ; Interleukin-8 ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; metabolism ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

BACKGROUNDThe release of Weibel-Palade Bodies (WPB) is a form of endothelial cell activation. But the signal transduction pathway leading to WPB release is not yet defined. We hypothesized that small G-protein rac1 and reactive oxygen species (ROS) mediate the ligand induced release of Weibel-Palade Bodies.

METHODSWe tested this hypothesis by using wild-type and mutant adenoviral rac1 expression vectors, and by manipulating the production and destruction of superoxide and hydrogen peroxide in human aortic endothelial cells (HAEC).

RESULTSThrombin (1.0 Unit, 30 min) induced the increase of WPB release by 3.7-fold in HAEC, and that H2O2 (0.1 mmol/L, 30 min) induced by 4.5-fold. These results correlated with thrombin-stimulated activation of rac-GTP binding activity by 3.5-fold, and increase of ROS production by 3.4-fold. The dominant negative adenoviral rac-N17 gene transfer dramatically inhibited the release of WPB by 64.2% (control) and 77.3% (thrombin-stimulation), and decreased ROS production by 65.5% (control) and 83.6% (thrombin-stimulation) compared with non-infected cells, respectively. Anti-oxidants, catalase and N-acetyl-cysteine significantly decreased the release of WPB by 34% and 79% in control cells, and further decreased by 63.6% and 46.7% in rac-N17 transferred cells compared with non-infected cells. We also confirmed that rac1 was located upstream of ROS in the WPB release pathway.

CONCLUSIONSSmall G-protein rac1 medicates ligand-induced release of Weibel-Palade Bodies in human aortic endothelial cells, and the signal pathway of WPB release is a rac1-dependent ROS regulating mechanism.

Aorta ; ultrastructure ; Endothelial Cells ; ultrastructure ; Humans ; Reactive Oxygen Species ; Signal Transduction ; Thrombin ; pharmacology ; Weibel-Palade Bodies ; physiology ; rac1 GTP-Binding Protein ; physiology

The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.
Apoptosis ; physiology ; Cell Cycle ; physiology ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; rac1 GTP-Binding Protein ; biosynthesis ; genetics ; physiology