ABSTRACT
Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2). However, whether Otub1 regulates MDMX stability and activity is not clear. Here we report that Otub1 also suppresses MDM2-mediated MDMX ubiquitination in cells and in vitro, independently of its deubiquitinating enzyme activity. Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX. Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria-mediated apoptotic pathway. Knockdown of MDMX reduced Otub1-induced p53S46P, which was shown to be critical for p53's mitochondrial function and apoptotic activity. Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion. Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria-mediated apoptosis, providing an alternative mechanism of Otub1's role in apoptosis.
Subject(s)
Apoptosis/genetics , Cysteine Endopeptidases/genetics , Mitochondria/genetics , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Apoptosis/radiation effects , Cell Cycle Proteins , Cell Line, Tumor , Cysteine Endopeptidases/metabolism , Deubiquitinating Enzymes , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Mitochondria/metabolism , Nuclear Proteins/metabolism , Phosphorylation/radiation effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Serine/genetics , Serine/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Ubiquitination , Ultraviolet RaysABSTRACT
BACKGROUND: Exercise is beneficial for blood glucose metabolism. However, whether moderate aerobic exercise could improve impaired fasting glucose is unknown. And the mechanism is also needed to investigate. METHODS: A cross-sectional research was performed and 120 participants with impaired fasting glucose (IFG) were randomly assigned into active and controlled groups. Briefly, participants in active group were required to take moderate aerobic exercise at least 30 min for five times per week, whereas in controlled group, participants were also advised to take exercise but not mandatorily required the same degree as that of active group. At baseline and 3 month's follow-up, laboratory and demographic variables were compared. RESULTS: At baseline, no significant between-group differences were observed. Generally, leukocyte ROCK2 activity in the active and controlled groups were 58.7 ± 6.0 mg/mL and 60.2 ± 7.3 mg/mL, and daily average exercise time at baseline in both groups was extremely little, with 5.2 ± 3.8 min and 5.9 ± 3.5 min, respectively. After 3 months' follow-up, 52 and 56 participants in the active and controlled groups completed the whole program. Compared to baseline, leukocyte ROCK2 activity and daily average exercise time were improved in both groups. Nonetheless, compared to the controlled group, leukocyte ROCK2 activity was reduced more profoundly and the daily average exercise time was longer in the active group (37.5 ± 6.3 min versus 18.3 ± 7.2 min, p < 0.05). Moreover, the percentage of IFG in the active group was decreased more prominently than the controlled group (76.9% versus 82.1%, p < 0.05). Multivariate regression analyses revealed that exercise time and leukocyte ROCK2 activity was significantly associated with IFG, with OR of 0.836 (active group versus controlled group, 95% CI 0.825-0.852, p < 0.05) in exercise time, and 1.043 (controlled group versus active group, 95% CI 1.021-1.069, p < 0.05) in leukocyte ROCK2 activity. In addition, exercise time was significantly associated with leukocyte ROCK2 activity, with OR of 0.822 (active group versus controlled group, 95% CI 0.818-0.843, p < 0.05). CONCLUSION: In subjects with IFG, increased daily average exercise time is beneficial for improving fasting blood glucose metabolism, and the mechanism may be associated with its effects on attenuating leukocyte ROCK2 activity.
Subject(s)
Hyperglycemia/therapy , Adult , Blood Glucose , Cross-Sectional Studies , Exercise , Exercise Therapy , Female , Humans , Hyperglycemia/blood , Leukocytes/enzymology , Male , Middle Aged , Treatment Outcome , rho-Associated Kinases/metabolismABSTRACT
We sought to measure serum salusin-α levels in patients with coronary artery disease (CAD) and to assess their correlation with the severity of the disease. We enrolled 172 patients with CAD and 91 controls. We assessed the angiographic severity of CAD by coronary atherosclerosis index (CAI) and detected serum salusin-α levels by enzyme-linked immunosorbent assay (ELISA). We demonstrated that CAD patients had significantly lower serum salusin-α levels compared to controls. Moreover, serum salusin-α levels were independently and negatively correlated with the presence and severity of CAD. These findings indicated that salusin-α might serve as a potential biomarker for predicting the development and progression of CAD.
Subject(s)
Coronary Artery Disease/blood , Intercellular Signaling Peptides and Proteins/blood , Aged , Biomarkers/blood , C-Reactive Protein/metabolism , Case-Control Studies , Coronary Angiography , Coronary Artery Disease/diagnostic imaging , Female , Humans , Male , Middle Aged , Natriuretic Peptide, Brain/blood , Peptide Fragments/blood , Severity of Illness IndexABSTRACT
AIMS: Regadenoson is comparable to adenosine in pharmacologic radionuclide stress tests but has not been studied with stress myocardial contrast echocardiography. This study assessed the haemodynamic profile and ability of regadenoson, a novel selective A(2A) receptor agonist, to detect coronary artery stenosis during myocardial contrast echocardiography. METHODS AND RESULTS: Myocardial contrast echocardiography was performed to measure myocardial blood volume, myocardial blood flow velocity, and total regional myocardial blood flow before and after administration of regadenoson (5 µg kg(-1), 10 s bolus) in 10 open-chest dogs with mild-to-moderate coronary stenosis that was not flow limiting at rest. Regadenoson decreased blood pressure but did not change heart rate. It increased coronary blood flow significantly (P < 0.05) for 30 min, which was attenuated in proportion to coronary stenosis severity. Whereas myocardial blood volume maximally increased by 0.5-0.75-fold in the control region, it did not change in the region supplied by the non-flow limiting stenosis. Perfusion defects were visually and quantitatively detectable for as long as 10 min after administration of regadenoson. No arrhythmias were noted with regadenoson either prior to or during myocardial contrast echocardiography. CONCLUSION: Regadenoson can be used as a vasodilator stress agent with myocardial contrast echocardiography to detect the presence of physiologically significant coronary stenosis. The optimum time for image acquisition was 3-10 min after drug administration.
Subject(s)
Adenosine A2 Receptor Antagonists , Coronary Stenosis/diagnosis , Echocardiography/instrumentation , Myocardium/pathology , Purines , Pyrazoles , Analysis of Variance , Aorta/drug effects , Coronary Stenosis/diagnostic imaging , Coronary Stenosis/pathology , Heart Rate/drug effects , Hemodynamics/drug effects , Humans , Myocardial Perfusion Imaging , Stroke Volume , Vasodilator Agents , Ventricular Function, LeftABSTRACT
OBJECTIVE: Although the majority of coronary artery anomalies are found incidentally and not clinically significant, the interarterial course between the major vessels of the aberrant artery may be responsible for syncope, angina, arrhythmias or sudden death. There are only a few case reports describing the origination of all the coronary arteries from a single ostium. This anomaly occurs in only 0.024%-0.044% of the population. Left coronary artery originating from the right coronary is a rare coronary abnormality. Here we report a case of acute myocardial infarction in a patient with anomalous left coronary artery originating from the right coronary artery, as was confirmed by computerized tomography angiogram, which showed that only one single coronary artery stem originating from the right sinus of Valsalva trifurcated into a right coronary artery, left circumflex artery and a hypoplastic left anterior descending artery. Subsequent percutaneous coronary intervention (PCI) procedures were performed successfully. PCI procedures should be carried out with great caution in such cases, and this condition should be managed as a left main lesion.
Subject(s)
Coronary Angiography/methods , Coronary Vessel Anomalies/complications , Myocardial Infarction/complications , Coronary Vessel Anomalies/diagnostic imaging , Humans , Male , Middle Aged , Myocardial Infarction/diagnostic imaging , Myocardial Infarction/therapy , Percutaneous Coronary InterventionABSTRACT
OBJECTIVE: To investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α (Ad-HIF-1α-564/402/803). METHODS: Ad-HIF-1α-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-1α-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-1α-564/402/803 at these time points were examined for HIF-1α protein expression using Western blotting. RESULTS: No significant changes were observed in the effect of lyophilized Ad-HIF-1α-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-1α showed no structural alterations or significant changes in the expression level of HIF-1α protein in the transfected hMVECs (P>0.05). CONCLUSION: Lyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.
Subject(s)
Adenoviridae/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Mutant Proteins/metabolism , Adenoviridae/genetics , Antibodies, Monoclonal/genetics , Freeze Drying , Genetic Vectors , HEK293 Cells , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mutant Proteins/geneticsABSTRACT
Ribosomal proteins play an important role in p53 activation in response to nucleolar stress. Multiple ribosomal proteins, including L5, L11, L23, and S7, have been shown to bind to and inhibit MDM2, leading to p53 activation. However, it is not clear whether ribosomal protein regulation of MDM2 is specific to some, but not all ribosomal proteins. Here we show that L29 and L30, two ribosomal proteins from the 60 S ribosomal subunit, do not bind to MDM2 and do not inhibit MDM2-mediated p53 suppression, indicating that the ribosomal protein regulation of the MDM2-p53 feedback loop is specific. Interestingly, direct perturbation of the 60 S ribosomal biogenesis by knocking down either L29 or L30 drastically induced the level and activity of p53, leading to p53-depedent cell cycle arrest. This p53 activation was drastically inhibited by knockdown of L11 or L5. Consistently, knockdown of L29 or L30 enhanced the interaction of MDM2 with L11 and L5 and markedly inhibited MDM2-mediated p53 ubiquitination, suggesting that direct perturbation of 60 S ribosomal biogenesis activates p53 via L11- and L5-mediated MDM2 suppression. Mechanistically, knockdown of L30 or L29 significantly increased the NEDDylation and nuclear retention of L11. Knocking down endogenous NEDD8 suppressed p53 activation induced by knockdown of L30. These results demonstrate that NEDDylation of L11 plays a critical role in mediating p53 activation in response to perturbation of ribosomal biogenesis.
Subject(s)
Ribosomal Proteins/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Tumor Suppressor Protein p53/metabolism , Blood Coagulation Factors/genetics , Blood Coagulation Factors/metabolism , Cell Cycle/genetics , Cell Cycle/physiology , Cell Line, Tumor , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , Fluorescent Antibody Technique , Humans , Immunoblotting , Immunoprecipitation , Protein Binding , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Proteins/genetics , Ribosome Subunits, Large, Eukaryotic/genetics , Tumor Suppressor Protein p53/genetics , Ubiquitination/genetics , Ubiquitination/physiologyABSTRACT
OBJECTIVE: To investigate the effect of recombinant adenovirus-mediated hypoxia-inducible factor-1alpha (Ad-HIF-1alpha) at different doses on angiogenesis in a rabbit model of hind limb ischemia. METHODS: Left hind limb ischemia was induced in 45 Zealand white rabbits by ligation of the left femoral artery. The rabbits were randomly divided into 5 groups (n=9) to receive intramuscular injections of 0.5 ml saline, 2x10(10) PFU empty vector (Ad-null), or different doses of Ad-HIF-1alpha (2x10(9), 2x10(10) or 2x10(11) PFU) immediately after the operation. On the 7th day after the operation, real-time PCR was used to detect the expression of HIF-1alpha mRNA in the skeletal muscles. Immediately and on the 14th and 28th days after the operation, contrast enhanced ultrasound (CEU) was used to observe the blood perfusion of the hind limb. On the 28th day postoperatively, immunohistochemistry for CD31 was performed to evaluate the microvascular density (MVD). RESULTS: Real-time PCR showed that Ad-HIF-1alpha significantly increased the expression of HIF-1alpha mRNA (P<0.01) in a dose-dependent manner as compared with that in the saline and Ad-null groups (P<0.01). CEU revealed greater blood perfusion in the hind limb of rabbits in association with increased dose of Ad-HIF-1alpha (P<0.05 or P<0.01); similar changes in the MVD was observed following Ad-HIF-1alpha injections as shown by immunohistochemistry (P<0.05 or P<0.01). No significant differences were found either in the blood perfusion or MVD between saline and Ad-null groups (P>0.05). CONCLUSION: Ad-HIF-1alpha can dose-dependently promote the angiogenesis in the ischemic limb of rabbits.
Subject(s)
Hindlimb/blood supply , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ischemia/physiopathology , Neovascularization, Physiologic/drug effects , Recombinant Proteins/administration & dosage , Adenoviridae/genetics , Adenoviridae/metabolism , Animals , Female , Genetic Therapy , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Ischemia/drug therapy , Male , Rabbits , Random Allocation , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
OBJECTIVE: To explore the effect of erythropoietin (EPO) on reperfusion arrhythmias in rats and identify the possible mechanism involved. METHODS: Forty-five SD rats were randomized into a sham-operated group and 4 cardiac ischemia/reperfusion (IR) injury groups, which were further divided into IR group, LY294002 group, EPO group, and EPO+LY294002 group. Cardiac IR injury was induced in the 4 IR injury groups by ligating the left anterior descending branch of the coronary artery (LAD) for 30 min followed by reperfusion for 3 h, with subsequent treatments accordingly. The occurrence of arrhythmias was monitored and scored during experiment, and the levels of serum CK-MB and cTnI were detected. The content of MDA in the myocardium was determined by thiobarbituric acid (TBA) method, and the content of SOD by xanthine oxidase method. RESULTS: The arrhythmia score in EPO group was significantly lower than those in IR, LY294002 and EPO+ LY294002 groups (P<0.05). The levels of serum CK-MB and cTnI were significantly lower in EPO group than in the other 3 IR groups (P<0.001). The EPO group showed also significantly lower MDA content (P<0.001) and higher SOD content than the other 3 IR groups (P<0.001). CONCLUSION: EPO at the dose of 1000 mg/kg decreases the incidence of reperfusion arrhythmias in rats, and this effect can be attenuated by LY294002 pretreatment, suggesting that the cardioprotective effect of EPO involves antioxidation mediated by the phosphoinositide 3-kinase (PI3K) pathway.
Subject(s)
Arrhythmias, Cardiac/prevention & control , Coronary Occlusion/drug therapy , Erythropoietin/therapeutic use , Myocardial Reperfusion Injury/prevention & control , Animals , Arrhythmias, Cardiac/etiology , Chromones/therapeutic use , Coronary Occlusion/complications , Female , Male , Morpholines/therapeutic use , Myocardial Reperfusion Injury/complications , Myocardial Reperfusion Injury/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: To investigate the changes in plasma matrix metalloproteinases-2 and -9 (MMP2 and MMP9, respectively) levels in patients with different types of coronary heart diseases (CHD), and assess the value of MMP2/MMP9 detection in predicting acute coronary syndrome (ACS). METHODS: According to the findings by coronary angiography and the clinical manifestations, 118 patients were divided in ACS group including 30 patients with unstable angina pectoris (UAP) and 19 with acute myocardial infarction (AMI) and non-ACS group including 23 patients with stable angina pectoris (SAP) and 21 with chronic total occlusion (CTO) of the coronary artery. Twenty-five individuals with normal coronary artery (NCA) served as the control group. Plasma levels of MMP9 and MMP2 were determined in these subjects using enzyme-linked immunosorbent assay (ELISA). RESULTS: Both the ACS and non-ACS groups showed significantly higher MMP9 and MMP2 levels than the NCA group (P<0.05), and MMP2 and MMP9 levels were significantly higher in ACS group than in non-ACS group (P<0.05). Compared with the NCA group, the UAP, AMI and CTO subgroups showed obvious increases in plasma MMP2 and MMP9 levels (P<0.01). Significantly increased MMP9, but not MMP2 level was noted in AMI subgroup in comparison with SAP (P<0.01) and UAP subgroups (P<0.05); both MMP2 and MMP9 levels were elevated in CTO subgroup in comparison with those in SAP (P<0.001), UAP (P<0.01), and AMI subgroups (P<0.05). CONCLUSION: Increased MMP2 and MMP9 levels in patients with CHD suggest the instability of the atherosclerotic plaque in correlation to the severity of ACS, and may serve as good indicators for the prediction of ACS and diagnosis of CTO of the coronary artery.
Subject(s)
Acute Coronary Syndrome/blood , Coronary Occlusion/blood , Matrix Metalloproteinase 2/blood , Matrix Metalloproteinase 9/blood , Myocardial Infarction/blood , Acute Coronary Syndrome/diagnostic imaging , Aged , Angina, Unstable/blood , Angina, Unstable/diagnostic imaging , Chronic Disease , Coronary Angiography , Coronary Occlusion/diagnostic imaging , Female , Humans , Male , Metamorphosis, Biological , Middle Aged , Myocardial Infarction/diagnostic imagingABSTRACT
OBJECTIVE: To investigate the feasibility of bone marrow mesenchymal stem cell (MSC) transplantation with ultrasound-targeted microbubble destruction. METHODS: Twenty-one Wistar rats were divided into MSCs-iv group (MSCs-iv), ultrasound+MSCs-iv group (US+MSCs-iv), ultrasound+microbubble+MSCs-iv group (US+MB+MSCs-iv) with intravenous MSC transfer, ultrasound and microbubble treatment as indicated. The skeletal muscles were obtained from the rats for microscopic examination with HE staining. The hindlimb gracilis and semimembranosus muscles were sampled 7 days after MSC transplantation, and the transplanted MSCs were detected by immunohistochemistry. The vital organs were collected from rats in US+MB+MSCs-iv group for immunohistochemistry. RESULTS: In US+MB+MSCs-iv group, HE staining demonstrated the presence of red blood cell leakage into the tissue space in the gracilis and semimembranosus muscles, and immunohistochemistry identified large numbers of transplanted MSCs in the the gracilis and semimembranosus muscles and the spleen, whereas no labeled cells were detected in the skeletal muscles in other groups. CONCLUSION: Ultrasound-targeted microbubble destruction provides a useful means for enhancing the efficiency of stem cell transplantation.
Subject(s)
Bone Marrow Cells/cytology , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/cytology , Microbubbles , Ultrasonics , Animals , Cell Movement/radiation effects , Female , Male , Muscle, Skeletal/cytology , Rats , Rats, WistarABSTRACT
OBJECTIVE: To validate the efficacy of velocity vector imaging (VVI) and quantitative tissue velocity imaging (QTVI) for evaluating left ventricular diastolic function. METHODS: Fifty-one patients underwent left heart catheterization were included in this study. Mean of peak early diastolic velocity (Em), EF and the ratio of early (E) to late (A) mitral valve flow velocity (E/A) were measured by echocardiography and the ratio of E to Em (E/Em) was calculated. Left ventricular end diastolic pressure (LVEDP) was measured during catheterization examination. RESULTS: E/Em derived from VVI or QTVI was significantly correlated with LVEDP (r = 0.808, P < 0.01 and r = 0.692, P < 0.01, respectively) and the correlation coefficient between VVI and LVEDP was significantly higher than that between QTVI and LVEDP (Z = 2.246, P = 0.025). Em derived from VVI and QTVI also negatively correlated with LVEDP (r = -0.740, P < 0.01 and r = -0.567, P < 0.01) and the correlation coefficient between VVI and LVEDP was significantly higher than that between QTVI and LVEDP (Z = 2.595, P = 0.009). However, there was no correlation between E/A and LVEDP (r = 0.117, P = 0.415). CONCLUSION: E/Em and Em derived from VVI and QTVI are valuable parameters for evaluating LV diastolic function.
Subject(s)
Diastole/physiology , Echocardiography/methods , Ventricular Function, Left/physiology , Blood Flow Velocity , Cardiac Catheterization , Humans , Mitral Valve/diagnostic imaging , Mitral Valve/physiology , Reproducibility of ResultsABSTRACT
OBJECTIVE: To evaluate the value of the myocardial biochemical markers including creatine kinase MB isoenzyme (CK-MB), cardiac isoform of Tropnin-T (cTnT) and N-termimal pro-brain natriuretic peptide (NT-proBNP) and electrocardiogram (ECG) in monitoring the cardiotoxicity of recombinant human endostatin (rh-endostatin) in cancer patients. METHODS: Forty cancer patients were divided into two groups and received rh-endostatin in addition to chemotherapy (group A, n=24) or chemotherapy only (Group B, n=24). Serum CK-MB, cTnT levels and plasma NT-proBNP levels were measured and the ECG was recorded in all the patients before and after each of the two therapy cycles. RESULTS: In group A, serum CK-MB, cTnT and plasma NT-proBNP levels were significantly increased after the treatment in comparison with the baseline levels (P<0.05), but such increment was not observed in group B (P>0.05). With comparable baseline levels of CK-MB, cTnT and NT-proBNP before the treatment (P>0.05), patients in group A showed significantly higher levels of the indices than those in group B after each therapy cycle (P<0.05). Increased ECG abnormality were observed after rh-endostatin treatment in Group A (P<0.05) at a rate significantly higher than that of Group B after the second treatment cycle (P<0.05). CONCLUSION: Rh-endostatin has definite cardiotoxicity, and detection of the myocardial biochemical markers of CK-MB, cTnT and NT-proBNP may help predict the occurrence of cardiotoxicity.
Subject(s)
Biomarkers, Tumor/blood , Endostatins/therapeutic use , Lung Neoplasms/blood , Lung Neoplasms/drug therapy , Adult , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/drug therapy , Creatine Kinase, MB Form/blood , Endostatins/adverse effects , Endostatins/genetics , Female , Humans , Male , Middle Aged , Natriuretic Peptide, Brain/blood , Ovarian Neoplasms/blood , Ovarian Neoplasms/drug therapy , Peptide Fragments/blood , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Risk Assessment , Troponin T/bloodABSTRACT
OBJECTIVE: To investigate the mechanism by which recombinant adenovirus (Ad)-mediated mutations of hypoxia inducible factor 1alpha (Ad-HIF-1alpha-Ala564-Ala803) regulates cell apoptosis. METHODS: 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. RESULTS: 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). CONCLUSION: HIF-1alpha may induce cell cycle arrest by up-regulating p21WAF1/CIP1 expressions to promote LoVo cell apoptosis.
Subject(s)
Adenoviridae/genetics , Apoptosis/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Mutation , Apoptosis/genetics , Blotting, Western , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , Genetic Vectors/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mutagenesis, Site-Directed , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Recombinant Proteins/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
OBJECTIVE: 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. METHODS: 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. RESULT AND CONCLUSION: 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.