To study the expression of hypoxia inducible factor-1alpha (HIF-1alpha) protein in prostate cancer (Pca) and its biological significance, the expression of HIF-1alpha was assayed by means of immunohistochemical technique in 42 prostate cancer, 12 prostatic intraepithelial neoplasm (PIN) and 9 normal prostate tissue (NP) specimens. Western blot was used to examine the expression of HIF-1alpha in prostate cancer cell line (PC-3M) induced by different oxygen tension. HIF-1alpha expression was positive in 33 Pca and 9 PIN specimens, and the positive rate of HIF-1alpha was higher in distant metastasis patients than in patients without metastasis of prostate cancer (P<0.05), while there was no expression of HIF-1alpha in NP. The level of HIF-1alpha in PC-3M significantly increased with the decrease of oxygen tension (P<0.01). Overexpression of HIF-1alpha is the preliminary event of the formation of Pca, which may induce carcinoma into malignant phenotype. Thus it may serve as an early diagnosis marker and the novel target for Pca treatment.
Adenocarcinoma/*metabolism ; Cell Line, Tumor ; Hypoxia-Inducible Factor 1, alpha Subunit/*biosynthesis ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics ; Prostatic Neoplasms/*metabolism ; Tumor Markers, Biological/*biosynthesis

OBJECTIVETo observe the distribution of hypoxia inducible factor-1alpha (HIF-1alpha) in different brain regions in aged rats and investigate the role of HIF-1alpha in the aging process of the nervous system.

METHODSThe Nissl bodies and HIF-1alpha expression in different brain regions were observed in rats aged 3 and 30 months using Nissl staining and immunohistochemical method, respectively.

RESULTSIn the 30-month-old rats, the neural cells in 4 different brain regions presented with large cell body and loose alignment, containing reduced Nissl bodies in the cytoplasm. Compared with the 3-month-old rats, the aged rats showed greater number of HIF-1alpha-positive cells in the brain (P < 0.01), and the number varied significantly between the different brain regions (P < 0.01). The CA3 region contained the greatest number of positive cells, which were fewer in the motor cortex and cerebellum.

CONCLUSIONThe capacity for protein synthesis in the neural cells is weakened but the expression of HIF-1alpha increased in aged rats, suggesting the important role that HIF-1alpha may play in the aging process of the nervous system, especially in hypomnesis.

Aging ; metabolism ; Animals ; Brain ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; Male ; Rats ; Rats, Sprague-Dawley

To study the expression of hypoxia inducible factor-1alpha (HIF-1alpha) protein in prostate cancer (Pca) and its biological significance, the expression of HIF-1alpha was assayed by means of immunohistochemical technique in 42 prostate cancer, 12 prostatic intraepithelial neoplasm (PIN) and 9 normal prostate tissue (NP) specimens. Western blot was used to examine the expression of HIF-1alpha in prostate cancer cell line (PC-3M) induced by different oxygen tension. HIF-1alpha expression was positive in 33 Pca and 9 PIN specimens, and the positive rate of HIF-1alpha was higher in distant metastasis patients than in patients without metastasis of prostate cancer (P<0.05), while there was no expression of HIF-1alpha in NP. The level of HIF-1alpha in PC-3M significantly increased with the decrease of oxygen tension (P<0.01). Overexpression of HIF-1alpha is the preliminary event of the formation of Pca, which may induce carcinoma into malignant phenotype. Thus it may serve as an early diagnosis marker and the novel target for Pca treatment.
Adenocarcinoma ; metabolism ; Biomarkers, Tumor ; biosynthesis ; Cell Line, Tumor ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Male ; Prostatic Neoplasms ; metabolism

OBJECTIVETo construct an adenovirus vector containing the double-mutant hypoxia-inducible factor-1alpha (HIF-1alpha) gene for exploring the therapeutic angiogenesis for coronary heart disease.

METHODSHuman double-mutant HIF-1alpha cDNA was obtained from PCR of pShuttle-2-HIF-1alpha containing the mutant HIF-1alpha gene (564). The recombinant adenoviral plasmid containing mutant HIF-1alpha cDNA was constructed by ligation of recombinant pShuttle2 containing double-mutant HIF-1alpha cDNA and Adeno-X viral DNA, followed by its identification and transfection into adenoviral packaging cells HEK293 via lipofectamine 2000.

RESULT AND CONCLUSIONThe recombinant pAdeno-HIF-1alpha was correctly constructed and verified by restriction endonuclease and DNA sequencing analyseis. This recombinant adenovirus containing the double-mutant HIF-1alpha gene may facilitate further investigation of mutant HIF-1alphagene therapy for coronary heart disease.

Adenoviridae ; genetics ; Cell Line, Tumor ; Coronary Disease ; therapy ; Genetic Therapy ; Genetic Vectors ; biosynthesis ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Plasmids

OBJECTIVETo investigate the expression and clinical significance of HIF-1a protein in hepatocellular carcinoma (HCC) tissues.

METHODSImmunohistochemistry (IHC), Western blotting and RT-PCR techniques were used to detect the expression of the HIF-1a gene protein in 35 HCC, 26 cirrhotic and 15 normal liver tissues. Their relationship with the pathological characteristics of the tumors were also analyzed.

RESULTSThe positive rates of HIF-1a expression in HCC tissues was 94%, which was similar to the positive rates of HIF-1a expression in liver cirrhosis tissues of 92%, but was higher than that in normal hepatic tissues of 7%, but the residual proliferatic hepatic trabeculae among the necrotic liver cells and the fibrotic tissues expressed HIF-1a strongly in comparison with the cirrhotic liver tissues. The expression intensity of HIF-1a protein of the cirrhotic liver tissues was stronger than that in HCC; the results by Western blotting and RT-PCR were in accordance to that by IHC. In addition, the expression intensity in HCC had a negative correlation in differentiation degree and a positive correlation to intrahepatic and extrahepatic metastases but no correlation was found between HIF-1a expression and the existence of portal vein tumor emboli, prognosis and the status of HBsAg.

CONCLUSIONHIF-1 protein was expressed in HCC and cirrhotic liver tissues, and was only affected by the factor of hypoxia. The expression of HIF-1a protein is associated with the differentiation of the tumor and its intrahepatic and extrahepatic metastases but was not related to the existence of portal vein tumor emboli, prognosis and the status of HBsAg. This phenomenon may provide a new idea for the treatment of liver cancer.

Adolescent ; Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; Neoplasm Metastasis

OBJECTIVETo investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1&alpha; (Ad-HIF-1&alpha;-564/402/803).

METHODSAd-HIF-1&alpha;-564/402/803 was amplified from HEK293A cells and purified by ultracentrifugation in CsCl gradient solutions. The infection efficiency was observed by X-gal staining. The lyophilized adenovirus was prepared under appropriate conditions. Before and after lyophilization, the effect of Ad-HIF-1&alpha;-564/402/803 on hMVEC proliferation was evaluated by MTS assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis before and 1 day, 6 months and 12 months after lyophilization, and hMVECs infected with Ad-HIF-1&alpha;-564/402/803 at these time points were examined for HIF-1&alpha; protein expression using Western blotting.

RESULTSNo significant changes were observed in the effect of lyophilized Ad-HIF-1&alpha;-564/402/803 on hMVECs proliferation at the optimal multiplicity of infection of 100 pfu/cell (P>0.05). At the 4 time points, the recombinant adenovirus HIF-1&alpha; showed no structural alterations or significant changes in the expression level of HIF-1&alpha; protein in the transfected hMVECs (P>0.05).

CONCLUSIONLyophilized Ad-HIF-1&alpha;-564/402/803 can maintain its biological activities for a long time.

Adenoviridae ; genetics ; metabolism ; Antibodies, Monoclonal ; genetics ; Freeze Drying ; Genetic Vectors ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Mutant Proteins ; genetics ; metabolism

OBJECTIVETo investigate the feasibility of stable transfection of human hypoxia inducible factor-1alpha (HIF-1alpha) gene into fibroblasts cells and the effects of supernatant from the transfected cell culture on hair follicle cells.

METHODSPcDNA-HIF1alpha was stably transfected into fibroblasts cells with lipofectamine 2000. Expression of HIF-1alpha was observed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The supernatant was obtained to detect the expression of vascular endothelial growth factor (VEGF) by ELISA. The mRNA expression of basic fibroblast growth factor (bFGF) was detected by RT-PCR. MTT was used to detect the activity of fibroblasts cells and dermal sheath cells added with supernatant.

RESULTSPcDNA-HIF1alpha was successfully transfected into fibroblasts cells. HIF-1alpha could be detected by RT-PCR and Western blot. The expression of VEGF in the supernatant of cells transfected with PcDNA-HIF1alpha was detected. The mRNA expression of bFGF was significantly higher than in the control group (P < 0.01). MTT showed the activity of cells added with supernatant was enhanced (P < 0.05).

CONCLUSIONPcDNA-HIF1alpha can stably transfected into fibroblasts cells, and the expressed HIF-1alpha induces the expression of VEGF and bFGF, and the expressed VEGF enhances the activity of cells.

Cell Culture Techniques ; Fibroblast Growth Factor 2 ; biosynthesis ; Fibroblasts ; metabolism ; Hair Follicle ; cytology ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo investigate the effect of hypoxia on chronic alcoholic liver disease.

METHODSTwenty four male Sprague-Dawley rats were randomly into two groups. The alcohol group (n=12) was fed 56% (v/v) of ethanol once per day by gastric infusion at 8 g/kg body weight for 24 weeks. The control group (n=12) was gastrically infused with normal saline with the same dose. At the end of 24 weeks, a blood sample was collected for determination of hepatic enzymes and then the rat was killed. Liver specimens were obtained for immunohistochemical staining and frozen at -80 degrees C used for RT-PCR.

RESULTSSerum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity increased significantly compared to the control group. A significant elevation in the expression of HIF1-alpha in liver of alcohol group was found compared to the control group.

CONCLUSIONHypoxia inducible factor 1-alpha expression was activated by ethanol-induced injury. This information suggested that hypoxia was involved in mechanism of alcoholic liver disease.

Animals ; DNA-Binding Proteins ; biosynthesis ; genetics ; Hypoxia ; complications ; metabolism ; Hypoxia-Inducible Factor 1 ; Hypoxia-Inducible Factor 1, alpha Subunit ; Liver Diseases, Alcoholic ; complications ; metabolism ; pathology ; Male ; Nuclear Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; biosynthesis ; genetics

To investigate the influence of HIF-1alpha overexpression on the differentiation of endothelial progenitor cells (EPCs) ex vivo, EPCs were isolated from human peripheral blood by density gradient centrifugation, overexpressed HIF-1alpha was transfected to EPCs by electroporation; HIF1alpha, HIF1beta, vascular endothelial growth factor (VEGF) mRNA level were measured with RT-PCR; HIF-1alpha protein was detected with immunohistochemistry in a time course. CD31(+) cells were measured with flow cytometry. Cell morphology was observed after transfection. The results showed that the transfection efficiency of HIF-1alpha to EPCs was about 20%. HIF-1alpha and its controlled target gene VEGF were markedly induced by HIF-1alpha vector (P < 0.05). HIF1beta had its same level as it before interference (P > 0.05). HIF-1alpha protein was induced by HIF-1alpha transfection after 12 hours but was undetectable at 24 hours. After 7 - 14 days cultured in 21% oxygen pressure, fluorescence-trace experiments revealed that CD31 + EPCs/EC could be generated more efficiently from overexpressed HIF-1alpha than that from pEGFP transfected group (P > 0.05). EPC morphology was observed by light microscopy. HIF-1alpha-transfected cells under normoxia sprouted more rapidly from the EPC colonies than the untransfected cells or cells transfected with an GFP vector, which essentially maintained the original colony formation. HIF-1alpha transfected cells took on an array-like arrangement rather than random dispersal, suggesting that they were in an advanced state of differentiation. It is concluded that the utility of overexpression of HIF-1alpha can induce target genes which have influence on cell differentiation. HIF-1alpha transfection was found to give a prospected way to do the insight research on ischemic treatment in vivo.
Cell Differentiation ; drug effects ; Endothelial Cells ; cytology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Plasmids ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Stem Cells ; cytology ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo investigate the transcription level and protein expression of HIF-1alpha and VEGF in SW480 cell line and colorectal adenocarcinoma, and to determine whether HIF-1alpha plays a role in angiogenesis through its regulation of VEGF.

METHODSHIF-1alpha mRNA expression was analyzed by in situ hybridization. HIF-1alpha and VEGF protein expressions were determined by immunochemical streptavidin/peroxidase (SP) in SW480 cells and colorectal carcinoma tissue samples and Western blot, using proteins extracted from SW480 cells. Tumor tissue microvessel density (MVD) was determined by CD34 immunostaining of colorectal carcinomas.

RESULTSThe levels of HIF-1alpha mRNA changed significantly in response to different oxygen concentrations and an addition of genistein in SW480 cells. Immunocytochemistry revealed that the levels of HIF-1alpha, VEGF protein expression in SW480 cells were significantly higher under hypoxia than those in nomoxia (P < 0.01, P < 0.05 respectively). However, addition of genistein, an inhibitor of HIF-1alpha, suppressed such responses to hypoxia. Western blot analysis showed that SW480 cells exposed to hypoxia expressed a high level of HIF-1alpha protein, compared to a weak expression in nomoxia. The addition of genistein in hypoxia suppressed the over-expression of HIF-1alpha. The positive rates of HIF-1alpha mRNA by in situ hybridization in colorectal adenomas and adenocarcinomas were 38.9% (7/18) and 67.7% (42/62), respectively. The percentage of HIF-1alpha mRNA positive cells varied significantly from colorectal adenomas to adenocarcinomas at different Duke stages (P < 0.05), and HIF-1alpha mRNA was higher in adenocarcinomas than in adenomas (P < 0.01). The positive rates of HIF-1alpha and VEGF protein expression in adenocarcinomas were 43.5% (27/62) and 37.1% (23/62), respectively. The expression of VEGF elevated as the Duke tumor staging increased. The conformation rate of HIF-1alpha and VEGF was 74.2% (46/62). MVD was significantly higher in HIF-1alpha and/or VEGF positive tumors than those without (P < 0.01 and P < 0.05 respectively). Among the four groups, i.e. HIF-1alpha+/VEGF+, HIF-1alpha+/VEGF-, HIF-1alpha+/VEGF- and HIF-1alpha-/VEGF-, the difference of MVD was highly significant (P < 0.01). HIF-1alpha expression was correlated significantly with VEGF expression and microvessel density.

CONCLUSIONSThese findings suggest hypoxia induces the expression of HIF-1alpha and VEGF in colorectal adenocarcinoma. HIF-1alpha may play an important role in angiogenesis and tumor progression by regulating the expression of VEGF in human colorectal carcinoma.

Adenocarcinoma ; blood supply ; metabolism ; pathology ; Colorectal Neoplasms ; blood supply ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Microcirculation ; pathology ; Neovascularization, Pathologic ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics