Copies Of Hypoxia Response Element Research Articles

Molecular and functional evaluation of a novel HIF inhibitor, benzopyranyl 1,2,3-triazole compound.

Hypoxia occurs in a variety of pathological events, including the formation of solid tumors. Hypoxia-inducible factor (HIF)-1α is stabilized under hypoxic conditions and is a key molecule in tumor growth and angiogenesis. Seeking to develop novel cancer therapeutics, we investigated small molecules from our in-house chemical libraries to target HIF-1α. We employed a dual-luciferase assay that uses a luciferase (Luc) reporter vector harboring five copies of hypoxia-responsive element (HRE) in the promoter. Under hypoxic conditions that increased Luc reporter activity by four-fold, we screened 144 different compounds, nine of which showed 30–50% inhibition of hypoxia-induced Luc reporter activity. Among these, “Compound 12, a benzopyranyl 1,2,3-triazole” was the most efficient at inhibiting the expression of HIF-1α under hypoxic conditions, reducing its expression by 80%. Under hypoxic conditions, the half maximal IC50 of the compound was 24 nM in HEK-293 human embryonic kidney cells, and 2 nM in A549 human lung carcinoma cells. Under hypoxic conditions, Compound 12 increased hydroxylated HIF-1α levels and HIF-1α ubiquitination, and also dose-dependently decreased HIF-1α target gene expression as well as vascular endothelial growth factor (VEGF) secretion. Furthermore, this compound inhibited VEGF-induced in vitro angiogenesis in human umbilical vein endothelial cells (HUVECs), and in vivo, it inhibited chick chorioallantoic membrane angiogenesis. In allogaft assays, cotreatment with Compound 12 and gefitinib significantly inhibited tumor growth and angiogenesis. Compound 12 can be a novel inhibitor of HIF-1α by accelerating its degradation, and shows much potential as an anti-cancer agent through its ability to suppress tumor growth and angiogenesis.

Prevention of hypoxic pulmonary hypertension by hypoxia-inducible expression of p27 in pulmonary artery smooth muscle cells.

Hypoxia-induced proliferation of pulmonary artery smooth muscle cells (SMCs) is important in the development of hypoxic pulmonary hypertension (HPH). We constructed a lentivirial vector containing a smooth muscle-specific promoter and six copies of hypoxia response element to co-drive the expression of p27, the key cyclin-dependent kinase inhibitor that blocks the G1 to S phase transition in cell cycle progression, in pulmonary artery SMCs in hypoxia. Then in vivo we examined the prevention effects of the vector on HPH in mice and in vitro the specificity on the hypoxia-inducible expression of p27 in pulmonary artery SMCs. Hypobaric hypoxia for 4 weeks resulted in significant increases in the right ventricular systolic pressure, the ratio of right ventricle to left ventricle plus septal weight and the muscularization of pulmonary vessels in mice. Administration of the vector before hypoxia significantly prevented the effects of hypoxia. In vitro, the vector exhibited hypoxic inducibility and relatively specific expression in pulmonary artery SMCs, inhibited the hypoxia-induced proliferation of pulmonary artery SMCs and arrested more cells at G0/G1 phase. These results demonstrate that the hypoxia-inducible p27 expression prevents the development of HPH in mice.

Neuroprotection of neurotrophin-3 against focal cerebral ischemia/reperfusion injury is regulated by hypoxia-responsive element in rats

Exogenous delivery of the neurotrophin-3 (NT-3) gene may provide a potential therapeutic strategy for ischemic stroke. To investigate the neuroprotective effects of NT-3 expression controlled by 5HRE after focal cerebral ischemia, we constructed a recombinant retrovirus vector (RV) with five copies of hypoxia-responsive elements (5HRE or 5H) and NT-3 and delivered it to the rat brain. Three groups of rats received RV-5H-NT3, RV-5H-EGFP or saline injection. Three days after gene transfer, the rats underwent 90min of transient middle cerebral artery occlusion (tMCAO), followed by 1–28days of reperfusion. Three days after tMCAO, brain NT-3 expression was significantly increased in the RV-5H-NT3-transduced animals compared with the RV-5H-EGFP or saline group, and brain infarct volume was smaller in the RV-5H-NT3-transduced group than the RV-5H-EGFP or saline group. The percentage of TUNEL-positive cells was reduced in RV-5H-NT3-transduced brains compared with the RV-5H-EGFP or saline group 3 and 7days after tMCAO. Furthermore, the neurological status of RV-5H-NT3-transduced rats was better than that of RV-5H-EGFP- or saline-transduced animals from 1day to 4weeks after tMCAO. Our results demonstrated that 5HRE could modulate NT-3 expression in the ischemic brain environment and that the up-regulated NT-3 could effectively improve neurological status following tMCAO due to decreased initial damage. To avoid unexpected side effects, 5HRE-controlled gene expression might be a useful tool for gene therapy of ischemic disorders in the central nervous system.

Abstract 2051: Hypoxia-induced triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to GCV and 5-FC and radiosensitization in human colorectal cancer cells

Abstract The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissues, may provide an opportunity for cancer-specific gene theapy. We constructed two vectors, both under the control of nine copies of hypoxia responsive element (HRE): (1) a herpes simplex virus type 1 thymidine kinase (TK) and enhanced green fluorescent protein (eGFP) fusion gene, and (2) a triple-fusion-gene, cytosine deaminase (CD), uracil phosphoribosyltransferase (UPRT) and monomeric DsRed (mDsRed). Human colorectal cancer cells (HCT8) were transfected with these plasmids, singly or in combination. Stable HCT8-HRE cells were established that contained and expressed the hypoxia-inducible vectors (i.e. HRE-TK/eGFP, HRE-CD/UPRT/mDsRed, or both HRE-TK/eGFP and HRE-CD/UPRT/mDsRed), as assessed by RT-PCR, Western blots and fluorescence microscopy analysis. In in vitro studies, we showed that hypoxia-induced TK/eGFP and CD/UPRT/mDsRed expression not only increased cytotoxicity to GCV and 5-FC, but also led to significant radiosensitization. In HCT8-HRE tumors transplanted in athymic mice, the distribution of TK/eGFP and CD/UPRT/mDsRed co-localized with that of the hypoxia marker pimonidazole. In addition, the administration of GCV and 5-FC in combination with local irradiation resulted in significant tumor regression, greater than that observed with drug or radiation treatment alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple gene therapy has the ability to specifically target hypoxic cancer cells and significantly improve tumor control in combination with radiotherapy. This novel model is a valuable tool for studying tumor hypoxia and hypoxia-targeted radio-gene therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2011-2051

Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice

The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein-protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP:dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP:dMODC or TKGFP and determined by optical imaging and PET. Unlike TKGFP, NESTKGFP:dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP:dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP:dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP:dMODC expression showed that NESTKGFP:dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. In translational molecular-genetic imaging, NESTKGFP:dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.

Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice

Some gene expression may be regulated by hypoxia-responsive element (HRE) that is bound by hypoxia-inducible factor-1 (HIF-1) which is up-regulated during cerebral ischemia. To explore ischemia/hypoxia-controlled expression and the neuroprotective effects of brain-derived neurotrophic factor (BDNF) after ischemic brain injury, an adenoviral vector using five copies of hypoxia response element (HRE) in the vascular endothelial growth factor gene to regulate the expression of BDNF gene (Ad5HRE:BDNF) was constructed, and its efficacy was verified for driving BDNF expression in cultured Hela cells under hypoxic condition by ELISA. We found that the concentration of BDNF in the Ad5HRE:BDNF-transfected culture media was 28-fold greater in a hypoxic condition than under normoxia. To examine the effect of Ad5HRE:BDNF on ischemic brain injury in vivo, Ad5HRE:BDNF was injected into right caudate putamen of adult mice 7 days prior to 60 min transient middle cerebral artery occlusion (MCAO). It was found that exogenous BDNF expression was increased in the Ad5HRE-BDNF-treated group and infarct volume of the Ad5HRE:BDNF-treated group at 3 days after MCAO was significantly smaller than that of vehicle- or AdNull-treated groups. Moreover, Ad5HRE:BDNF injection resulted in significantly improved sensorimotor scores 7 days after MCAO and induced a reduction in the number of Fluoro-Jade B-positive neurons and TUNEL-positive cells, compared with vehicle- or AdNull-injection. Our findings suggest that BDNF expression could be regulated in hypoxia/ischemia condition with five copies of HRE and ameliorates ischemic brain injury in a mouse focal cerebral ischemia model.

Visualization of Hypoxia-Inducible Factor-1 Transcriptional Activation in C6 Glioma Using Luciferase and Sodium Iodide Symporter Genes

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor of hypoxic response in cancer cells and is associated with tumor progression, angiogenesis, metastasis, and resistance to therapy. We assessed whether the human sodium iodide symporter (NIS) reporter systems can be used to visualize transcriptional activation of HIF-1 in C6 glioma. Two types of plasmid-expressing human NIS or luciferase (Luc) genes, controlled by 5 copies of hypoxia response element (5HRE), were constructed: p5HRE-NIS or p5HRE-Luc. C6 glioma cells were stably transfected with p5HRE-NIS or p5HRE-Luc plasmids (C6-5HRE-NIS or C6-5HRE-Luc). Hypoxic conditions were modeled by exposing culture medium to desferrioxamine (DFO) or a low oxygen atmosphere (<1% O(2)) in a hypoxic chamber. HIF-1 transcription activity was assessed by measuring cellular (125)I uptake and luminescent intensities. Reverse-transcription polymerase chain reaction and Western blotting were performed to observe the messenger RNA and protein levels of reporter and target genes under hypoxic or normoxic conditions. C6, C6-cytomegalovirus (CMV)-NIS, or C6-CMV-Luc and C6-5HRE-NIS or C6-5HRE-Luc cells were injected subcutaneously into nude mice (the NIS and Luc groups, respectively). Two weeks after tumor challenge, bioluminescence and (99m)Tc scintigraphic images were acquired before and after intraperitoneal DFO administration. Natural hypoxia in tumors was induced by growing tumors for 3 wk. Ex vivo studies, such as biodistribution, immunohistochemistry, and (99m)Tc autoradiography, were performed. Time- and concentration-dependent increases of (125)I uptake and bioluminescence were observed in hypoxically stressed reporter cells. Also, messenger RNA and protein levels of reporter and target genes increased under hypoxic conditions. (99m)Tc uptake and bioluminescence signals from C6-5HRE-NIS and C6-5HRE-Luc tumors increased during hypoxia. In the biodistribution study, a larger amount of (99m)Tc accumulated in C6-5HRE-NIS tumors than in the other tumors not containing 5HRE (P<0.005). In the Luc group, immunostaining showed similar distribution patterns for luciferase and pimonidazole, and in the NIS group, autoradiography of C6-5HRE-NIS tumors showed a distribution similar to that observed for pimonidazole immunostaining. The transcriptional activation of HIF-1 induced by hypoxia or DFO was visualized by both bioluminescence and scintigraphic reporter gene systems.

A novel chimeric promoter that is highly responsive to hypoxia and metals

To develop a potent hypoxia-inducible promoter, we evaluated the usefulness of chimeric combinations of the (Egr-1)-binding site (EBS) from the Egr-1 gene, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 gene. In transient transfection assays, combining three copies of HRE (3 x HRE) with either EBS or MRE significantly increased hypoxia responsiveness. When a three-enhancer combination was tested, the EBS-MRE-3 x HRE (E-M-H) gave a hypoxia induction ratio of 69. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. Gel shift assays together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1alpha, metal transcription factor-1 and Egr-1 may be associated with the high inducibility of the E-M-H chimeric promoter. E-M-H was also induced by hypoxia mimetics such as Co2+ and deferoxamine (DFX) and by hydrogen peroxide. Gene expression from the E-M-H was reversible as shown by the reduced expression of the transgene upon removal of inducers such as hypoxia and DFX. In vivo evaluation of the E-M-H in ischemic muscle revealed that erythropoietin secretion and luciferase and LacZ expression were significantly higher in the E-M-H group than in a control or H group. With its high induction capacity and versatile means of modulation, this novel chimeric promoter should find wide application in the treatment of ischemic diseases and cancer.

Hypoxia-Induced BAX Overexpression and Radiation Killing of Hypoxic Glioblastoma Cells

One major challenge in treating glioblastoma multiforme (GBM) has been the presence of radiation-resistant hypoxic cells. The pro-apoptosis protein BAX has been reported to be a possible radiation sensitizer of cancer cells; however, to our knowledge, no studies have reported on the effects of BAX on radiation sensitivity under hypoxic conditions. Therefore, in this study, we specifically overexpressed murine Bax in hypoxic cells in an attempt to enhance radiation-induced cell killing. We have previously stably transfected U-251 MG and U-87 MG human GBM cells with constructs containing murine Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible factor 1 (HIF1) forms and binds to HRE; this binding facilitates the transcription of downstream genes. In the experiments reported here, two protocols were used. In the first protocol, parent and clone cells were exposed to graded doses of X rays under hypoxic conditions, left hypoxic for 0, 4, 16 or 24 h, and then assayed for clonogenic cell survival. In the second protocol, cells were incubated under hypoxic conditions for 20 h, irradiated with graded doses under hypoxia, then left in hypoxic conditions for 4 h before being assayed for clonogenic cell survival. Western blots showed that we had successfully increased Bax expression in both U-251 MG and U-87 MG Bax clone cells after 16 h of hypoxic incubation, yet dose-response curves showed no difference in radiation-induced cell killing between control non-Bax-expressing pNeo clone cells and the U-251 MG Bax clone cells using either protocol. In U-87 MG cells, the first protocol showed no difference in radiation response between control pNeo clone cells and the Bax clone, similar to the results obtained in U-251 cells. However, the second protocol revealed that Bax overexpression did render these cells more sensitive to radiation under hypoxic conditions. Therefore, we conclude that whether Bax is a radiation enhancer under hypoxia not only is cell line-dependent but also depends on when the Bax overexpression occurs.

1052. A Versatile Chimeric Enhancer Vector That Is Highly Inducible by Hypoxia and Metals

To develop a potent hypoxia-inducible vector, we evaluated the usefulness of chimeric combinations of the early growth response factor-1 (Egr-1)-binding site (EBS) from the Egr-1 promoter, the metal-response element (MRE) from the metallothionein gene, and the hypoxia-response element (HRE) from the phosphoglycerate kinase 1 (PGK1) gene. In transient trasfection assays, combination of three copies of HRE (3xHRE) with either EBS or MRE resulted in a significant increase in hypoxia-responsiveness. With three-enhancer combination such as the EBS-MRE-3xHRE (E-M-H), a hypoxia induction ratio of 69 was achieved. The expression induced from E-M-H-pGL3 was 2.4-fold higher than that induced from H-pGL3 and even surpassed the expression from a human cytomegalovirus (HCMV) promoter-driven vector. The high inducibility of E-M-H was confirmed by validation studies in different cells and by expressing other cDNAs. The E-M-H was also tested for induction by hypoxia mimetics and other stresses such as heat, low glucose, low pH, and hydrogen peroxide. Co2+, deferoxamine, and Ni2+turned out to be potent inducers whereas hydrogen peroxide worked as a moderate inducer. Gel shift assay together with functional overexpression studies suggested that increased levels of hypoxia-inducible factor 1|[alpha]| (HIF-1|[alpha]|), metal transcription factor-1 (MTF-1), and Egr-1 may be associated with the high inducibility of E-M-H enhancer. In vivo evaluation of the E-M-H vector in ischemic calf muscle revealed that luciferase expression was significantly higher in E-M-H group than in control, H, or HCMV group. With its high induction capacity and versatile means of modulation, the novel chimeric enhancer should find wide application in the treatment of ischemic diseases and cancer.

Hypoxia-responsive element-mediated soluble Tie2 vector exhibits an anti-angiogenic activity in vitro under hypoxic condition

Hypoxia-inducible factor-1 (HIF-1) is one of the key mammalian transcription factors and shows increased levels in both protein stability and intrinsic transcriptional activity during low oxygen tension. Hypoxia-activated functional HIF-1 protein binds to hypoxia-responsive elements (HRE) in the enhancers of several genes including VEGF, the major player in angiogenesis, and initiates their mRNA expression. The molecular mechanisms regulating the gene expression under hypoxic conditions could increase the therapeutic window of tumor-specific delivery systems. In this study, to examine hypoxia-specific production of anti-angiogenic therapeutic gene, we constructed 5 copies of HRE (5xHRE) of human VEGF linked to soluble Tie2 (sTie2) driven by minimal SV40 promoter (5xHRE/SV40/sTie2). Our data showed that under hypoxia the secreted sTie2 selectively inhibited tube formation and migration capacities of endothelial cells in vitro. Hence, we propose that the vector system, 5xHRE/SV40/sTie2, might be a useful tool for down-regulating tumor angiogenesis under hypoxic condition.

192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

A problem associated with angiogenic gene therapy is that uncontrolled gene expression of angiogenic factors may cause several side effects, including: hemangioma formation, retinopathy, occult tumor growth and atherosclerotic plaque progression. Previously, we used the hypoxia response element (HRE) isolated from the erythropoietin (Epo) gene enhancer to render vascular endothelial growth factor (VEGF) expression responsive to hypoxia induction in vitro and in ischemic myocardium in vivo. We found in our experiment that adeno-associate viral vector (AAV) can be detected in other organs in addition to the heart after intra-myocardial injection. Thus, unwanted effects may still occur even with HRE controlled VEGF expression, since hypoxia in other tissues in patients with heart failure may results in extra-cardiac VEGF gene expression. To ensure the safety of angiogenic therapy for ischemic heart disease, it is necessary that the gene expression is cardiac specific and hypoxia responsive. In this study, we explored the feasibility of using the cardiac myosin light-chain-2v (MLC-2v) promoter and Epo HRE to mediate cardiac-specific and hypoxia-responsive VEGF gene expression. We constructed an AAV vector with MLC-2v promoter and 9 copies of Epo HRE driving VEGF (MLCVEGF) expression. Gene expression mediated by this vector was studied in vitro by transfection of this vector into a rat cardiac muscle cell line (H9c2), a rat skeletal muscle cell line (L6) and a mouse fibroblast cell line (NIH3T3), and in vivo by direct injection of the vector to normal and ischemic mouse hearts. AAV vectors with CMV driving VEGF (CMVVEGF), and 9HRE plus minimum SV40 promoter driving VEGF (H9VEGF) were used as controls. RESULTS: With MLCVEGF infection, VEGF expression is higher in H9c2 than the other two non-cardiac cell lines, and the expression is hypoxia inducible. In vivo studies indicated that MLCVEGF increased VEGF expression by 74% in ischemic heart compared to normal heart. VEGF expression was detected in liver, kidney, spleen and gonad of CMVVEGF inoculated mice by RT-PCR, but no VEGF expression was detected in organs other than heart and liver of MLCVEGF inoculated mice. CONCLUSION: Our result indicates that MLC-2v promoter and 9 copies of HRE can mediate cardiac-specific and hypoxia-inducible gene expression in vitro and in vivo. Induction of angiogenesis and improvement of cardiac function by this vector, studies currently in progress, may provide a safe approach for the treatment of ischemic heart disease.

A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy

The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HIRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

Hypoxia-inducible expression of BAX: application in tumor-targeted gene therapy

Object The presence of hypoxic cells in human brain tumors contributes to the resistance of these tumors to radiation therapy. However, because normal tissues are not hypoxic, the presence of hypoxic cells also provides the potential for designing cancer-specific gene therapy. Suicide genes can be expressed specifically in hypoxic conditions by hypoxia-responsive elements (HREs), which are activated through the transcriptional complex hypoxia-inducible factor–1 (HIF-1). Methods The authors have transfected the murine BAX–green fluorescent protein (GFP) fusion gene under the regulation of three copies of HRE into U-87 MG and U-251 MG cells and selected stably transfected clones. Even though BAX was expressed under both oxic and anoxic conditions in these clones, cell survival assays demonstrated increased cell killing under anoxic as compared with oxic conditions. Cells obtained from most of these clones did not grow in vivo, or the tumors exhibited highly variable growth rates. However, cells obtained from the U-251 MG clone A produced tumors that grew as well as tumors derived from parental cells, and examination of the tumor sections under fluorescent microscopy revealed GFP expression in localized regions. Western blot analyses confirmed an increased BAX expression in these tumors. Analysis of the results suggests that HRE-regulated BAX can be a promising tool to target hypoxic brain tumor cells. However, there are measurable levels of BAX-GFP expression in this three-copy HRE–mediated expression system under oxia, suggesting promoter leakage. In addition, most clones did not show significant induction of BAX-GFP under anoxia. Therefore, the parameters of this HRE-mediated expression system, including HRE copy number and the basal promoter, need to be optimized to produce preferential and predictable gene expression in hypoxic cells.

Development of a hypoxia-responsive vector for tumor-specific gene therapy.

We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. As vascular endothelial growth factor (VEGF) is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with five copies of hypoxia-responsive elements (HREs) derived from the 5'-untranslated region (UTR) of the human VEGF showed excellent transcriptional activation at low oxygen tension relevant to tumor hypoxia. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. In addition, we also investigated whether the 3' UTR of the VEGF gene would confer increased post-transcriptional mRNA stability under hypoxic conditions. However, despite increases in the hypoxic/aerobic ratio of luciferase activity, gene expression with 3' UTR was lower due to mRNA destabilization by AU-rich elements (AREs). Thus, we found no benefit from the inclusion of the 3' UTR in our vectors. Of all the vectors tested, the combination of 5HRE and a CMV minimal promoter exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter. We propose that this vector would be useful for tumor selective gene therapy.