Influences Of The P53 Status On Hypoxia-induced Gene Expression & Adenovirus-mediated Gene Therapy Targeting Tumor Hypoxia

Hypoxia is an important microenvironment in solid tumors. Hypoxic tumor cells exhibit increased resistance to radiotherapy and chemotherapy. The existence of hypoxic cells is one of the most important reasons for treatment failure, relapse and metastasis. Upon hypoxia stimuli, a lot of genes are activated, including angiogenesis related genes, glucose transport related genes, glycolysis related genes, erythropoiesis related genes and apoptosis related genes. Hypoxia inducible factor 1 (HIF-1), the most characterized hypoxia-regulated transcriptional factor, mediates the hypoxia-dependent transcription of these genes.p53 is mutated in many types of solid tumors. The inactivation of p53 is associated with malignant phenotype of tumor cells, which could be enhanced by hypoxia. It was believed that wild-type p53 could inhibit HIF-1-mediated gene transcription, for example the hypoxia-dependent expression of vascular endothelial growth factor (VEGF). It has been reported that exogenous p53 could inhibit HIF-1-mediated transcription from the VEGF promotor constructs or erythropoietin (EPO) promotor constructs. Wild-type p53, butnot mutated p53, displayed inhibitory effects on endogenous expression of VEGF. A transduction with an adenoviral vector containing the wild-type p53 gene could down-regulate VEGF expression in human colon cancer cell lines with p53 mutations. However, these in vitro experiments were performed without hypoxic culture conditions. Moreover, others have argued that there is no evidence for a casual relationship between the loss of p53 activity and increased VEGF expression. They show that hypoxia could increase endogenous VEGF mRNA levels in either the presence or absence of functional p53. Recent studies show that HIF-l either directly bound to p53 or indirectly via binding to Mdm2, suggesting functional interactions between p53 and HIF.Hypoxia cells display differentially expressed gene profiles comparing to normoxia cells, which provides possibilities for hypoxia-targeted therapy in solid tumor. Many hypoxia-dependent genes contain hypoxia-responsive elements (HRE) in their regulatory region. HRE is the binding site for HIF-l. HIF-l is inactivated under normoxia for its a subunit is degraded by proteosome. Under hypoxia, degradation process is inhibited, which allow accumulation of a subunit and activation of HIF-l. All these data suggest that HRE might mediate hypoxia-dependent expression of some interesting genes. It was first demonstrated in 1997 that heterologous gene expression driven by a HRE from the mouse phosphoglycerate kinase could be activated in hypoxic tumor cells. Hereafter, the HRE from different hypoxia-dependent genes was successfully used in hypoxia-targeted gene expression. However, the anti-tumor effects of the hypoxia-targeted gene therapy are very limited, which needs further explorations.Aims:1. To explore the effects of p53 status on hypoxia-induced gene expression.2. To construct a hypoxia-activated adenovirus vector expressing suicide gene BCD and to evaluate the anti-tumor effects of this hypoxia-targeted suicide gene therapy system with or without combination of radiotherapy.Methods:Part I : Influences of the p53 status on hypoxia-induced gene expression1. Inhibition of gene expression by wt-p53 in transfection assay in SaOS2 cells1) Construction of tetracycline-regulated plasmid pTRE2wt-p53 : a cDNA fragment encoding wild type p53 gene was obtained by an RT-PCR method. The amplified fragment was digested, subcloned into the sites of a pTRE2hyg expression vector. The correct construction of pTRE2wt-p53 was confirmed by enzyme digestion and a sequencing analysis.2) Establishment of stable SaOS2 Tet-Off clones : SaOS2 cells were tranfected with pTet-Off vector using the Superfect reagent. The cells were plated for clonal selection of stable transfectants in 400(ig/ml G-418. To obtain good clones that showed a robust responsiveness to vari...