| Radio-genetic therapy is a novel strategy for cancer treatment, in which an ionizing radiation-inducible regulatory sequence is linked with an adjuvant tumor-therapeutic cytokine sequence, and transfected into tumor cells. The expression of therapeutic gene, therefore, will be induced by radiation in cancer radiotherapy. The transfected cancer cells will be destroyed by both radiation and the radiation-inducible gene. Although some beneficial effects for tumor therapy have been reported in a recent clinical trial, a few shortcomings still exist in radio-genetic therapy, which include the expression level of therapeutic genes induced by radiation is too low to completely eradicate the tumor, especially under the routine clinical dose of radiation; and the sensitivity to radiation of radio-inducible sequences in in vivo tumor tissues is less than that of in vitro cultured cells. These shortcomings will decrease the therapeutic effects of radio-genetic therapy on tumors.The cellular adaptive response to hypoxia involves a series of modulations of biochemical and pathophysiological processes such as glucose homeostasis, angiogenesis, vascular permeability and inflammation. HIF-1(hypoxia inducible factor-1) is the core mediator of hypoxic responses. To modulate related gene expression, HIF-1 specifically binds to hypoxia-responsive elements(HREs), enhancers containing the same core sequence in several hypoxia-regulated genes. The HRE/HIF regulatory system was shown to be common to all mammalian cells including human cells tested to date, and HIF-1α subunit was found to be overexpressed in 68% of the human tumors analysed. The high frequency of HIF-1 expression in numerous human tumors with diverse tissue origins provides a possible therapeutic target for HRE-directed gene therapy of tumor cells in hypoxic environments. It has been demonstrated that expression of promoters which were linked with HRE could be enhanced under hypoxic conditions. Solid tumors are characterized by hypoxia which can activate the HRE effectively. Therefore, we want to utilize the HRE to enhance the transcriptional activity of radiation-inducible promoters inthe hypoxic environment of tumors to try to solve the problems with radio-genetic therapy mentioned above.Cellular responses to ionizing radiation include the transcriptional induction of immediate early genes such as early growth response-1(Egr-1) which may enhance cell death after ionizing radiation. The radiosensitivity of the Egr-1 promoter has been demonstrated, and widely used in radio-genetic therapy. To address the feasibility of hypoxia-specific enhancement of expression of the Egr-1 promoter induced by ionizing radiation, experiments have been carried out as follows:①First, a reporter vector containing the luciferase gene under the control of the Egr-1 promoter was constructed, and transfected into the human adenocarcinoma cell line A549. After exposure to doses of 2,4,6,8 or 10 Gy γ-radiation using a 60Co source, the expression level of luciferase in cell extracts at 0h,3h,8h,12h,24h and 36h post-irradiation was analysed. The expression level of luciferase and H2O2 production in the cells was also detected at 8h post-radiation in trasfected cells with exposure to above doses of radiation under 0.1%,0.5%,1%,2.5%,5% or 10% oxygen concentration. ②An HRE-Egr chimeric promoter was constructed, in which a 0.3 kb fragment of the human vascular endothelial growth factor 5`-flanking sequence containing HRE was fused to the Egr-1 promoter. The chimeric promoter was then inserted into the upstream of the luciferase gene in a reporter vector. Luciferase activity was determined in cell extracts after exposure to the same doses of radiation and oxygen concentrations as shown above. Furthermore, the dynamic alterations in HRE-Egr promoter activity were observed after the cells were incubated in 1% O2 and irradiated. ③The OSM(Oncostatin M) expression vectors pEO and pHEO, in which the Egr-1 promoter or HRE-Egr-1 promoter was inser...
|
PDF Full Text Request