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The purpose of the present study was to determine the therapeutic potential of combining radiotherapy with tumor necrosis factor (TNF)-alpha-based gene therapy in the human prostate cancer PC-3 xenograft. PC-3 cells are highly resistant to TNF-alpha-induced cytotoxicity in vitro. A modest enhancement of radiation killing was observed with the addition of TNF-alpha in clonogenic survival assays. Combined treatment with Ad.Egr-TNF, a replication-deficient adenovirus modified to express TNF-alpha following the exposure of infected cells to ionizing radiation (40 Gy administered at 5 Gy per fraction) in vivo, resulted in increased tumor control, as defined by a reduction of tumor volume, when compared with treatment with Ad.Egr-TNF alone or with radiation alone (P.03). The improvement in tumor control was achieved without increasing acute normal tissue damage when compared with tissue injury from radiation alone. The results of these studies support further development and clinical application of genetic radiotherapy for human prostate cancer.

Gene therapy combined with radiation therapy to enhance selectively radiation cytotoxicity in malignant cells represents a new approach for cancer treatment. We investigated the efficacy of adenoviral (Ad5)-directed cytosine deaminase/5-fluorocytosine (CD/5-FC) enzyme/prodrug gene therapy to enhance selectively the tumoricidal action of ionizing radiation in human cancer xenografts derived from a human squamous carcinoma cell line (SQ-20B). Tumor xenografts grown in hindlimbs of nude mice were transfected with an adenoviral vector (Ad.CMV.CD) containing the cytosine deaminase (CD) gene under the control of a cytomegalovirus (CMV) promoter. Mice were injected i.p. with 800 mg/kg of 5-FC for 12 days, and tumors were treated with fractionated radiation at a dose of 5 Gy/day to a total dose of 50 Gy. In larger tumors with a mean volume of 1069 mm3, marked tumor regression to 11% of the original tumor volume was observed at day 21 (P = 0.01). The volumetric regression of smaller tumors with a mean volume of 199 mm3, which received the same combined treatment protocol, was significant at day 12 (P = 0.014). However, unlike large tumors, regression of the smaller tumors continued until day 36 (P = 0.01), with 43% cured at day 26. No cures or significant volumetric reduction in size was observed in tumors treated with radiation alone; Ad.CMV.CD with or without radiation; or with Ad.CMV.CD and 5-FC. These results suggest that the CD/5-FC gene therapy approach is an effective radiosensitizing strategy and may lead to substantial improvement in local tumor control that would translate into improved cure rates and better survival.

Intratumoral injection of an adenoviral vector containing radiation-inducible DNA sequences of the early growth response gene (Egr-1) promoter ligated to a cDNA encoding tumor necrosis factor-alpha (TNF-alpha; Ad.Egr-TNF) increases the radiation killing of a human radioresistant xenograft (SQ-20B). Viral dose-escalation experiments demonstrated that SQ-20B growth inhibition correlated with viral titer. Injection of 5 x 10(8) PFU Ad.Egr-TNF produced regression to a mean volume of 22 +/- 13% of the original tumor volume, 1 x 10(8) PFU to a mean of 62 +/- 24%, and 5 x 10(7) PFU to a mean of 67 +/- 27%. No regression was observed when tumors were injected with 1 x 10(7) PFU Ad.Egr-TNF or with the null viral vector (Ad.null). When two injections of vector (2 x 10(8) PFU Ad.Egr-TNF) were combined with 50 Gy, a significant increase in tumor regression was observed compared with injection of buffer, Ad.Egr-TNF, or 50 Gy. The interactive killing between TNF and radiation was enhanced significantly (P = 0.05) when the number of injections was increased from two to five while maintaining a constant viral titer (2 x 10(8) PFU Ad.Egr-TNF) and a constant radiation dose (50 Gy). Significant TNF-alpha levels were present in irradiated vs. unirradiated tumors following injection with Ad.Egr-TNF. Taken together, these data suggest that the volumetric reduction produced by the combined effects of Ad.Egr-TNF and radiation is enhanced with increasing vector concentration and the number of vector injections.

Intratumoral injection of an adenoviral vector containing radiation-inducible DNA sequences of the Egr-1 promoter linked to a cDNA encoding tumor necrosis factor (TNF) alpha (Ad.Egr-TNF) enhances the tumoricidal action of ionizing radiation in a human epidermoid carcinoma xenograft (SQ-20B). The dominant histopathological feature in tumor-bearing animals treated with Ad.Egr-TNF and irradiation is extensive intratumoral vascular thrombosis and tumor necrosis. Thrombosis and necrosis are not observed in animals treated with either the viral construct encoding TNF-alpha or radiation and did not occur in irradiated normal tissues adjacent to tumor in animals injected with Ad.Egr-TNF. To determine if the occlusive effects of Ad.Egr-TNF and X-irradiation were specific for tumor vessels, non-tumor-bearing mice were irradiated after receiving i.m. injection of Ad.Egr-TNF at viral titers 20-100 times greater than titers injected intratumorally. No vascular thrombosis was observed in the treated normal tissues. Combined Ad.Egr-TNF and radiation produced occlusion of tumor microvessels without significant normal tissue damage. Taken together, these data suggest that the interaction between radiation inducible TNF-alpha and X-irradiation occurs selectively within the tumor vessels.

We report that radiation enhances gene therapy of a radioresistant tumor by upregulating the induction of a chimeric gene encoding a radiosensitizing protein, tumor necrosis factor alpha (TNF-alpha). We ligated the radiation-inducible CArG elements of the radiation-inducible Egr-1 promoter/enhancer region upstream to the transcriptional start site of the human TNF cDNA (pE425-TNF). This construct was transfected using cationic liposomes into the variant murine fibrosarcoma cell line, P4L. The P4L cell line was both radioresistant (D0 = 188) and resistant to TNF. After a single intratumoral injection of 10 micrograms of pE425-TNF in cationic liposomes and two 20-Gy doses of irradiation, mean tumor volumes were significantly reduced in P4L tumors as compared to those receiving either pE425-TNF in liposomes or radiation alone (P = 0.01). TNf protein in P4L tumors was induced by radiation as high as 29 times control levels and remained detectable for 14 days. Our data indicate that combined gene therapy using liposomes, together with ionizing radiation to locally activate the induction of a radiosensitizing protein, is successful at overcoming resistance to both TNF and radiation.

Transcriptional regulation of the promoter/enhancer region of the Egr-1 gene is activated by ionizing radiation. We linked DNA sequences from the promotor region of Egr-1 to a complementary DNA sequence which encodes human tumor necrosis factor (TNF) alpha, a radiosensitizing cytokine. The Egr-TNF construct was transfected into a human cell line of hematopoietic origin, HL525, which was used in an experimental animal system. HL525 (clone 2) cells containing the Egr-TNF construct which exhibits radiation induction of TNF-alpha were injected into human xenografts of the radioresistant human squamous cell carcinoma cell line SQ-20B. Animals treated with radiation and clone 2 demonstrated an increase in tumor cures compared with animals treated with radiation alone or unirradiated animals given injections of clone 2 alone. No increase in local or systemic toxicity was observed in the combined treatment group. The combination of gene therapy and radiation therapy enhances tumor cures without increasing normal tissue toxicity and is a new paradigm for cancer treatment.