ABSTRACT
<p><b>OBJECTIVE</b>To investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis.</p><p><b>METHODS</b>LoVo cells were infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 and control virus Ad-lacZ under normoxia condition. Real-time PCR was used to detect HIF-1alpha and p21WAF1/CIP1 mRNA expressions at different time points. Western blotting was employed to verify HIF-1alpha and p21WAF1/CIP1 protein expression. Hoechst 33342 flourescein staining was performed to observe the ratio of apoptotic LoVo cells.</p><p><b>RESULTS</b>The expression levels of HIF-1alpha mRNA and protein increased after infection with Ad-HIF-1alpha- Ala564-Ala803, accompanied by an increase in p21WAF1/CIP1 mRNA and protein expressions. The apoptotic ratio was significantly higher in LoVo cells infected with recombinant Ad-HIF-1alpha-Ala564-Ala803 (16.2%) than in the control cells (5.5%, P=0.00).</p><p><b>CONCLUSION</b>HIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Apoptosis , Genetics , Physiology , Blotting, Western , Cell Line, Tumor , Colonic Neoplasms , Genetics , Metabolism , Pathology , Cyclin-Dependent Kinase Inhibitor p21 , Genetics , Genetic Vectors , Genetics , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Mutagenesis, Site-Directed , Mutation , RNA, Messenger , Genetics , Recombinant Proteins , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To construct an adenovirus vector containing the double-mutant hypoxia-inducible factor-1alpha (HIF-1alpha) gene for exploring the therapeutic angiogenesis for coronary heart disease.</p><p><b>METHODS</b>Human 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.</p><p><b>RESULT AND CONCLUSION</b>The 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.</p>