The present study aimed to investigate the effect of proto-oncogene c-src on the viability of rat spermatogonial stem cells from 9 day-old rat in vitro. MTT method was used to observe the viability of the spermatogonial stem cells treated with antisense c-src oligodeoxynucleotides (ODNs) in vitro; RT-PCR was utilized to observe the expression of c-src mRNA and Western blot was used to observe the protein expressions of pp60c-src and phosphorylated signal transducer and activator of transcription-3 (p-STAT3). Compared with that in control group, the viability of spermatogonial stem cells decreased by 8.1% (P<0.05) and the expression of c-src mRNA decreased significantly after treatment with 10 μmol/L antisense c-src ODNs for 12 h. Compared with that in the control group, the protein expressions of pp60c-src and p-STAT3 decreased by 33.8% and 45.3% (both P<0.01), respectively, in the spermatogonial stem cells after being transfected with antisense c-src ODNs. The results suggest that proto-oncogene c-src regulates the viability of rat spermatogonial stem cells through p-STAT3.
Animals ; Cells, Cultured ; Genes, src ; Male ; Phosphorylation ; Proto-Oncogene Proteins pp60(c-src) ; metabolism ; RNA, Messenger ; Rats ; STAT3 Transcription Factor ; metabolism ; Spermatogonia ; cytology ; metabolism ; Stem Cells ; cytology ; metabolism ; Transfection

The present study was to determine the effect of c-SRC on the viability of human cervical cancer HeLa cells and the expression of phosphorylated signal transducer and activator of transcription-3 (p-STAT3) of the cell. Post-transfection of c-SRC RNA interference vector, RT-PCR and Western blot were utilized to observe the contents of c-SRC mRNA and protein, respectively, in HeLa cells. The MTT was used to observe the viability of the cells. Cell cycle was observed by flow cytometry. The content of p-STAT3 in the cells was also investigated after knockdown of c-SRC. Knockdown of c-SRC significantly decreased the contents of c-SRC mRNA and protein in the cells. The viability of the cells decreased by 23.1%, 29.3%, 38.6% and 45.0% (all P < 0.05), respectively, after the cells were transfected with c-SRC RNA interference vector for 24, 48, 72, and 96 h. The number of S-phase cells decreased by 5.6%, 10.0%, 15.2% and 19.9% (all P < 0.05), respectively, after transfection of c-SRC RNA interference vector for 24, 48, 72, and 96 h. The content of p-STAT3 also decreased when c-SRC was knockdowned. Compared with the control group, after treatment of HeLa cells with STAT3 inhibitor Piceatannol for 24, 48, 72, and 96 h, the cell viability decreased by 23.8%, 29.7%, 37.3% and 45.4% (all P < 0.05), respectively, while increase of c-SRC content could not reverse the inhibitory effect. These results suggest that the inhibited viability of HeLa cells caused by knockdown of c-SRC is associated with the decreased content of p-STAT3 protein.
Cell Survival ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Genes, src ; genetics ; HeLa Cells ; Humans ; Phosphorylation ; RNA, Messenger ; biosynthesis ; genetics ; STAT3 Transcription Factor ; genetics ; metabolism ; Transfection

PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.
Cell Line ; Cell Proliferation ; Genes, ras ; Humans ; Hydroquinones ; Lipids ; NIH 3T3 Cells ; Parents ; Phenotype ; Proteins ; Proto-Oncogene Proteins c-raf ; RNA, Small Interfering ; src-Family Kinases ; Transfection ; Tyrosine

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.
Adenocarcinoma/*metabolism/pathology/*secondary ; Antigens, Surface ; Cell Adhesion/genetics ; Cell Aggregation/genetics ; Gene Expression Regulation ; Genes, Tumor Suppressor ; Genes, src ; Human ; Male ; Membrane Glycoproteins/genetics/*metabolism ; Prostatic Neoplasms/*metabolism/pathology/*secondary ; Signal Transduction/genetics ; Tumor Cells, Cultured ; src-Family Kinases/genetics/metabolism

Rhei Rhizoma is a Chinese medicine with multiple botanical origins. There is a problem to identify it with conventional methods. To compare the characteristics of chloroplast matK gene sequences of different Rheum species and authenticate inspected species, the matK gene sequences of different species from different origins were amplified, cloned, and sequenced. Genomic DNA of Rheum plants was extracted using modified DNA extracted Kit and matK gene sequences were analyzed by ContingExpress, DNAman and MEGA5.0. The length of matK gene sequences of Rheum palmatum, R. tanguticum and R. officinale were 1 518 bp containing 57 variable loci. According to the mutation sites, R. palmatum, R. tanguticum and R. officinale were divided into different genotypes separately. Based on the established method according to the loci 587, 707, 838, we successfully identified the genuine Rheum species from its adulterants.
Amino Acid Sequence ; Base Sequence ; DNA, Plant ; genetics ; Drug Contamination ; Genes, Chloroplast ; Genes, Plant ; Genotype ; Molecular Sequence Data ; Mutation ; Phylogeny ; Plants, Medicinal ; genetics ; Proto-Oncogene Proteins pp60(c-src) ; genetics ; Rheum ; classification ; genetics ; Rhizome ; genetics ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVECloning and characterization of a novel gene by exon trapping and exon linking at chromosome 8p22.

METHODSA novel gene was cloned using exon trapping and exon linking, and its expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot.

RESULTSA sequence containing 3 exons was found. The sequence is homologous with the putative gene AK024799 which consists of 2880 bp cDNA with 1362 bp open reading frame and codes 454 amino acids with an SH(2) domain. The gene was named SH(2)A at chromosome 8p22. SH(2)A gene is ubiquitously expressed in various tissues with three transcripts. The aberrant expression of SH(2)A gene in some cancers was detected.

CONCLUSIONSH(2)A is a novel docking protein of SH(2) signaling protein family, which may play an important role in cellular signal transduction. It relates to the pathogenesis of tumor.

Animals ; Blotting, Northern ; COS Cells ; Carrier Proteins ; Chromosome Mapping ; Chromosomes, Human, Pair 8 ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Exons ; Gene Expression ; Genes ; genetics ; Guanine Nucleotide Exchange Factors ; Humans ; Introns ; Membrane Proteins ; genetics ; Molecular Sequence Data ; RNA ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; rac1 GTP-Binding Protein ; src Homology Domains ; genetics