ABSTRACT
Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate whether progesterone receptor B (PRB) can be sumoylated by SUMO-2/3 and the effect of sumoylation on PRB transcriptional activity.</p><p><b>METHODS</b>SUMO-2/3 cDNA was amplified from MCF-7 cDNA and cloned into the eukaryotic expression vector pcDNA3-FLAG. The plasmid pXJ40-myc-PRB was cotransfected with pcDNA3FLAG-SUMO2, pcDNA3FLAG-SUMO3 or the mock control into 293T cells, and PRB sumoylation was detected by immunoprecipitation and Western blotting. The effect of PRB sumoylation on its transcriptional activity was determined using reporter luciferase assay.</p><p><b>RESULTS</b>pcDNA3FLAG-SUMO2 and pcDNA3FLAG-SUMO3 vectors were successfully constructed. SUMO-2/3 could bind covalently to PRB and increase its transcriptional dependent on the presence of progesterone.</p><p><b>CONCLUSION</b>PRB can be sumoylated by SUMO-2/3 and its function is regulated by this modification.</p>
Subject(s)
Animals , Humans , Cell Line , Plasmids , Genetics , Receptors, Progesterone , Genetics , Metabolism , Small Ubiquitin-Related Modifier Proteins , Genetics , Metabolism , Transcription, Genetic , Transfection , Ubiquitination , Ubiquitins , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To identify the expression of Drosophila Eyes Absent Homologue 2 (EYA2) in non-small cell lung cancer (NSCLC) and to investigate its correlation with clinical parameters.</p><p><b>METHODS</b>59 fresh specimens of lung cancer and paired normal lung tissue were obtained from 59 NSCLC cases treated in the department of thoracic surgery in our hospital from June 2006 to October 2007. Western blotting and immunohistochemistry were used to assay the specimens with goat anti-human EYA2 polyclone antibody. Clinicopathological parameters were collected and the correlation with EYA2 expression was subsequently analyzed.</p><p><b>RESULTS</b>The expression of EYA2 was detected in cytoplasm and nucleus of the cancer cells, but mostly in cytoplasm. Western blotting and immunohistochemistry showed the expression of EYA2 in NSCLC was increased and correlated with pathological type, but not with gender, age, pTNM stage, histological differentiation and lymph node metastasis. EYA2 expression was significantly up-regulated in adenocarcinoma, while not changed in lung squamous cell carcinoma.</p><p><b>CONCLUSION</b>The results of this study suggest that expression of EYA2 in lung adenocarcinoma is augmented. EYA2 is likely participating in the development of lung adenocarcinoma as a transcriptional activator.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Adenocarcinoma , Metabolism , Pathology , Carcinoma, Non-Small-Cell Lung , Metabolism , Pathology , Carcinoma, Squamous Cell , Metabolism , Pathology , Cytoplasm , Metabolism , Intracellular Signaling Peptides and Proteins , Metabolism , Lung , Metabolism , Lung Neoplasms , Metabolism , Pathology , Lymphatic Metastasis , Neoplasm Staging , Nuclear Proteins , Metabolism , Protein Tyrosine Phosphatases , Metabolism , Up-RegulationABSTRACT
<p><b>OBJECTIVES</b>To study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).</p><p><b>METHODS</b>Both HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.</p><p><b>RESULTS</b>Both HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.</p><p><b>CONCLUSIONS</b>HBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.</p>
Subject(s)
Humans , Histone Deacetylase 1 , Metabolism , Hydroxamic Acids , Metabolism , Immunoprecipitation , Plasmids , Protein Interaction Mapping , Trans-Activators , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the features of HBx protein distributed in liver cells and its expression in E. coli.</p><p><b>METHODS</b>The expression vectors encoding the full length HBx and its mutants were constructed by the routine molecular cloning method. HBx protein expression was detected using Western blotting. The distribution feature of HBx protein in liver cells was examined using the fluorescence confocal microscopy. A series of purified HBx fusion proteins were obtained by glutathione-sepharose 4B affinity chromatography.</p><p><b>RESULTS</b>The expression vectors were successfully constructed for the full length HBx and its mutants. HBx was found distributed uniformly in the nuclei but granularly in the cytoplasm of the liver cells. Under optimal conditions, the mutant GST-HBx (72-120aa) was easily degraded.</p><p><b>CONCLUSION</b>This study may provide a basis for further study on the biological function of HBx at the protein level.</p>
Subject(s)
Humans , Carcinoma, Hepatocellular , Pathology , Cell Line , Cloning, Molecular , Escherichia coli , Metabolism , Genetic Vectors , Glutathione Transferase , Genetics , Hepatocytes , Cell Biology , Metabolism , Liver , Cell Biology , Liver Neoplasms , Pathology , Mutation , Recombinant Fusion Proteins , Genetics , Trans-Activators , Genetics , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the effects of exogenous ER beta on the growth of breast cancer MCF-7 cells under different treatment.</p><p><b>METHODS</b>An eukaryotic expression vector containing 1.6 kb of human entire coding sequence of ER beta (pCDNA3-ER beta) was transfected into human breast cancer MCF-7 cells using lipofectamine 2000. The biological activity of ER beta was detected with the luciferase reporter containing estrogen responsive element (ERE) and the expression of ER beta protein by Western blot. The growth properties of MCF-7, pCDNA 3-transfected MCF-7 and pCDNA 3-ER beta-transfected MCF-7 cells under different treatment, including E2 (17beta-estradiol) and 4-OHT (4-hydroxytamoxifen), were observed.</p><p><b>RESULTS</b>A stronger activation of the reporter by ER beta in the presence of E2 was observed in the pCDNA 3-ER beta-transfected MCF-7 cells than in the pCDNA 3-transfected MCF-7 and in MCF-7 cells. Western blot analysis showed that the protein level of ER beta in the pCDNA 3-ER beta-transfected MCF-7 cells was markedly increased. Exogenous ER beta expression did not change the growth properties and the morphology of MCF-7 cells under normal condition. The pCDNA 3-ER beta-transfected MCF-7 cells proliferated at the same rate as naive cells in the presence of 4-OHT, whereas a strong inhibition of the proliferation of the pCDNA 3-ER beta-transfected MCF-7 cells in the presence of E2 was observed.</p><p><b>CONCLUSION</b>Exogenous ER beta expression does not increase the resistance to 4-OHT, and a strong inhibition of the proliferation may occur in the presence of E2.</p>
Subject(s)
Female , Humans , Breast Neoplasms , Metabolism , Pathology , Cell Line, Tumor , Cell Proliferation , Estradiol , Pharmacology , Estrogen Antagonists , Pharmacology , Estrogen Receptor beta , Genetics , Metabolism , Tamoxifen , Pharmacology , TransfectionABSTRACT
The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.
ABSTRACT
Estrogen receptor alpha (ERalpha) has been a primary target of treatment as well as a prognostic indicator for breast cancer. The level of human X-box binding protein 1 (XBP-1) mRNA was related with that of ERalpha in breast tumors and was over-expressed in some breast tumors. These previous studies suggested that XBP-1 may interact with ERalpha. XBP-1 has two isoforms, XBP-1S and XBP-1U, as the result of unique splicing. GST pull-down assay showed that both XBP-1S and XBP-1U bound to ERalpha in vitro. The binding of XBP-1S to ERalpha was stronger than that of XBP-1U to ERalpha. Co-immunoprecipitation revealed that the binding was in a ligand-independent manner. XBP-1S and XBP-1U interacted with the region of ERalpha that contains a DNA-binding domain. The ERalpha-interacting regions on XBP-1S and XBP-1U have been mapped to two regions, the N-terminal basic region leucine zipper domain (bzip) and the C-terminal activation domain. These findings suggest that XBP-1S and XBP-1U may participate in ERalpha signaling pathway through the mediation of ERalpha.
Subject(s)
Female , Humans , Breast Neoplasms , Genetics , Metabolism , Cell Line, Tumor , DNA-Binding Proteins , Genetics , Metabolism , Estrogen Receptor alpha , Genetics , Metabolism , Protein Interaction Domains and Motifs , Physiology , RNA, Messenger , Genetics , Regulatory Factor X Transcription Factors , Signal Transduction , Transcription Factors , Genetics , Metabolism , X-Box Binding Protein 1ABSTRACT
<p><b>OBJECTIVE</b>To construct an ERbeta expression vector and study its expression and function in different cancer cells.</p><p><b>METHODS</b>Standard PCR was used to amplify the full-length coding sequence of ERbeta. The amplified ERbeta gene was cloned into the eukaryotic expression vector pCDNA3, generating pCDNA3-ERbeta. The ERbeta expression was detected by Western blot and in vitro translation. The biological activity of ERbeta was detected by transfecting the pCDNA3-ERbeta into SV40-transformed embryonic kidney cell line 293T,breast cancer cell lines MDA-MB-435, MDA-MB-436, SKBR3, and prostate cancer cell line PC-3, with reporters containing estrogen response elements.</p><p><b>RESULTS</b>The recombinant plasmid pCDNA3-ERbeta was confirmed by restriction analysis to contain the ERbeta gene. The 63 000 ERbeta expression was shown by Western blot and further confirmed by in vitro translation. The ERbeta expression in different cancer cells was demonstrated to stimulate the expression of the reporters containing estrogen response elements, ERE and C3.</p><p><b>CONCLUSION</b>ERbeta protein is successfully expressed and has biological activity, laying solid foundation for further study on its role in cancer cells.</p>
Subject(s)
Female , Humans , Male , Breast Neoplasms , Metabolism , Pathology , Cell Line , Cell Line, Tumor , Embryo, Mammalian , Epithelial Cells , Estrogen Receptor beta , Genetics , Metabolism , Genes, Reporter , Genetics , Genetic Vectors , Kidney , Cell Biology , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , Recombinant Proteins , Genetics , Metabolism , Response Elements , Genetics , TransfectionABSTRACT
Breast cancer susceptibility gene 1(BRCA1) plays an important role in breast cancer development and progression. BRCA1 encodes a 1863-amino acid protein with two BRCA1 C-terminal (BRCT) domains at its C-terminus, BRCT1 and BRCT2. Many cancer-predisposing mutations are located in the BRCT domains, which have been shown to induce chromatin unfolding by use of an approach that allows visualization of large-scale chromatin structure through lac repressor/lac operator recognition. To map the important region of BRCT domain (amino acid residues 1642-1736), six deletion mutant constructs were made. The chromatin structure assay showed that amino acid residues 1691-1721 are involved in the induction of chromatin unfolding. To further localize the critical amino acid residues, ten alanine scanning mutant constructs were made. The chromatin structure assay demonstrated that the 1707IAGGK1711 region is critical for the chromatin unfolding activity. Based on the mapped important region, Blast analysis identified a novel homologous protein. Mapping of the BRCT1 domain may aid in the presymptomatic risk assessment and provide a valuable tool for further study on the BRCT1 structure and function.