OBJECTIVETo achieve high expression of human renal cell carcinoma-associated antigen G250 in Escherichia coli.

METHODSThe gene fragments encoding the protein obtained by PCR was cloned into prokaryotic expression vector pET32a(+) and expressed in E. coli Rosseta. The immunogenicity of the recombinant protein was evaluated by Western blotting.

RESULTSThe plasmid pET32a(+)/G250 was constructed and expressed in E. coli Rosseta successfully. Western blot analysis showed that the recombinant protein could be specifically recognized by monoclonal antibody M75.

CONCLUSIONEfficient G250 expression is achieved in prokaryotic expression system, which may facilitate further functional study of the protein and its monoclonal antibody preparation.

Antibodies, Monoclonal ; immunology ; Antibody Specificity ; immunology ; Antigens, Neoplasm ; genetics ; immunology ; metabolism ; Biomarkers, Tumor ; genetics ; immunology ; metabolism ; Blotting, Western ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; immunology ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo investigate the expression of MN/CAIX in patients with renal cell carcinoma (RCC) and assess the value of MN/CAIX in the diagnosis of RCC.

METHODSRT-PCR was employed to detect MN/CAIX mRNA in the carcinoma tissue and peripheral blood of 62 patients with RCC, using normal renal tissue and peripheral blood sample from 32 patients without RCC as control. Immunohistochemistry was used to detect MN/CAIX protein in the tissue specimens of clear cell RCC (n=36), non-clear cell renal neoplasm (n=17) and normal kidney (n=16).

RESULTSThe positivity rate of MN/CAIX mRNA was 82.3% (51/62) in renal carcinoma tissues and 54.8% (34/62) in the peripheral blood from patients with RCC, significantly higher than the rates in the control cases (P<0.05). In cases of clear cell renal cell carcinoma, the positivity rate of MN/CAIX mRNA was 98% (49/50) in the carcinoma tissues and 66% (33/50) in the peripheral blood, significantly higher than the rates in cases of non-clear cell type of RCC (P<0.05). Immunohistochemistry showed a significantly higher positivity rate of MN/CAIX protein in clear cell RCC tissues [97.2% (35/36)] than in non-clear cell renal neoplasm and normal renal tissues (P<0.05).

CONCLUSIONMN/CAIX is specifically overexpressed in RCC, especially in clear cell RCC, suggesting its potential in the diagnosis and prognostic and therapeutic evaluation of RCC.

Adult ; Aged ; Antigens, Neoplasm ; genetics ; metabolism ; Biomarkers, Tumor ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; metabolism ; Carcinoma, Renal Cell ; diagnosis ; metabolism ; Female ; Humans ; Kidney Neoplasms ; diagnosis ; metabolism ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Young Adult

Adult ; Azoospermia/pathology* ; Carbonic Anhydrases/genetics* ; Congenital Abnormalities/genetics* ; Epithelial Sodium Channels/genetics* ; Gene Expression Regulation/genetics* ; Genome, Human ; Humans ; Infertility, Male/genetics* ; Male ; Male Urogenital Diseases/genetics* ; Mutation ; Vas Deferens/abnormalities*

This study was aimed to investigate the effects of bortezomib on VEGF gene expression of endothelial cell line HMEC-1, and to determine the changes of the transcriptional regulation activity of hypoxia-inducible factor 1 (HIF-1alpha) and expression intensity of Annexin A2, so as to analyze the possible mechanisms of the above expression of VEGF gene. Expression intensity of VEGF gene was determined by real-time quantitative PCR; the relative proliferation activity of cells was assayed by cell count kit CCK-8; the expression intensity of carbonic anhydrase IX (CA IX) gene was detected by RT-PCR; expression of Annexin A2 at gene and protein levels were determined by real-time quantitative PCR and Western blot respectively. The results showed that after being treated by bortezomib with 2.5, 5.0, 10 nmol/L for 12 hours, the expression intensity of VEGF gene of endothelial cell line HMEC-1 was as follows: 0.730 +/- 0.106, 0.673 +/- 0.153, 0.767 +/- 0.090 (as 1.0 was made in 0 nmol/L) (p < 0.05); the proliferation activity of cells was not significantly suppressed by bortezomib in 2.5, 5.0 nmol/L (p > 0.05), while that was significantly suppressed by bortezomib of 10 nmol/L (p = 0.024), The results from RT-PCR showed that expression intensity of CA IX gene was conspicuously down-regulated by bortezomib in different concentrations, which suggested that the transcriptional regulation activity of HIF-1alpha was inhibited by bortezomib. And down-regulated expression of Annexin A2 protein by bortezomib in different concentrations was confirmed by real-time quantitative PCR and Western blot. It is concluded that low doses of bortezomib has no significant inhibition effect on the activity of proteasome. Bortezomib may down-regulate the expression of VEGF gene of endothelial cell through regulating the activity of HIF-1alpha and the expression of Annexin A2.
Annexin A2 ; genetics ; metabolism ; Antigens, Neoplasm ; genetics ; metabolism ; Boronic Acids ; pharmacology ; Bortezomib ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; genetics ; metabolism ; Cell Line ; Down-Regulation ; Endothelial Cells ; drug effects ; metabolism ; Gene Expression ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Pyrazines ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.

METHODSA mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.

RESULTSMammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.

CONCLUSIONA vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

Antigens, Neoplasm ; metabolism ; Cancer Vaccines ; immunology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; secretion ; Interleukin-12 ; genetics ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

OBJECTIVETo demonstrate the impact of hypoxia on ER-alpha in both breast cancer tissue and cell line, and its relationship with hypoxia-related parameters.

METHODSExpression of ER-alpha in 51 breast cancer patients with ER positive determined by ligand-binding assay was examined by immunohistochemistry and compared with CA-IX and Glut-1. Impact of hypoxia on breast cancer cell line MCF-7 (ER-alpha positive) was observed by Western Blot and RT-PCR.

RESULTSOf 51 breast cancer patients, 49 were ER-alpha positive. Regional decrease of ER-alpha expression was consistently observed in peri-necrotic regions as compared to distant regions in both in-situ carcinomas (n=29, P <0.0001) and invasive carcinomas (n=20, P=0.0001), which was closely associated with the induction of CA-IX and Glut-1 in hypoxia (P <0.0001). The decreased expression of ER-alpha protein and mRNA in breast cancer cell lines were attributed to hypoxia and not to other stress factors, such as reduced glucose, low pH, and products released from necrotic or hypoxic cells. Chronic intermittent hypoxia could cause persistent down-regulation of ER-alpha in the MCF-7 breast cancer cell line.

CONCLUSIONRegional hypoxia in breast cancer is associated with the reduced ER-alpha expression, and intermittent hypoxia can cause persistent down-regulation. Hypoxia may therefore contribute to the progression of ER-alpha negative status and potentially to the development of resistance to endocrine therapy.

Antigens, Neoplasm ; metabolism ; Breast ; metabolism ; pathology ; Breast Neoplasms ; metabolism ; pathology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Carcinoma in Situ ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Down-Regulation ; Estrogen Receptor alpha ; genetics ; metabolism ; Female ; Glucose Transporter Type 1 ; Humans ; Hypoxia ; metabolism ; Monosaccharide Transport Proteins ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

Plasmodium falciparum (P. falciparum) is responsible for the majority of life-threatening cases of human malaria, causing 1.5-2.7 million annual deaths. The global emergence of drug-resistant malaria parasites necessitates identification and characterization of novel drug targets and their potential inhibitors. We identified the carbonic anhydrase (CA) genes in P. falciparum. The pfCA gene encodes anα-carbonic anhydrase, a Zn(2+)-metalloenzme, possessing catalytic properties distinct from that of the human host CA enzyme. The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes. A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions. The structure of the groups substituting the aromatic-ureido- or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides. One derivative, that is, 4- (3, 4-dichlorophenylureido)thioureido-benzenesulfonamide (compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor, and was also the most effective antimalarial compound on the in vitro P. falciparum growth inhibition. The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei, an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria.
Animals ; Antimalarials ; pharmacology ; therapeutic use ; Carbonic Anhydrase Inhibitors ; pharmacology ; therapeutic use ; Carbonic Anhydrases ; chemistry ; genetics ; metabolism ; Catalysis ; Genome, Protozoan ; Genomics ; Humans ; Life Cycle Stages ; Malaria, Falciparum ; drug therapy ; parasitology ; Parasites ; drug effects ; enzymology ; Plasmodium falciparum ; drug effects ; enzymology ; genetics ; growth & development ; Protein Conformation ; Sulfonamides ; pharmacology ; therapeutic use

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.
Animals ; Azacitidine/*analogs & derivatives/pharmacology ; Carbonic Anhydrases/metabolism ; *DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; Mice ; Neoplasm Invasiveness/genetics ; Neoplasm Metastasis/genetics/*pathology ; Neoplasms, Experimental ; Neoplasms, Glandular and Epithelial/genetics/*metabolism/pathology ; Ovarian Neoplasms/genetics/*metabolism/pathology ; Phenotype ; Promoter Regions, Genetic ; RNA, Messenger/metabolism