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Obliteration of the tumor vasculature is an effective means of achieving tumor regression. Antiangiogenic agents have begun to enter cancer clinical trials. Ionizing radiation activates the inflammatory cascade and increases the procoagulative state within blood vessels of both tumors and normal tissues. These responses are mediated through oxidative injury to the endothelium, leading to induction of cell-adhesion molecules and exocytosis of stored proteins from the endothelial cytoplasm. Agents that activate homeostatic responses in the endothelium can enhance thrombosis and vasculitis of irradiated tumor blood vessels. Proinflammatory and prothrombotic biological response modifiers given concurrently with ionizing radiation are known to induce vascular obliteration and necrosis of tumors. Other mechanisms of interaction between antiangiogenic agents and ionizing radiation include the direct cytotoxic effects of these agents. Interactions between drugs and radiation therapy might therefore occur at the level of the vascular endothelium. The importance of this paradigm is that the endothelium might not develop resistance to drugs or radiation because of lessened potential for mutagenesis and clonogenesis. The future design of clinical trials must consider the effects of radiation therapy on the vascular endothelium.

Ionizing radiation induces the inflammatory response in part through leukocyte binding to cell adhesion molecules that are expressed on the vascular endothelium. We studied the effects of X radiation on the pattern of immunohistochemical staining of CD62P (P-selectin). P-selectin was localized within cytoplasmic granules in the untreated vascular endothelium. Immunohistochemical staining of P-selectin was observed at the luminal surface of vascular endothelium within 1 h of irradiation. Increased P-selectin staining at the blood-tissue interface occurred primarily in pulmonary and intestinal blood vessels. To determine whether localization of P-selectin at the vascular lumen occurs through exocytosis of endothelial cell stores in addition to platelet aggregation, we removed the vascular endothelium from the circulation and irradiated endothelial cells in vitro. In this system, we studied the mechanisms by which ionizing radiation induced translocation of P-selectin by using immunofluorescence of human umbilical vein endothelial cells (HUVEC) and confocal microscopy. Prior to irradiation, P-selectin is localized in cytoplasmic reservoirs of HUVEC. After irradiation of HUVEC, P-selectin was translocated to the cell membrane, where it remained tethered. The lowest dose at which we could expect translocation of P-selectin to the cell membrane was 2 Gy. To determine whether P-selectin in Weibel-Palade bodies requires microtubule-dependent membrane transport, we added two microtubule-depolymerizing agents, Colcemid and nocodazole. Microtubule-depolymerizing agents prevented radiation-induced trans- location of P-selectin to the cell membrane. Thus P-selectin accumulates in irradiated blood vessels through both platelet aggregation and microtubule-dependent exocytosis of storage reservoirs within the vascular endothelium.

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.

X-ray-induced expression of inflammatory mediators has been proposed to contribute to radiation injury in normal tissues. Radiation-inducible inflammatory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1. Nuclear factor (NF)kappaB is activated by X-rays and may participate in the transcriptional regulation of each of these inflammatory mediators. To determine whether NFkappaB inhibition abrogates X-ray induction of inflammatory mediators, we used two experimental approaches including NFkappaB inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells were treated with the NFkappaB inhibitors ALLN, PDTC, NAC, and MG132. After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and ICAM-1 expression was measured by use of immunofluorescence and fluorescence-activated cell-sorting analysis. E-selectin expression increased 7-fold, and ICAM-1 expression increased 4-fold after irradiation. All of the inhibitors attenuated E-selectin expression after irradiation. ALLN and MG132 attenuated radiation-induced ICAM expression. However, PDTC and NAC induced increased expression of ICAM-1 in HUVECs. Inhibition of X-ray induction of ICAM by these agents could not be demonstrated. In separate experiments, the NFkappaB dominant negative genetic construct was cotransfected with the promoter-reporter constructs by means of Lipofectin reagent. The ICAM promoter-reporter construct consists of the 1.2-kb segment of the human ICAM promoter upstream of the transcriptional start site linked to the luciferase reporter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-selectin promoter linked to the human growth hormone reporter gene (pE525-GH). Endothelial cells transfected with the ICAM-1 promoter-reporter construct showed a 3-fold induction after irradiation. Likewise, cells transfected with pE525-GH showed a 7-fold induction after irradiation. When cotransfected with the CAM reporter-promoter constructs, the NFkappaB dominant negative genetic construct abolished X-ray-induced transcriptional activation of the E-selectin and ICAM-1 promoters. NFkappaB inhibition is, therefore, a means of abrogating radiation-induced expression of CAMs.

P-selectin is a cell adhesion molecule that is sequestered in Weibel-Palade storage reservoirs within the vascular endothelium and alpha granules in platelets. P-selectin is rapidly translocated to the vascular lumen after tissue injury to initiate the adhesion and activation of platelets and leukocytes. We studied the histological pattern of P-selectin expression in irradiated tumor blood vessels. We observed that P-selectin was localized within the endothelium of tumor vessels prior to treatment. At 1-6 h following irradiation, P-selectin was mobilized to the lumen of blood vessels. To determine whether radiation-induced vascular lumen localization of P-selectin was tumor type specific or species specific, we studied tumors in rats, C3H mice, C57BL6 mice, and nude mice. P-selectin localization to the vascular lumen was present in all tumors and all species studied. Irradiated intracranial gliomas showed P-selectin localization to the vascular lumen within 1 h, whereas blood vessels in normal brain showed no P-selectin staining in the endothelium and no localization to the irradiated vascular lumen. Radiation-induced P-selectin localization to the vascular lumen increased in time-dependent manner, until 24 h after irradiation. P-selectin in platelets may account for the time-dependent increase in staining within the vascular lumen after irradiation. We therefore used immunohistochemistry for platelet antigen GP-IIIa to differentiate between endothelial and platelet localization of P-selectin. We found that GP-IIIa staining was not present at 1 h after irradiation, but it increased at 6 and 24 h. P-selectin localization to the vascular lumen at 6-24 h was, in part, associated with platelet aggregation. These findings indicate that radiation-induced P-selectin staining in the vascular lumen of neoplasms is associated with aggregation of platelets. Radiation-induced localization of P-selectin to the vascular lumen is specific to the microvasculature of malignant gliomas and is not present in the blood vessels of the irradiated normal brain.

We evaluated the antitumor effects of ionizing radiation and tumor necrosis factor-alpha (TNF-alpha) gene therapy in human malignant glioma (D54) xenografts. An adenoviral vector (Ad5) containing DNA sequences of the Egr-1 promoter was linked to a cDNA encoding the TNF-alpha gene (Ad. Egr-TNF). Athymic nude mice bearing D54 xenografts received intratumoral injections of Ad.Egr-TNF or the null vector (Ad.null), with and without fractionated radiation, 5 gray (Gy) per day for 6 days, a total dose of 30 Gy. Administration of Ad.Egr-TNF and 30 Gy resulted in complete tumor regression in 71% of xenografts compared with xenografts treated with radiation alone (7.4%, P = 0.006), Ad.Egr-TNF alone (0%, P = 0.012) or Ad.null with 30 Gy (0%, P = 0.002). Combined treatment with Ad.Egr-TNF and 30 Gy significantly reduced mean fractional tumor volumes compared with radiation alone (P = 0.002), Ad.Egr-TNF alone (P = 0.002) and Ad.null plus 30 Gy (P = 0.018). Histopathologic analyses of glioma xenografts treated with Ad.Egr-TNF and radiation revealed tumor vessel thrombosis by day 4 and necrosis by day 7. Thrombosis was not observed in tumors treated with Ad.Egr-TNF alone and was significantly reduced in all other treatment groups. These studies suggest that in the D54 glioma xenograft model, the antitumor effects of combining radiation and Ad.Egr-TNF are mediated, in part, by the destruction of the tumor microvasculature.

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.

Ionizing radiation causes histological changes in normal tissues that resemble those resulting from the inflammatory response. Inflammation is a multistep process requiring expression of adhesion molecules on the surface of endothelial cells which results in leukocyte extravasation. E-selectin is an adhesion molecule that mediates leukocyte "rolling" on the endothelium and is required for the inflammatory response. We quantified E-selectin expression and selectin-dependent adhesion of leukocytes to human endothelial cells after X irradiation to determine whether E-selectin participates in the radiation-mediated inflammation-like response. Immunofluorescence staining of irradiated endothelial cells demonstrated expression of E-selectin on the cell surface similar to that elicited by treatment with interleukin-1 (IL-1). Radiation-mediated expression of E-selectin was dependent on dose and time and occurred at doses as low as 0.5 Gy. Furthermore, the increased adhesion of leukocytes to irradiated endothelial cells was prevented by an E-selectin-blocking antibody. Sialyl Lewis X is one of the molecules on the surface of leukocytes that adheres to E-selectin. The anti-inflammatory agents glycyrrhizin and carminic acid, which are structural analogues of sialyl Lewis X, attenuated adhesion of leukocytes to endothelial cells treated with X rays or IL-1. These data implicate a new class of anti-inflammatory agents in the prevention of adhesions of leukocytes to the irradiated vascular endothelium.

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.

Tumor necrosis factor-alpha enhances x-ray killing of human tumor cells in vitro and enhances tumor control when combined with radiation in animal tumor models. To determine the toxicity and maximal tolerated dose of tumor necrosis factor-alpha given daily during radiotherapy, we conducted a phase I dose-escalation study combining tumor necrosis factor-alpha and radiation.Thirty-one patients, including 14 patients with locally advanced primary tumors and 17 patients with metastatic sites, were entered into this study. Recombinant human tumor necrosis factor-alpha was administered intravenously at doses ranging from 10 microg/m2 to 150 microg/m2 4 hours prior to each radiation therapy session, which was given on consecutive days for a minimum of 2 weeks. Radiation was prescribed to localized fields, with dose fractions ranging from 150 to 300 cGy/day for palliation or control of locally advanced tumors.Major toxicity requiring withdrawal from the study was independent of tumor necrosis factor-alpha dose and occurred in seven patients. Symptoms included angina in two patients, and hypotension, respiratory distress, atrial fibrillation, allergic reaction, and progressive leukopenia in one patient each. A tumor necrosis factor-alpha dose of 150 microg/m2 was the maximum dose administered. No single dose-limiting toxicity was observed and a maximal tolerated dose could not be defined. There was no obvious increase in in-field toxicity. Response to treatment was assessed in 20 patients. Complete regression within the irradiated field was achieved in four patients, partial regression in four, and a minimal response in four. A trend toward a greater response rate at higher doses of tumor necrosis factor-alpha was observed.The maximal tolerated dose of tumor necrosis factor-alpha when given with radiotherapy is at least 150 microg/m2 and a dose-limiting toxicity was not observed. Future studies will show whether responses to treatment are increased over those expected with radiation alone. Tumor localization of tumor necrosis factor-alpha by gene therapy combined with radiation therapy may eliminate the systemic toxicity we observed.

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.

Calcium is required as a cofactor by primer recognition proteins involved in DNA synthesis and by protein kinase C (PKC), which is activated by ionizing radiation. Because these processes may be involved in radiation-mediated regulation of the progression of cells through the phases of the cell cycle, we studied the effects of the intracellular Ca2+ chelator, acetoxymethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (AM-BAPTA), on PKC activation, expression of c-jun and Gadd45 and distribution of cells in the phases of the cell cycle after irradiation. AM-BAPTA prevented ionizing-radiation-induced activation of PKC and expression of c-jun in cells of human tumor cell lines. Conversely, calcium chelation had no effect on X-ray-induced expression of the Gadd45 gene. To determine whether changes in the intracellular Ca2+ concentration ([Ca2+]i) occurred during irradiation, we measured [Ca2+]i in single cells using fura-2-based microfluorimetry. There was no increase in [Ca2+]i during or after irradiation of cells of the human tumor cell lines RIT-3, SQ-20B or HL-60 or normal human fibroblast strain IMR-90. The percentage of human tumor cells crossing the G1/S-phase border was reduced by pretreatment with AM-BAPTA. These data indicate that calcium is required for ionizing radiation-induced cell cycle regulation and PKC activation, but that increases in [Ca2+]i do not occur in cells of the cell lines irradiated in this study.

The promoter regions of several radiation-inducible genes contain AP-1 cis-acting regulatory elements that are dependent upon protein kinase C signaling. We analyzed nuclear protein from irradiated human tumor cell lines for binding to the AP-1 consensus sequence. The increase in nuclear protein binding following irradiation was specific for the AP-1 sequence and was reduced by antibodies to c-Jun and c-Fos. The AP-1 DNA binding sequence was found to regulate transcription in irradiated cells and mutation of the AP-1 site within the c-jun promoter abolished transcriptional induction by radiation. The gene encoding the chimeric transcription factor Gal4-Jun5-253, which includes the DNA binding region of Gal4 and the transcriptional regulatory region of c-Jun, was cotransfected with the reporter plasmid with Gal4 binding sequences (G5B-CAT). Transfection of RIT-3 and HeLa cells revealed that the regulatory region of Jun was sufficient to activate transcription following irradiation. Conversely, Hep G2 cells, which do not contain the cell type-specific Jun repressor, were not responsive to radiation-induced Jun activation. The c-Jun repressor was found to regulate Jun activation by experiments using the expression vector CMV-jun, which competes for Jun inhibitor and eliminates radiation-induction of Jun. We propose transcription factor dissociation from inhibitor proteins may participate in the initiation of cellular responses to ionizing radiation.

Cytokine induction following ionizing radiation exposure occurs through transcriptional activation in specific proteins studied to date. This process can be blocked by inhibition of radiation-induced signaling pathways, which may potentially modify certain deleterious effects of radiotherapy, e.g., effects related to production of TNF or TGF-beta. bFGF is another radiation-inducible protein that may participate in repair and protection of irradiated endothelial cells following a complex interaction with cellular receptors. The cytokines such as IL-6, TNF, and IL-1 may be useful to protect hematopoietic cells from radiation, while TNF may enhance the killing of tumor cells. Our understanding of cytokine/radiation interactions is only beginning to be understood, but knowledge of the biology of these interactions may prove clinically useful.

Our laboratory has cultured cells from 23 patients with head and neck carcinoma and 13 patients with soft tissue sarcoma. The data suggest that, in some patients, inherent tumor cell radioresistance contributes to radiotherapy failure. This paper examines possible mechanisms that lead to increased cell survival following radiation exposure, and their potential as targets to overcome the radioresistant patient population.

Protein kinase C (PKC) is activated rapidly and transiently following ionizing radiation exposure and is postulated to activate downstream nuclear signal transducers. Inhibition of this enzyme attenuates radiation-mediated expression of the c-jun and Egr-1/zif-268 genes which are associated with cellular proliferation. To investigate further the role of PKC in the radiation response of human tumor cell lines, two human squamous cell carcinoma cell lines, SQ-20B and JSQ-3, were exposed to graded doses of X rays in the presence of staurosporine, sangivamycin, or H7, all PKC inhibitors. The protein kinase inhibitors staurosporine and sangivamycin produced dose-dependent cytotoxicity in cells of the SQ-20B and JSQ-3 cell lines while H7 did not. Nontoxic concentrations of sangivamycin (10 nM) and staurosporine (1 nM), added to cell cultures from 1 to 7 h before X irradiation, enhanced cell killing by radiation in both cell lines. Maximal sensitization of killing occurred when inhibitors were added 1 h prior to irradiation. The enhanced radiation-induced cell killing was not due to any measurable alteration in the induction or rejoining of DNA single- or double-strand breaks as determined by alkaline and neutral filter elution assays. These data suggest that protein kinase activity is important for cell survival following radiation exposure, although the specific role of PKC in radiation responses is unknown.

Twenty-one patients with tumor stage mycosis fungoides (MF) with or without lymph node (LN) involvement, were treated with total skin electron beam irradiation (TSEB) followed by six monthly cycles of systemic chemotherapy (CT) of either mechlorethamine (HN2) or cyclophosphamide (CTX) with vincristine (VCR), procarbazine, and prednisone (PRD) (COPP or MOPP). All patients had complete clearing of the skin after TSEB. However, while receiving chemotherapy, two patients developed visceral involvement and eight patients relapsed with limited cutaneous plaques (LCP). The median duration of remission was 12 months from the completion of TSEB, and all patients relapsed with cutaneous plaques within 25 months. Complete remission was again achieved using additional electron irradiation and maintenance therapy in all but one patient. Multiple cutaneous recurrences occurred in all patients. Median survival from the initiation of TSEB is 6 years. Five patients are living beyond 8 years (four off treatment without disease for 1 to 7 years). LN involvement did not influence initial response or survival. Combined modality therapy for tumor stage MF using TSEB followed by systemic CT and subsequent maintenance therapy may lead eventually to prolonged disease-free survival (DFS) in selected patients.