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

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

To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin (FN), cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression, the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively, Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR. The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin. When RPMI8226 cells had been adhered to FN or BSA-coated wells for 1, 6 and 12 hours, the adhesion rates were (43.71 +/- 2.18)%, (55.63 +/- 1.56)%, and (63.42 +/- 2.46)% respectively. After treatment with STI571 20 micromol/L, the adhesion rates decreased to (15.12 +/- 1.04)%, (17.58 +/- 1.32)% and (17.24 +/- 1.59)% respectively (P < 0.05). The experiment revealed that growth of RPMI8226 cells adhered to FN-coated plates had a significant advantage over growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period, and the mean IC(50) value for FN-adhered cells was (1.46 +/- 0.04) micromol/L while mean IC(50) value for BSA control was (0.78 +/- 0.03) micromol/L (P < 0.05). Following treatment with 20 micromol/L STI571, the mean IC50 values for FN and BSA adhered cells were (0.81 +/- 0.05) micromol/L, (0.74 +/- 0.02) micromol/L respectively, there was no significant difference between them (P > 0.05). RT-PCR demonstrated that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20 micromol/L STI571. It is concluded that STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin, reverse cell adhesion mediated adriamycin-resistance, and downregulate Rac1 mRNA level.
Benzamides ; Cell Adhesion ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Fibronectins ; metabolism ; Humans ; Imatinib Mesylate ; Multiple Myeloma ; metabolism ; pathology ; Piperazines ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Tumor Cells, Cultured ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

OBJECTIVETo study the expressions and activities of Rho GTPases in hypoxia and its relationship with tumor angiogenesis.

METHODSThree tumor cell lines were used in this study: gastric cancer cell lines AGS, SGC7901 and hepatocellular carcinoma cell line HepG2. Expression level of Rac1 mRNA was detected by semi-quantitative RT-PCR. Activity of Rac1 was determined by pull-down assay and expression of HIF-1alpha, VEGF, p53 and PTEN protein was detected by Westernblot.

RESULTSThe expression level of Rac1 mRNA was significantly increased in hypoxia compared to normoxia. Pull-down assay showed that hypoxia-induced activity of Rac1 was elevated in a time-dependent manner and climaxed at 3 hours. The expressions of HIF-1alpha and VEGF protein were up-regulated, while those of PTEN and p53 protein were down-regulated.

CONCLUSIONThese results indicate that hypoxia enhances Rac1 expression which might be involved in tumor angiogenesis by reacting with hypoxia-responsive genes.

Cell Hypoxia ; Cell Line, Tumor ; Hepatoblastoma ; blood supply ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Liver Neoplasms ; blood supply ; metabolism ; pathology ; Neovascularization, Pathologic ; PTEN Phosphohydrolase ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Stomach Neoplasms ; blood supply ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics ; rac1 GTP-Binding Protein ; biosynthesis ; genetics ; rho GTP-Binding Proteins ; biosynthesis ; genetics

OBJECTIVETo construct lentiviruses carrying dominant negative mutant of Rac1-GTPase (Rac1N17) or the constitutive active mutant of Rac1-GTPase (Rac1L61) and expressing enhanced green fluorescent protein (EGFP) bicistronically.

METHODSThe lentiviral expression plasmid Plenti6/v5-Rac1N17 and Plenti6/v5-Rac1L61 were constructed and identified by restriction enzyme digestion and DNA sequence analysis. The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses Rac1L61 and Rac1N17, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different Rac1 mutants were evaluated and the morphology of the transfected PFCs was observed.

RESULTSThe results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction. The packaged lentiviral titer was 1x10(6) TU/ml. Analysis of Rac1 biological activity showed that Rac1N17 lentivirus particles infection significantly inhibited epidermal growth factor-stimulated Rac1 activity in the PFCs, while Rac1L61 lentivirus particles enhanced the Rac1 activity. The transfection efficiency of these Rac1 mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses.

CONCLUSIONThe lentiviruses carrying Rac1 dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs and lend important support for the study of Rac1-GTPase.

3T3 Cells ; Animals ; Animals, Newborn ; Cerebral Cortex ; cytology ; GTP Phosphohydrolases ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; metabolism ; Mice ; Neurons ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; Transfection ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

OBJECTIVETo study the role of T lymphoma invasion/metastasis gene 1 (Tiam1) and protein in ovarian tumor cells.

METHODSExpressions of Tiam1 mRNA, Rac1 mRNA, and Tiam1 protein in four ovarian tumor cells A2780, Caov3, Skov3, and SW626 were studied by using RT-PCR and Western blot, respectively. The cell migration ability was analyzed by in vitro invasion assay.

RESULTSExpressions of Tiam1 mRNA and protein, as well as Rac1 mRNA were detected in all four ovarian tumor cells. There was a strong direct correlation between the levels of Tiam1 and Rac1 mRNA expression and migration potentials of all four ovarian cancer cells in vitro experiments. The increased expressions of Tiam1 mRNA were coincident with those of Rac1 mRNA, with a parallel relationship (P = 0.003, r = 0.874). Levels of Rac1 mRNA expression were significantly correlated with the potentials of tumor cell migration (P = 0.042, r = 0.814).

CONCLUSIONTiam1-Rac1 signaling pathway plays a positive role in assessing tumor cell invasion and metastasis and provides a new target for gene therapy of ovarian cancer.

Cell Movement ; Female ; Gene Expression Regulation, Neoplastic ; Guanine Nucleotide Exchange Factors ; Humans ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Protein Biosynthesis ; Proteins ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphoma Invasion and Metastasis-inducing Protein 1 ; Tumor Cells, Cultured ; rac1 GTP-Binding Protein ; biosynthesis ; genetics

Tumor cells are known to produce larger amounts of reactive oxygen species (ROS) than normal cells. Although numerous reports have indicated the importance of ROS in urokinase plasminogen activator (uPA) production, the precise mechanisms remain controversial. In our study, we investigated the effect of ROS on uPA generation in human hepatoma cells, HepG2 and Hep 3B. We determined the effects of hepatocyte growth factor (HGF) on the regulation of ROS, which resulted in suppression of ROS production, as measured with the fluorescent probe, 2'-7'-dichlorofluorescein diacetate. The role of HGF in modulating ROS production, particularly that regulated by Rac-1, was determined. HGF suppressed the increment in Rac-1-regulated ROS in both cell lines. Treatment with 200 microM of H2O2 showed a 1.6-2.1 fold increment in HGF, but a little increment occurred at 500 microM of H2O2. It looks no dose dependent manner. Combined treatment with H2O2 and HGF, resulted in a slightly increased production of HGF compared to no treatment (control). Also, H2O2 upregulated uPA expression in both hepatoma cell lines. To identify the downstream pathways regulated by ROS, we treated cells with PD 98059, an MEK inhibitor, and SB 203580, a p38 inhibitor, after treatment with H2O2, and showed negative control between ERK and p38 kinase activities for uPA regulation. We found that HGF modulate Rac-1-regulated ROS production through activation of Akt and ROS regulates uPA production via MAP kinase, which provides a novel clue to clarify the mechanism underlying hepatoma progression.
Cell Line, Tumor ; Fluorescent Dyes/chemistry ; Hepatocyte Growth Factor/pharmacology/*physiology ; Humans ; Hydrogen Peroxide/pharmacology ; Imidazoles/pharmacology ; Liver Neoplasms/drug therapy ; Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism ; Pyridines/pharmacology ; Reactive Oxygen Species/*metabolism ; Recombinant Proteins/pharmacology ; Urokinase-Type Plasminogen Activator/*biosynthesis ; rac1 GTP-Binding Protein/metabolism

Triptolide, a diterpenoid triepoxide from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f., is a potential treatment for autoimmune diseases as well a possible anti-tumor agent. It inhibits proliferation of coloretal cancer cells in vitro and in vivo. In this study, its ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK1, IL6R and phosphorylated STAT3 were all reduced by triptolide treatment. Triptolide prohibited Rac1 activity and blocked cyclin D1 and CDK4 expression, leading to G1 arrest. Triptolide interrupted the IL6R-JAK/STAT pathway that is crucial for cell proliferation, survival, and inflammation. This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development.
Animals ; Cell Transformation, Neoplastic/*drug effects ; Colitis/complications ; Colonic Neoplasms/chemically induced/*drug therapy/metabolism/pathology ; Dextran Sulfate/toxicity ; Dimethylhydrazines/toxicity ; Diterpenes/*administration & dosage ; Epoxy Compounds/administration & dosage ; Humans ; Interleukin-6/biosynthesis ; Janus Kinases/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred ICR ; Mice, Nude ; Neoplasm Transplantation ; Phenanthrenes/*administration & dosage ; STAT3 Transcription Factor/metabolism ; Signal Transduction/*drug effects ; Tumor Burden/drug effects ; rac1 GTP-Binding Protein/*biosynthesis