Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.
Amino Acid Sequence ; Animal ; Base Sequence ; Cloning, Molecular ; Comparative Study ; Cross Reactions ; DNA, Complementary/genetics ; Human ; Molecular Sequence Data ; Rats ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; cdc42 GTP-Binding Protein/immunology ; cdc42 GTP-Binding Protein/genetics*

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
*Aging ; Animal ; Brain/metabolism ; Calcium/pharmacology ; Cattle ; Comparative Study ; GTP-Binding Proteins/*metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Phosphorylation/drug effects ; Rats ; Rats, Sprague-Dawley ; Synaptic Membranes/*metabolism ; Synaptosomes/*metabolism ; cdc42 GTP-Binding Protein/biosynthesis/metabolism ; rab3A GTP-Binding Protein/metabolism ; rab5 GTP-Binding Proteins/metabolism ; rhoA GTP-Binding Protein/biosynthesis/metabolism

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
*Aging ; Animal ; Brain/metabolism ; Calcium/pharmacology ; Cattle ; Comparative Study ; GTP-Binding Proteins/*metabolism ; Guanosine 5'-O-(3-Thiotriphosphate)/metabolism ; Molecular Weight ; Phosphorylation/drug effects ; Rats ; Rats, Sprague-Dawley ; Synaptic Membranes/*metabolism ; Synaptosomes/*metabolism ; cdc42 GTP-Binding Protein/biosynthesis/metabolism ; rab3A GTP-Binding Protein/metabolism ; rab5 GTP-Binding Proteins/metabolism ; rhoA GTP-Binding Protein/biosynthesis/metabolism

OBJECTIVETo test the effect of CDC42 (a member of Rho family of small GTPases) knockdown mediated by a CDC42 short-hairpin RNA (shRNA) on the morphology of colorectal cancer SW480 cells in vitro.

METHODSFour CDC42 siRNA fragments targeting CDC42 were designed and the most efficient siRNA for CDC42 knockdown was selected to construct the shRNA vector for transfection of colorectal cancer SW480 cells. The interference efficiency in the stably transfected cells (sw480.shCDC) was detected using real-time PCR and Western blotting, and the morphological changes of the transfected cells were observed.

RESULTSWestern blotting result showed that siCDC42-3 was the most efficient fragment for CDC42 knockdown, which caused CDC42 knockdown by over 50%. DNA sequencing confirmed successful construction of the CDC42 shRNA vector. Transfection of the cells with the vector significantly reduced CDC42 expressions at both the mRNA and protein levels. The transfected cells exhibited reduced filopodia and cell size with smooth cell margins.

CONCLUSIONshRNA-mediated CDC42 knockdown can reduce the cytoskeleton dynamics of colorectal cancer cells to lower their invasiveness. This shRNA construct facilitates further study of the role of CDC42 in the tumorigenesis and progression of colorectal cancer.

Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; Gene Knockdown Techniques ; Humans ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection ; cdc42 GTP-Binding Protein ; genetics

OBJECTIVETo investigate the effect of Cdc42-shRNA plasmid to proliferation, migration, invasion and other malignant biological behavior in hepatoma SMMC-7721 cells.

METHODSCdc42-shRNA interfering vector transfected to SMMC-7721 cells with liposome method. The growth curve of transfected cells SMMC-7721, U6-control, Cdc42-shRNA2 was detected by MTT. The cells mobility was detected by wound healing experiment. Transwell chamber experiments to observe the cell migration and invasion. Detected AFP and PCNA expression level by Western blot.Human hepatoma SMMC-7721 transplanted subcutaneously in nude mouse, detected the expression of Cdc42 of the tumor by immunohistochemistry.t test was used to analyze the data between two groups.

RESULTSThe doubling time of Cdc42-shRNA2, U6-control and SMMC7721 was 42.7 h, 34.9 h and 35.1 h. The relative migration distance of Cdc42-shRNA2 and U6-control on 36 h was (47.1 ± 4.1)% and (86.6 ± 5.3)% (t=-10.21, P<0.05). Transwell chamber experimental methods showed the numbers of permeating cells were 18.2 ± 2.1(Cdc42-shRNA2) and 41.0 ± 3.5 (U6-control) (t=-9.67, P<0.05) on 24 h. The AFP and PCNA expression of hepatoma cells is significantly inhibited after the Cdc42-shRNA2 was transfected compared with U6-control group.The tumor average weight of group Cdc42-shRNA2 was (335.1 ± 178.2) mg, which was much lighter than that of SMMC-7721 group ((925.3 ± 241.4) mg) and U6-control group ((910.5 ± 225.6) mg) (t=-4.47, -4.39; P<0.05) and the Cdc42 expression was also weak positive.

CONCLUSIONCdc42 interfere with plasmid significant changes in human malignant biological behavior of hepatocellular carcinoma cells, and reduces liver cancer cell growth, invasion and metastasis of capacity.

Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Humans ; Liver Neoplasms ; Mice ; Mice, Nude ; Plasmids ; RNA, Small Interfering ; Transfection ; Tumor Burden ; cdc42 GTP-Binding Protein

OBJECTIVETo compare the change of lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice in acute lung injury.

METHODSThe mice with vascular endothelial cell-specific expression of cre recombinase were crossed with cdc42(flox/flox) mice. The cdc42(flox/+)Cre(+/-) F1 offspring mice were crossed back with cdc42(flox/flox) mice, resulting in the F2 generation mice with three genotypes, namely cdc42(flox/+)Cre(+/-), cdc42(flox/flox)Cre(-/-) and cdc42(flox/+)Cre(+/-). The heterozygous mice with cdc42(flox/+)Cre(+/-) genotype were selected as the model mice, with the other two genotype groups as the control. After intratracheal instillation of 2 mg/kg LPS to induce acute lung injury, the mice were sacrificed to examine the lung pathologies, lung wet/dry ratio and lung microvascular permeability.

RESULTSThe heterozygous mice with cdc42 gene knockout (cdc42(flox/+)Cre(+/-)) showed no significant differences from the two control groups in the lung pathological score, lung wet/dry ratio or the lung microvascular permeability coefficient.

CONCLUSIONThere were no significant difference on lung tissue and vasopermeability between the vascular endothelial cells special cdc42-deficient heterozygous mice and the non-knockout mice.

Acute Lung Injury ; pathology ; Animals ; Capillary Permeability ; Endothelial Cells ; pathology ; Integrases ; genetics ; Lung ; blood supply ; pathology ; Mice ; Mice, Knockout ; cdc42 GTP-Binding Protein ; genetics

Influenza virus assembly requires the completion of viral protein and vRNP transport to the assembly site at the plasma membrane. Therefore, efficient regulation of intracellular transport of the viral proteins and vRNPs to the surface of the host cell is especially important for virus morphogenesis. Influenza A virus uses the machineries of host cells to transport its own components including ribonucleoproteins (vRNPs) and three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and matrix 2 protein (M2). It has been shown that newly synthesized vRNPs are associated with active form of Rab11 and accumulate at recycling endosomes adjacent to the microtubule organizing center (MTOC) following nuclear export. Subsequently, they are transported along the microtubule network toward the plasma membranes in cargo vesicles. The viral transmembrane proteins are translated on the rough endoplasmic reticulum and transported to the virus assembly site at the plasma membrane. It has been found that several host factors such as ARHGAP21 and GTPase Cdc42 are involved in regulation of intracellular trafficking of influenza A virus transmembrane proteins including NA. In this review, we will highlight the current knowledge about anterograde transport and its regulation of the influenza A virus transmembrane proteins and genome in the host cytoplasm.
Cytoplasm ; metabolism ; GTP Phosphohydrolases ; metabolism ; GTPase-Activating Proteins ; metabolism ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; metabolism ; Humans ; Influenza A virus ; genetics ; pathogenicity ; physiology ; Neuraminidase ; metabolism ; Protein Transport ; Ribonucleoproteins ; metabolism ; Viral Matrix Proteins ; metabolism ; cdc42 GTP-Binding Protein ; metabolism

BACKGROUND AND OBJECTIVEMicroRNAs have emerged as post-transcriptional regulators that are critically involved in the biologic behavior of cells. This study was designed to investigate the effect of members of the microRNA-29 family on the expression of cell division cycle 42 (Cdc42) and their roles on proliferation, migration, and invasion of gastric cancer cells.

METHODSWe detected microRNA-29s and Cdc42 expression in gastric cancer cells by real-time polymerase chain reaction (PCR) and Western blot analysis. Negative controlled RNA (ncontrol), microRNA-29 family members (microRNA-29a, -29b, and -29c), and Cdc42-specific small interfering RNA (si-Cdc42) were chemically synthesized and transfected into SGC7901 and BGC823 gastric cancer cells, which have a relatively low expression of microRNA-29s and a relatively high expression of Cdc42. The expression of Cdc42 and the phosphorylation of its downstream molecular PAK1 expressions were determined by Western bolt analysis. Cell Counting Kit-8 was used to measure cell proliferation, and wound-healing and invasion assays were used to examine the abilities of migration and invasion.

RESULTSSimilar to si-Cdc42, the ectopic expression of microRNA-29 family members significantly reduced the expression of Cdc42 and its downstream molecular PAK1 phosphorylation levels. Consistently, ectopic expression of microRNA-29s inhibited proliferation and migration in gastric cancer cells. Invasive cell counts of the SGC7901, ncontrol/SGC7901, si-Cdc42/SGC7901, microRNA-29a/SGC7901, microRNA-29b/SGC7901, and microRNA-29c/SGC7901 cell groups were 84.0+/-4.2, 71.7+/-4.6, 16.3+/-3.2, 15.7+/-3.8, 16.3+/-3.0, and 16.7+/-3.1, respectively. The invasive cell counts of the BGC823, ncontrol/BGC823, si-Cdc42/BGC823, microRNA-29a/BGC823, microRNA-29b/BGC823, and microRNA-29c/BGC823 cell groups were 199.0+/-10.5, 146.3+/-9.7, 72.7+/-8.2, 86.7+/-8.5, 86.0+/-8.5, and 73.3+/-8.3, respectively (P<0.05).

CONCLUSIONSMembers of the microRNA-29 family can obviously inhibit cell proliferation, migration, and invasion of gastric cancer cells by targeting Cdc42.

Animals ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; MicroRNAs ; genetics ; metabolism ; NIH 3T3 Cells ; Neoplasm Invasiveness ; Phosphorylation ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; cdc42 GTP-Binding Protein ; metabolism ; p21-Activated Kinases ; metabolism

BACKGROUNDHigh microvascular permeability plays an essential role in pathological process of multiple diseases such as septic shock, acute lung injury and acute respiratory distress syndrome, and burns. Inhibiting hyperpermeability is significant for controlling these conditions. Cdc42, as a main member of the small Rho GTPase family, plays a critical role in controlling and regulating the endothelial junctional permeability. We aimed to generate and identify endothelial specific cdc42-deficient mice by the Cre/loxp recombination approach, for examination in an animal model of the contribution of the cdc42 gene in the microvascular barrier function.

METHODSWe crossed cdc42(Flox/Flox) mice with mice expressing endothelial cell-specific Cre recombinase, and the offspring with the genotype cdc42(Flox/+)Tie2Cre(+/-) were back-crossed with the cdc42(Flox/Flox) mice. The cdc42(Flox/Flox)Tie2Cre(+/-) mice in the F2 generation were the target mice. If the cdc42 deficient mice did not survive, we would observe the cdc42 deficient mice embryos, and compare them with wild-type mice embryos.

RESULTSCdc42(flox/+)Cre(+/-) mice were mated with the cdc42(Flox/Flox) mice and among the living offspring there were no cdc42(Flox/Flox)Cre(+/-) target mice. We found the endothelial special cdc42 deficient embryos at the E7.5-E16.5 stage. We observed that cdc42 deficient embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos.

CONCLUSIONSEndothelial specific knockout of cdc42 caused embryonic lethality and the mice did not survive to birth. The target embryos were much smaller, had fewer vessels and were a little more swollen compared with the wild-type embryos. These results demonstrated that the cdc42 plays an important role in development of embryos and in development of microvessels as well as microvascular permeability.

Animals ; Embryo, Mammalian ; blood supply ; metabolism ; Endothelium, Vascular ; embryology ; metabolism ; Female ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Neovascularization, Physiologic ; genetics ; physiology ; cdc42 GTP-Binding Protein ; genetics ; metabolism

OBJECTIVETo study differential expression proteins associated with colorectal cancer genesis and hepatic metastasis with proteomic techniques.

METHODSUsing isoelectric focusing/SDS acrylamide gel two-dimensional electrophoresis to analyse differential expression protein spots among normal colorectal mucosa, primary cancer lesion and hepatic metastasis. Peptide mass fingerprinting was used to identify the differential proteins.

RESULTSSignificant differences of protein expression were found on two-dimensional electrophoresis. Nine differential protein spots were analysed and identified. Calmodulin, ribonuclease 6 precursor and protein XP_040720 (mannosidase-alpha) were detected in normal colorectal mucosa, but lost in primary cancer lesion and hepatic metastasis. Proapolipoprotein was expressed progressively from normal mucosa to primary cancer and hepatic metastasis. Expression of beta-globin was found in normal mucosa and hepatic metastasis, but not in primary cancer lesion. Cdc42 was a differential expression protein in hepatic metastasis. Peptide mass fingerprints of differential protein spot C4, M7 and M9 had low homology with database proteins, they were candidates of associated proteins with colorectal cancer genesis and hepatic metastasis.

CONCLUSIONLoss of calmodulin, ribonuclease 6 precursor and mannosidase-alpha expression are associated with colorectal cancer genesis. Enhancement expression of proapolipoprotein is related with colorectal genesis and hepatic metastasis. Cdc42 and beta-globin are associated proteins with hepatic metastasis of colorectal cancer.

Calmodulin ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; Electrophoresis, Gel, Two-Dimensional ; Female ; Globins ; metabolism ; Humans ; Isoelectric Focusing ; Liver Neoplasms ; metabolism ; secondary ; Male ; Peptide Mapping ; Proteome ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; cdc42 GTP-Binding Protein ; metabolism