losartan-potassium and Glioma

Topics: Brain Neoplasms; Diagnosis, Differential; Erythropoietin; Female; Glioma; Hemangiosarcoma; Humans; Immunohistochemistry; Lectins; Male; Meningioma; Plant Lectins; Receptors, Cell Surface; Receptors, Thrombin; von Willebrand Factor

EphA2 is a key factor underlying invasive propensity of gliomas, and is associated with poor prognosis of tumors. We aimed to develop a radiomics-based imaging index for predicting EphA2 expression in diffuse gliomas, and further estimating its value for grading of tumors.. A total of 182 patients with diffuse gliomas were included. All subjects underwent pre-operative MRI and post-operative pathological diagnosis. EphA2 expression of tumors was scored on pathological sections with immunohistochemical staining using monoclonal EphA2 antibody. MRI radiomics features were extracted from three-dimensional contrast-enhanced T1-weighted imaging and diffusion kurtosis imaging. Predictive models were constructed using machine learning-based radiomics features selection and three classifiers for predicting EphA2 expression and tumor grade. Features of best EphA2 expression model were subsequently used to construct another model of tumor grading. For each model, 146 cases (80%) were randomly picked as training and the rest 36 (20%) were testing cohorts. EphA2 expression was further correlated to the radiomics features in both grade models using Spearman's correlation.. Logistic regression model presented highest performance for predicting EphA2 expression (AUC: 0.836/0.724 in training/validation set). Tumor gradings model guided by features from EphA2 expression model demonstrated comparable performance (AUC: 0.930/0.983) to that constructed directly using imaging radiomics features (AUC: 0.960/0.977). Two radiomics features which included in both LR-grade models showed strong correlation (P < 0.05) with EphA2 expression.. The expression of EphA2 in gliomas could be predicted by radiomics features extracted from diffusion kurtosis MRI, which could also be used to assist tumor grading.

Topics: Brain; Brain Neoplasms; Carcinoma, Hepatocellular; Erythropoietin; Glioma; Humans; Liver Neoplasms; Magnetic Resonance Imaging; Receptors, Erythropoietin; Retrospective Studies

Although erythropoietin (EPO) has been proven to significantly promote the proliferation of cancer cells, the mechanism for promoting glioma proliferation is poorly understood. Here, we examined the functional role of the AKT/GSK-3β/β-catenin signaling pathway in the EPO-mediated proliferation of glioma.. The distribution of EPO and Ki-67 among clinical samples with different WHO grades was plotted by Immunological Histological Chemistry analysis. U87 and U251 glioma cell lines were treated with short hairpin RNA targeting (shEPO), recombinant human erythropoietin (rhEPO) and/or AKT-specific inhibitor (MK-2206). The changes in phosphorylated AKT, nuclear β-catenin, cyclin D1 and p27kip1 expression were detected. Cell cycle distributions and glioma proliferation in vitro and in vivo were analyzed.. The expression level of EPO was significantly elevated with the increase of WHO grade and Ki67 in clinical glioma specimens. In vitro, knockdown of endogenous EPO in U87 and U251 cells effectively block the phosphorylation of AKT and GSK-3β and the expression of nuclear β-catenin. shEPO treatment also significantly decreased the expression of cyclin D1 and increased the expression of p27kip1. The cell cycle transition then slowed down and the proliferation of glioma cells or mouse xenograft tumors both decreased. Treatment of cells or tumors with extra rhEPO reversed the above biological effects mediated by shEPO. rhEPO-induced activation of the AKT/GSK-3β/β-catenin pathway and proliferation were abolished by MK-2206.. Our study identified the AKT/GSK-3β/β-catenin axis as a critical mediator of EPO-induced glioma proliferation and further provided a clinically significant dimension to the biology of EPO.

Topics: Animals; Apoptosis; beta Catenin; Biomarkers, Tumor; Brain Neoplasms; Cell Proliferation; Erythropoietin; Female; Gene Expression Regulation, Neoplastic; Glioma; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-akt; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Malignant gliomas are aggressive spinal cord tumors. In this study, we hypothesized that combination therapy using an anti-angiogenic agent, bevacizumab, and hypoxia-inducible glioblastoma-specific suicide gene could reduce tumor growth.. In the present study, we evaluated the effect of combination therapy using bevacizumab and pEpo-NI2-SV-TK in reducing the proliferation of C6 cells and tumor growth in the spinal cord. Spinal cord tumor was generated by the injection of C6 cells into the T5 level of the spinal cord. Complexes of branched polyethylenimine (bPEI)/pEpo-NI2-SV-TK were injected into the spinal cord tumor. Bevacizumab was then administered by an intraperitoneal injection at a dose of 7 mg/kg. The anti-cancer effects of combination therapy were analyzed by histological analyses and magnetic resonance imaging (MRI). The Basso, Beattie and Bresnahan scale scores for all of the treatment groups were recorded every other day for 15 days to assess the rat hind-limb strength.. The complexes of bPEI/pEpo-NI2-SV-TK inhibited the viability of C6 cells in the hypoxia condition at 5 days after treatment with ganciclovir. Bevacizumab was decreased in the cell viability of human umbilical vein endothelial cells. Combination therapy reduced the tumor size by histological analyses and MRI. The combination therapy group showed improved hind-limb function compared to the other groups that were administered pEpo-NI2-SV-TK alone or bevacizumab alone.. This study suggests that combination therapy using bevacizumab with the pEpo-NI2-SV-TK therapeutic gene could be useful for increasing its therapeutic benefits for intramedullary spinal cord tumors.

Topics: Angiogenesis Inhibitors; Animals; Bevacizumab; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Endothelial Cells; Enhancer Elements, Genetic; Erythropoietin; Genes, Reporter; Genes, Transgenic, Suicide; Glioblastoma; Glioma; Hindlimb; Injections, Intraperitoneal; Magnetic Resonance Imaging; Rats; Spinal Cord Neoplasms

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of erythropoiesis. In response to hypoxia-ischemia, hypoxia-inducible factor 1 induces EPO production, which, in turn, inhibits apoptosis of erythroid progenitor cells. By the same mechanism and acting through other signaling pathways, EPO exerts neuroprotective effects. Increased resistance to hypoxia and decreased apoptosis are thought to be important mechanisms for tumor progression, including malignant glioma. Because recent studies have demonstrated that EPO and its receptor (EPOR) are expressed in several tumors and can promote tumor growth, in the present study, we investigated EPO and EPOR expression in human glioma and the effect of EPO administration in a rat model of glioma implantation.. Using Western blotting and immunohistochemical analysis, we examined the expression of EPO, EPOR, platelet endothelial cell adhesion molecule, and Ki-67 in human glioma specimens and experimentally induced glioma in rats. In the experimental setting, a daily dose of recombinant human EPO (rHuEPO) or saline solution were administered for 21 days in Fischer rats subjected to 9L cell line implantation.. In both human and animal specimens, we found an increase in EPOR expression as long as the lesion presented with an increasing malignant pattern. A significant direct correlation was found between the expression of EPOR and Ki-67 and EPOR and platelet endothelial cell adhesion molecule in low- and high-grade gliomas. The rats treated with rHuEPO presented with significantly larger tumor spread compared with the saline-treated rats.. The results of our study have shown that the EPO/EPOR complex might play a significant role in the aggressive behavior of high-grade gliomas. The larger tumor spread in rHuEPO-treated rats suggests a feasible role for EPO in the aggressiveness and progression of malignant glioma.

Topics: Adult; Aged; Animals; Blotting, Western; Brain Neoplasms; Cell Line, Tumor; Disease Models, Animal; Erythropoietin; Female; Glioma; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Neoplasm Transplantation; Platelet Endothelial Cell Adhesion Molecule-1; Rats, Inbred F344; Receptors, Erythropoietin; Recombinant Proteins; Tumor Burden

To determine whether erythropoietin (EPO) promotes rapid proliferation of glioma through Akt pathway.. We detected the expression of EPO in human glioma tissues using immunohistochemistry. A nude mouse model bearing human glioma U87 cell xenograft was established and given intraperitoneal injection of EPO or saline every other day, and the tumor growth was observed. In the in vitro experiment, U87 cells were treated with PBS (control), EPO, or EPO with Akt inhibitor, and the expression of p-Akt and cyclin D1 was detected using Western blotting; the cell proliferation rate was determined using cell counting kit-8 and clone formation assay, and the cell cycle changes were analyzed with flow cytometry.. Compared with low-grade glioma tissues, high-grade glioma tissues exhibited a significantly increased EPO expression (P=0.0002). In the tumor-bearing mice, EPO treatment significantly increased the expression of EPO (P=0.0006) and p-Akt (P=0.0003) in the tumor and obviously increased the tumor volume (P<0.0001) and weight (P=0.0003). In U87 cells cultured in vitro, EPO treatment obviously accelerated the cell proliferation (P=0.020 on day 3 and 0.028 on day 5), promoted clone formation (P=0.0010), and increased proliferation index (P=0.0028); EPO significantly enhanced the protein expression of p-Akt (P=0.0020) and cyclin D1 (P=0.0022). The application of Akt inhibitor significantly suppressed the effect of EPO in enhancing cyclin D1 and p-Akt expression (both P<0.0001) and promoting cell proliferation.. EPO can significantly accelerate the proliferation of glioma through Akt pathway.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Erythropoietin; Glioma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Proto-Oncogene Proteins c-akt; Signal Transduction

In this study, the extent of angiogenesis, evaluated as microvascular density, and the immunoreactivity of tumor cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human glioma specimens, and the effect of anti-Epo antibody on glioma-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) has been investigated. Results show that: (1) Epo/EpoR expression correlates with angiogenesis, (2) in the CAM assay, tumor bioptic specimens induce a strong angiogenic response, comparable to that induced by VEGF, and (3) an anti-Epo antibody co-administered with tumor bioptic specimens significantly inhibits the angiogenic response. These findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by glioma tumor cells and it affects glioma vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner. Moreover, as demonstrated by in vivo experiments, Epo is responsible for the strong angiogenic response induced by human glioma bioptic specimens, because an anti-Epo antibody is able to significantly inhibit this response.

Topics: Angiogenesis Inducing Agents; Animals; Biomarkers, Tumor; Brain Neoplasms; Chick Embryo; Chorioallantoic Membrane; Erythropoietin; Glioma; Humans; Immunoenzyme Techniques; Neovascularization, Pathologic; Prognosis; Receptors, Erythropoietin; Retrospective Studies

Hypoxia has been shown to be one of the major events involved in EPO expression. Accordingly, EPO might be expressed by cerebral neoplastic cells, especially in glioblastoma, known to be highly hypoxic tumours. The expression of EPOR has been described in glioma cells. However, data from the literature remain descriptive and controversial. On the basis of an endogenous source of EPO in the brain, we have focused on a potential role of EPOR in brain tumour growth. In the present study, with complementary approaches to target EPO/EPOR signalling, we demonstrate the presence of a functional EPO/EPOR system on glioma cells leading to the activation of the ERK pathway. This EPO/EPOR system is involved in glioma cell proliferation in vitro. In vivo, we show that the down-regulation of EPOR expression on glioma cells reduces tumour growth and enhances animal survival. Our results support the hypothesis that EPOR signalling in tumour cells is involved in the control of glioma growth.

Topics: Animals; Astrocytes; Brain Neoplasms; Caudate Nucleus; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Culture Media, Conditioned; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Glioma; Hep G2 Cells; Humans; Male; MAP Kinase Signaling System; Mice; Mice, Nude; Phosphorylation; Rats; Rats, Inbred F344; Receptors, Erythropoietin; RNA, Small Interfering; Sequence Deletion; Signal Transduction; Survival Analysis; Xenograft Model Antitumor Assays

Despite beneficial effects of irradiation/chemotherapy on survival of glioblastoma (GBM) patients, collateral damage to intact neural tissue leads to "radiochemobrain" and reduced quality of life in survivors. For prophylactic neuroprotection, erythropoietin (EPO) is a promising candidate, provided that concerns regarding potential tumor promoting effects are alleviated.. Human GBM-derived cell lines U87, G44, G112, and the gliosarcoma-derived line G28 were treated with EPO, with and without combinations of irradiation or temozolomide (TMZ). Responsiveness of glioma cells to EPO was measured by cell migration from spheroids, cell proliferation, and clonogenic survival. Implantation of U87 cells into brains of nude mice, followed 5 days later by EPO treatment (5,000 U/kg intraperitoneal every other day for 2 weeks) should reveal effects of EPO on tumor growth in vivo. Reverse transcriptase-polymerase chain reaction was performed for EPOR, HIF-1alpha, and epidermal growth factor receptor (EGFR)vIII in cell lines and 22 human GBM specimens.. EPO did not modulate basal glioma cell migration and stimulated proliferation in only one of four cell lines. Importantly, EPO did not enhance tumor growth in mouse brains. Preincubation of glioma cells with EPO for 3 h, followed by irradiation and TMZ for another 24 h, resulted in protection against chemoradiation-induced cytotoxicity in three cell lines. Conversely, EPO induced a dose-dependent decrease in survival of G28 gliosarcoma cells. In GBM specimens, expression of HIF-1alpha correlated positively with expression of EPOR and EGFRvIII. EPOR and EGFRvIII expression did not correlate.. EPO is unlikely to appreciably influence basal glioma growth. However, concomitant use of EPO with irradiation/chemotherapy in GBM patients is not advisable.

Topics: Animals; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Division; Cell Line, Tumor; Cell Movement; Cell Survival; Combined Modality Therapy; Dacarbazine; Erythropoietin; Glioma; Gliosarcoma; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Temozolomide; Transplantation, Heterologous

Relationship between haemoglobin levels and tumour oxygenation has been already reported. The purpose of this work was to compare in human malignant glioma-bearing mice the sensitivity of two well established techniques of tumour hypoxia assessment, especially their ability to detect expected weak variations of tumour oxygenation status associated to haemoglobin level modifications. The relationship between tumour hypoxia and glucose metabolism was also investigated. Experiments were performed on a human malignant glioma (GBM Nan1) xenografted into nude mice. Twenty-four hours after tumour implantation, animals were randomized into three groups: 'Anaemia' for mice subjected to repeated blood samplings, 'Control', and 'rHuEPO' for mice receiving recombinant human erythropoietin. Once the tumours reached a volume of 300+/-100 mm(3), tumour hypoxia was assessed both using the pO(2)-Histograph, Eppendorftrade mark and the pimonidazole binding assay. Glucose metabolism was evaluated by (18)F-FDG autoradiography and compared with the pimonidazole binding distribution pattern. Repeated blood samplings significantly reduced mean haemoglobin levels (10.9+/-2.0 g/dl), inducing chronic anaemia in mice, while daily administration of rHuEPO led to increase of haemoglobin levels (15.8+/-2.0 g/dl). Oxygenation status evaluated by a microelectrode was worsened in anaemic mice (mean pO(2) in tumour = 6.9+/-0.8 mmHg) and improved in rHuEPO-treated animals (mean pO(2)in tumour = 11.4+/-1.2 mmHg). No correlation was observed between the oxygen-sensitive probe and pimonidazole labelling results: both techniques give different but complementary information about tumour hypoxia. Areas of high pimonidazole binding and areas of high (18)F-FDG uptake superimposed well. Present results confirm that modification of haemoglobin levels leads to alteration of tumour oxygenation status. These variations were detectable using the oxygen-sensitive electrode but not the pimonidazole binding assay. The strong correlation between pimonidazole labelling and (18)F-FDG uptake suggests a positive relationship between hypoxia and increased glucose metabolism in this tumour model.

Topics: Anemia; Animals; Autoradiography; Electrodes; Erythropoietin; Fluorodeoxyglucose F18; Glioma; Hemoglobins; Humans; Hypoxia; Mice; Mice, Nude; Muscle, Skeletal; Neoplasm Transplantation; Nitroimidazoles; Recombinant Proteins; Transplantation, Heterologous

The hypoxia-inducible pleiotropic hormone, erythropoietin (EPO), has recently been found to promote the development and survival of neurons and astrocytes. Since hypoxia has been implicated in the malignant progression of some human cancers, the authors investigated whether EPO signaling influenced the malignant properties of human astrocytoma cells.. Reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemical studies were used to measure EPO and its receptor (EPOR). Cell viability, Matrigel invasion assays, metalloprotease assays, EPO neutralizing antibodies, and EPOR overexpression were used to study the biological actions of EPO. Expression of both EPO and EPOR was observed in the hypoxic regions and invasive margins of glioma specimens obtained at biopsy, and expression of EPOR correlated with the stage of the tumor. The EPOR was also functionally upregulated by hypoxia in cultured glioblastoma multiforme (GBM) cells. Both hypoxia and EPO protected cultured GBM cells from cisplatin cytotoxicity and promoted the invasiveness of GBM cells through Matrigel by potentiating metalloprotease activity. Hypoxia-enhanced cell invasion was attenuated in cells that overexpressed a nonfunctional EPOR.. Hypoxia-inducible autocrine and paracrine EPO signaling participates in the malignant progression of GBMs.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cisplatin; Epoetin Alfa; Erythropoietin; Glioma; Hematinics; Humans; Neoplasm Invasiveness; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Signal Transduction

Although a significant negative prognostic factor, tumor hypoxia can be exploited for gene therapy. To maximize targeting within the tumor mass, we have developed synthetic gene promoters containing hypoxia-responsive elements (HREs) from the erythropoietin (Epo) gene as well as radiation-responsive CArG elements from the early growth response (Egr) 1 gene. Furthermore, to achieve high and sustained expression of the suicide gene herpes simplex virus thymidine kinase (HSVtk), our gene therapy vectors contain an expression amplification system, or 'molecular switch', based on Cre/loxP recombination. In human glioma and breast adenocarcinoma cells exposed to hypoxia and/or radiation, the HRE/CArG promoter rapidly activated Cre recombinase expression leading to selective and sustained HSVtk synthesis. Killing of transfected tumor cells was measured after incubation with the prodrug ganciclovir (GCV; converted by HSVtk into a cytotoxin). In vitro, higher and more selective GCV-mediated toxicity was achieved with the switch vectors, when compared with the same inducible promoters driving HSVtk expression directly. In tumor xenografts implanted in nude mice, the HRE/CArG-switch induced significant growth delay and tumor eradication. In conclusion, hypoxia- and radiation-activated 'molecular switch' vectors represent a promising strategy for both targeted and effective gene therapy of solid tumors.

Topics: Adenocarcinoma; Adenoviridae; Animals; Antiviral Agents; Brain Neoplasms; Breast Neoplasms; Cell Death; Cell Hypoxia; Combined Modality Therapy; Early Growth Response Protein 1; Erythropoietin; Ganciclovir; Gene Expression Regulation, Neoplastic; Genes, Switch; Genes, Transgenic, Suicide; Genetic Engineering; Genetic Therapy; Genetic Vectors; Glioma; Humans; Mice; Mice, Nude; Neoplasms; Oncolytic Virotherapy; Promoter Regions, Genetic; Simplexvirus; Thymidine Kinase; Tumor Cells, Cultured

Our study investigated the influence of recombinant human erythropoietin (rHuEPO) treatment, inducing raised hemoglobin levels in nonanemic mice, on intratumor oxygenation before and during fractionated irradiation. Furthermore, the consequences of rHuEPO administration on tumor response to fractionated radiotherapy (RT) were evaluated.. Experiments were performed on two human malignant glioma (GBM Nan1 and U87) xenografted in nude mice. RHuEPO was daily delivered (0.3 IU/g/day, 5 days/week). Tumor hypoxia was assessed before (T1) and during (T6) fractionated irradiation using (1) pO(2)-Histograph (Eppendorf, Hamburg, Germany) and (2) the EF5-binding assay. Vascular density was determined using type IV collagen immunostaining. To assess RT efficacy, the irradiation schedule was 20 fractions of 2 Gy, once daily, 5 days/week over 4 weeks.. At T1, hemoglobin levels in rHuEPO-treated mice were significantly increased. Percentage of pO(2) values <2.5 mm Hg was reduced in rHuEPO-treated tumors as compared with control groups (37.1 +/- 19.1% vs. 58.5 +/- 27.0%; p = 0.009 for GBM Nan1; 81.6 +/- 13.4% vs. 91.5 +/- 8.3%; p = 0.035 for U87). The decrease of viable hypoxic tumor cells fraction after rHuEPO was confirmed by the EF5-binding assay. Vascular density was not altered after rHuEPO treatment. At T6, rHuEPO reduced the hypoxic fraction by about 20% (p = 0.036 and p = 0.171) in GBM Nan1 and U87 irradiated tumors. RHuEPO did not influence tumor growth by itself. RT alone or combined with rHuEPO induced a significant tumor growth delay. Finally, rHuEPO significantly enhanced RT efficacy (p = 0.012 in GBM Nan1 and p = 0.037 in U87), resulting in radiopotentiation ratios of 1.21 and 1.54 for respective models.. Our results indicate that rHuEPO, by enhancing blood oxygen-carrying capacity, decreases intrinsic tumor hypoxia and maintains its effect during fractionated irradiation in malignant glioma xenografts. Therefore, rHuEPO contributes to radiosensitize these tumors.

Topics: Animals; Cell Hypoxia; Erythropoietin; Glioma; Hemoglobins; Humans; Mice; Mice, Nude; Oxygen; Radiation Tolerance; Recombinant Proteins; Transplantation, Heterologous

Recent studies suggest that erythropoietin plays an important role in the process of neoplastic transformation and malignant phenotype progression observed in malignancy. To study the role of erythropoietin and its receptor (EPOR) on the response of cancer cells in vitro, we used two solid tumor cell lines, namely the human malignant glioma cell line U87 and the primary cervical cancer cell line HT100. All experiments were done with heat-inactivated fetal bovine serum in order to inactivate any endogenous bovine erythropoietin. The expression of the EPOR in these cells was confirmed with immunoblot techniques. The addition of exogenous recombinant human erythropoietin (rhEPO) induces the cancer cells to become more resistant to ionizing radiation and to cisplatin. Furthermore, this rhEPO-induced resistance to ionizing radiation and to cisplatin was reversed by the addition of tyrphostin (AG490), an inhibitor of JAK2. Our findings indicate that rhEPO result in a significant, JAK2-dependent, in vitro resistance to ionizing radiation and to cisplatin in the human cancer cells lines studied in this report.

Topics: Animals; Antineoplastic Agents; Cattle; Cisplatin; Drug Resistance, Neoplasm; Erythropoietin; Female; Glioma; Humans; Janus Kinase 2; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Radiation Tolerance; Radiation, Ionizing; Receptors, Erythropoietin; Recombinant Proteins; Tumor Cells, Cultured; Tyrphostins; Uterine Cervical Neoplasms

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.

Topics: Cell Hypoxia; DNA-Binding Proteins; Endothelial Growth Factors; Erythropoietin; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Glioma; Helix-Loop-Helix Motifs; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphokines; Nuclear Proteins; Transcription Factors; Transfection; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors