Enhanced efficacy of radiation-induced gene therapy in mice bearing lung adenocarcinoma xenografts using hypoxia responsive elements.

The aim of the present study was to investigate whether the hypoxia responsive element (HRE) could be used to enhance suicide gene (HSV-tk) expression and tumoricidal activity in radiation-controlled gene therapy of human lung adenocarcinoma xenografts. A chimeric promoter, HRE-Egr, was generated by directly linking a 0.3-kb fragment of HRE to a 0.6-kb human Egr-1 promoter. Retroviral vectors containing luciferase or the HSV-tk gene driven by Egr-1 or HRE-Egr were constructed. A human adenocarcinoma cell line (A549) was stably transfected with the above vectors using the lipofectamine method. The sensitivity of transfected cells to prodrug ganciclovir (GCV) and cell survival rates were analyzed after exposure to a dose of 2 Gy radiation and hypoxia (1%). In vivo, tumor xenografts in BALB/c mice were transfected with the constructed retroviruses and irradiated to a total dose of 6 Gy, followed by GCV treatment (20 mg/kg for 14 days). When the HSV-tk gene controlled by the HRE-Egr promoter was introduced into A549 cells by a retroviral vector, the exposure to 1% O(2) and 2 Gy radiation induced significant enhancement of GCV cytotoxicity to the cells. Moreover, in nude mice bearing solid tumor xenografts, only the tumors infected with the hybrid promoter-containing virus gradually disappeared after GCV administration and radiation. These results indicate that HRE can enhance transgene expression and tumoricidal activity in HSV-tk gene therapy controlled by ionizing radiation in hypoxic human lung adenocarcinoma.

Cisplatin-controlled p53 gene therapy for human non-small cell lung cancer xenografts in athymic nude mice via the CArG elements.

Cisplatin, a commonly used chemotherapeutic agent, causes tumor cell death by producing DNA damage and generating reactive oxygen intermediates, which have been reported to activate the early growth response-1 (Egr-1) promoter through specific cis-acting sequences, termed CArG elements. The aim of this study was to construct an adenoviral vector containing CArG elements cloned upstream of the cDNA for human wt-p53, and to observe the effect of this vector on human non-small cell lung cancer (NSCLC) xenografts in athymic nude mice when combined with cisplatin treatment. The adenoviral vector AdEgr-p53 was generated by inserting CArG elements upstream of human wt-p53 cDNA. Two human NSCLC cell lines of varying p53 gene status, A549 (containing wild-type p53) and H358 (containing an internal homozygous deletion of the p53 gene) were used for in vitro and in vivo experiments. Wt-p53 production in cultured tumor cells and xenografts treated with the combination of AdEgr-p53 and cisplatin were detected by enzyme-linked immunosorbent assays. The antitumor responses in nude mice with the A549 or H358 xenografts following treatment with AdEgr-p53 and cisplatin were observed. We found that p53 was produced in tumor cells and xenografts treated with a combination of AdEgr-p53 and cisplatin. Furthermore, the Egr-1 promoter is induced by cisplatin, and this induction is mediated in part through the CArG elements. There was an enhanced antitumor response without an increase in toxicity following treatment with AdEgr-p53 and cisplatin, compared with either agent alone. Cisplatin-inducible p53 gene therapy may provide a means to control transgene expression while enhancing the effectiveness of commonly used chemotherapeutic agents. This is a novel treatment for human NSCLC.

Detecting normal cell radiosensitivity via assay of DNA damage in lymphocytes for individualizing radiotherapy in head and neck cancer patients.

PURPOSE: The purpose of this study was to determine whether the distribution of radiosensitivities in normal tissues of head and neck cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable reliable identification of patients with abnormal sensitivities. MATERIAL AND METHODS: Radiosensitivity was assessed in 307 lymphocyte samples from unselected head and neck cancer patients and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gray and per DNA unit (200 Mbp). RESULTS: The existence of an inter-individual variation in the radiosensitivity parameter is described by the range (0.41--9.38 dsb/Gy/DNA unit) of the values found. We detected 37 patients who developed severe skin reactions during radiotherapy treatment and we compared their radiosensitivity values with the remaining patients treated. Radiosensitivity values of >7.20 dsb/Gy/DNA unit should theoretically correspond to highly radiosensitive patients. CONCLUSIONS: Our results suggest that initial DNA damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. .

Correlation between DNA repair capacity in lymphocytes and acute side effects to skin during radiotherapy in nasopharyngeal cancer patients.

PURPOSE: Repair of radiation-induced DNA damage plays a critical role for both the susceptibility of patients to side effects after radiotherapy and their subsequent cancer risk. The study objective was to evaluate whether DNA repair data determined in vitro are correlated with the occurrence of acute side effects during radiotherapy. EXPERIMENTAL DESIGN: Nasopharyngeal cancer patients receiving radiation therapy were recruited in a prospective epidemiologic study. As an indicator for clinical radiosensitivity, adverse reactions of the skin were recorded. Cryopreserved lymphocytes from 100 study participants were gamma-irradiated with 5 Gy in vitro and analyzed using the alkaline comet assay. Reproducibility of the assay was determined by repeated analysis (n = 22) of cells from a healthy donor. A coefficient of variation of 0.24 was calculated. RESULTS: The various parameters determined to characterize the individual DNA repair capacity showed large differences between patients. Twenty-one patients were identified with considerably enhanced DNA damage induction, and 19 patients exhibited severely reduced DNA repair capacity after 15 and 30 minutes. Eight patients were considered as clinically radiosensitive, indicated by moist desquamation of the skin after a total radiation dose of 70 Gy. CONCLUSIONS: Using the alkaline comet assay as described here, nasopharyngeal cancer patients were identified showing abnormal cellular radiation effects, but this repair deficiency corresponded only at a very limited extent to the acute radiation sensitivity of the skin.

[HSV-TK gene therapy of lung adenocarcinoma xenografts using a hypoxia/radiation dual-sensitive promoter].

BACKGROUND & OBJECTIVE: Radio-genetic therapy is a novel strategy for cancer treatment; however, a limited success was shown due to lower sensitivity of tumor cells to radiation under hypoxia, which is a unique feature for solid tumors. In order to improve the efficacy of radiogenetic therapy for lung cancer, a hypoxia/radiation dual-sensitive promoter was constructed to enhance the expression of HSV-TK in transfected cells exposed to radiation under hypoxia. METHODS: The chimeric promoter HRE-Egr was generated by insertion of hypoxia response elements (HREs) upstream of the Egr-1 (early growth response gene-1) promoter. HSV-TK expression vector was constructed by cloning HRE-Egr promoter upstream of HSV-TK gene, which was transfected into A549 cells via liposome. The expression of HSV-TK in transfected cells exposed to irradiation (6 Gy) and/or hypoxia (1% O2) were analyzed by Northern blot, and the relative survival rate of cells in presence of prodrug ganciclovir (GCV) was tested using MTT method. To examine the efficacy of this HRE-Egr-TK gene therapy in vivo, the A549 adenocarcinoma xenografts were planted in BALB/c nude mice. The tumor volumes and the suppression rates were assayed in nude mice bearing xenografts infected with plasmids and exposed to radiation. RESULTS: HSV-TK gene expression in transfected cells was markedly increased in both radiation (227 U) and hypoxia (94 U) groups compared with control group (21 U). The HSV-TK expression (769 U) in transfected cells exposed to radiation under hypoxia is much more higher than the former groups. The survival rate of transfected cells exposed to radiation under hypoxia in the presence of GCV was obviously decreased with comparison of cells under normoxia (7.2%+/-1.8 % vs 32.7%+/-4.6 %). HRE-Egr promoter transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n=10), the tumor suppression rates was 91.2%. CONCLUSIONS: The hypoxia/radiation dual-sensitive promoter HRE-Egr can enhance the HSV-TK gene expression in solid tumors under hypoxia. Enhanced tumor suppression effect was observed in A549 xenografts infected with HRE-Egr promoter exposed to radiation.

[Oncostatin M gene therapy in mice bearing lung adenocarcinoma xenograft using a hypoxia/radiation dual-sensitive promoter].

OBJECTIVE: To improve the efficacy of radiogenetic therapy for lung cancer, a hypoxia/radiation dual-sensitive promoter was constructed to enhance the expression of oncostatin M (OSM) in transfected cells exposed to radiation under hypoxia. METHODS: The chimeric promoter HRE-Egr was generated by insertion of hypoxia response elements (HREs) upstream of the Early growth response gene-1 (Egr-1) promoter. OSM expression vector was constructed by cloning HRE-Egr promoter upstream of OSM gene, which was transfected into A549 cells. The expression of OSM in transfected cells exposed to irradiation and(or) hypoxia was analyzed, and the relative survival rate of transfected cells exposed to the above conditions was tested. To examine the efficacy of this HRE-Egr-OSM gene therapy in vivo, the tumor suppression effects were investigated in 40 nude mice bearing lung adenocarcinoma xenograft. RESULTS: Expression of OSM gene in transfected cells exposed 6 Gy irradiation was markedly increased under hypoxia. A gene therapy experiment in vitro showed that the survival rate of transfected cells exposed to radiation under hypoxia was obviously decreased with comparison of cells under normoxia. HRE-Egr promoter transfected tumors regressed significantly after a combination therapy of irradiation and HRE-Egr transfection in all mice (n = 10), and six tumors disappeared in 3 weeks without any side effects. CONCLUSION: The data indicate that tumor targeted expression of OSM gene under the control of a hypoxia/radiation dual-sensitive promoter represents a novel strategy for safe and effective gene therapy of lung carcinoma and might have clinical application in the future.

[Experimental study on lung carcinoma-targeted suicide gene therapy induced by irradiation].

OBJECTIVE: To improve the efficacy and selectivity of gene therapy for lung carcinoma, a strategy was designed for suicide gene therapy in conjunction with irradiation therapy, by constructing a plasmid tgEgr-HyTK in which HSV-TK gene was driven by the early growth responsive gene-1 (Egr-1) promoter. METHODS: The radio-inducible suicide gene was constructed by insertion of Egr-1 promoter upstream of the HSV-TK gene. The expressions of HSV-TK in lung carcinoma cell lines A549 which were infected with tgEgr-HyTK and exposed to different doses of gamma-ray irradiation were analyzed, and the relative survival rates of cells in presence of prodrug ganciclovir (GCV) were tested. The tumor suppression effects were investigated in 40 nude mice bearing lung tumors to examine the efficacy of this Egr-TK gene therapy in vivo. RESULTS: Expression of HSV-TK gene in lung carcinoma cells infected with tgEgr-HyTK plasmids was markedly increased in a radiation dose-dependent manner. A gene therapy experiment in vitro showed that tgEgr-HyTK transduced lung carcinoma cells became highly sensitive to GCV after irradiation, but not without irradiation. tgEgr-HyTK transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 10), and five tumors disappeared in 3 weeks without any side effect. CONCLUSION: The data indicate that tumor targeted expression of HSV-TK gene under the control of a radio-inducible promoter represents a novel strategy for safe and effective gene therapy of lung carcinoma, which may have clinical application in the future.