Radiation-inducible Promoter Research Articles

A Novel Gene Transfer Therapy Against Pancreatic Cancer (TNFerade) Delivered by Endoscopic Ultrasound (EUS) and Percutaneous Guided Fine Needle Injection (FNI)

A Novel Gene Transfer Therapy Against Pancreatic Cancer (TNFerade) Delivered by Endoscopic Ultrasound (EUS) and Percutaneous Guided Fine Needle Injection (FNI) Kenneth J. Chang, Neil Senzer, Theodore Chung, J. Randolph Hecht, Stephen Vogel, Alexander Rosemurgy, John Nemunaitis, John Gibbs, Milind Javle, Tony Reid, Jennifer Macko, Paul Kessler, Mitchell C. Posner, James Farrell, L. Shane Grundy, Roy Soetikno, Irving Waxman, Nader Hanna We published two trials utilizing EUS-guided FNI for the delivery of immune therapy (Cancer 2000) and oncolytic viral therapy (Clin Cancer Res 2003) against pancreatic cancer. We now present a novel gene transfer therapy, TNFerade, delivered by EUS and percutaneous approaches (PTA) guided by ultrasound or computerized tomography (CT), in the treatment of unresectable, locally advanced adenocarcinoma of the pancreas (LAPC). TNFerade is a replicationdeficient adenovector containing human TNFa gene, regulated by a radiationinducible promoter Egr-1. Purpose: 1) Assess safety and efficacy of TNFerade plus chemoradiation in pts with LAPC. 2) Compare EUS and PTA as delivery modalities. Methods: Dose-escalating run-in design. Five-week treatment of weekly intratumoral injections of TNFerade (43 10 particle units (pu) in 2mL) via EUS or PTA (each institution allowed one preferred technique), continuous iv 5-FU (200 mg/m/d 3 5 days/wk) and radiation (50.4 Gy). TNFerade was delivered with a single needle pass at a single site in the tumor for PTA, while up to 4 injections were given by EUS. Endpoints: Safety and tumor response on spiral CT (core lab). Results: Of 37 pts, 17 had EUS and 20 had PTA (similar TNFerade doses). EUS vs PTA: T4 staging = 88% vs. 75%; N1 staging = 29% vs. 40%, mean tumor area (cm)= 18 vs.19; mean CA19-9= 4,396 vs. 1,707, meanKPS= 88 vs. 90. (p=NS for all). 1 dose limiting toxicity (gr 3 hypotension) in a PTApt; all other adverse events (AEs) potentially related to TNFerade were grade 1-2. Procedure related AEs were all grade 1-2 and were similar, except for injection site pain: 35% PTA vs 0% EUS (p = .01). Tumor responses and disease control were similar (Table 1). 4 patients underwent resection; one, an EUS patient, had a complete pathologic response. Conclusions: 1) TNFerade plus chemoradiation was well tolerated with maximum tolerated dose not reached; 2) indications of clinical activity were demonstrated by local tumor control and progression-free survival at up to 3 months; 3) EUS and PTA appear to be similarly safe and effective for delivery of TNFerade.

Gene therapy and endovascular treatment of intracranial aneurysms.

Endovascular treatment of intracranial aneurysms is safe and effective but too often is followed by recurrences. Gene therapy may improve healing after embolization, and endovascular approaches may offer future in situ delivery systems designed to prevent aneurysm rupture. Advances in coil technology have focused on coating strategies designed to modify the biological reaction to the embolic agent. Gene therapy in cardiovascular applications is limited by low efficiency and transient gene expression. Current advances include the potential use of circulating progenitor cells for ex vivo genetic manipulations followed by in vivo delivery. Direct gene transfer may also be enhanced in situ by coils carrying antibody-tethered adenovirus or through the use of cell-specific or radiation-inducible promoters. Candidate genes that may be of value in promoting healing after endovascular treatment include growth factors and metalloproteinase inhibitors. A better understanding of the biology of aneurysm is necessary to conceive strategies designed to control the development of these lesions before their rupture. Many technical difficulties remain to be solved, but the combination of gene therapy and endovascular techniques offers multiple therapeutic possibilities in the future control of intracranial aneurysms.

A novel radiation/tissue-specific promoter for gene therapy of brain tumors

Purpose/Objective: Radiogenic therapy is a novel approach to treat cancer. It implies using genetic modification to alter sensitivity of tumor or normal tissue to the effect of radiation. Using recombinant adenovirus (Ad) as a vector, heterologous gene delivery with consequent phenotypic change in radiosensitivity has been shown with different therapeutic payloads. Radiation inducible promoters incorporated into Ad vectors have been used to deliver therapeutic genes. However, lack of tumor specificity and moderate levels of gene induction have been a concern. On the other hand, tissue specific promoters (TSP) have been widely developed and show both high selectivity and robust gene expression in transfected tumors. Of note, radiation also induces gene expression by transcriptional regulation of some of these promoters. Herein we hypothesize that radiotherapy may enhance the selectivity and level of expression of TSP.

Tumor-specific gene delivery using genetically engineered bacteria.

The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy. Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy. In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria. Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects. The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment. The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors. One of the most important strains in that context is Clostridium. Other bacteria have also been implicated in experimental anti-cancer settings. Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising. We will discuss the potential advantages and the pitfalls of this alternative delivery approach. We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy. We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors.

Inhibition of benzoyl peroxide and ultraviolet-B radiation induced oxidative stress and tumor promotion markers by cycloartenol in murine skin

The chemopreventive potential of cycloartenol on benzoyl peroxide and UVB radiation-induced cutaneous tumor promotion markers and oxidative stress in murine skin is assessed. Benzoyl peroxide treatment (20 mg/animal/0.2 ml acetone) and UVB radiation (0.420 J/m2/s) caused a decrease in the activities of cutaneous antioxidant enzymes namely, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, phase II metabolizing enzyme such as glutathione-S-transferase and quinone reductase and depletion in the level of cutaneous glutathione. There was also enhancement in cutaneous microsomal lipid peroxidation, xanthine oxidase activity, [14C]-ornithine decarboxylase activity and [3H]-thymidine incorporation into cutaneous DNA. Cycloartenol was topically applied prior to the application of benzoyl peroxide at dose levels of 0.2 mg and 0.4 mg/kg body weight in acetone, which resulted in significant inhibition of epidermal ornithine decarboxylase activity and DNA synthesis (P < 0.001). There was also significant reduction of lipid peroxidation and xanthine oxidase activity (P < 0.001). In addition, the depleted levels of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, were also recovered to a significant level (P < 0.001). The data indicate that cycloartenol is an effective chemopreventive agent in skin carcinogenesis.

TNFerade Biologic: Preclinical toxicology of a novel adenovector with a radiation-inducible promoter, carrying the human tumor necrosis factor alpha gene

TNFerade Biologic (TNFerade) is a second-generation (E1-, E3-, and E4-deleted) replication-deficient adenovector carrying the transgene encoding for human tumor necrosis factor alpha (TNFalpha), regulated by the radiation-sensitive promoter Early Growth Response (Egr-1). We hypothesized that intratumoral injection of TNFerade followed by radiation would result in potentially therapeutic levels of TNFalpha with minimal toxicity. Three preclinical studies were conducted, the purpose of which was to characterize the toxicity and pharmacokinetics of TNFerade in conjunction with radiation in nude as well as immune-competent (Balb/c) mice. A total of 80 mice in the nude mouse toxicology study, all bearing human squamous cell carcinoma xenografts, 120 mice in the Balb/c study, and 33 nude mice in the pharmacokinetic study were used. Doses ranging from 4x10(9) to 4x10(10) particle units (pu) (4x10(11) pu in the Balb/c study) were explored, with and without radiation. In the nude mice studies, TNFerade was injected intratumorally, whereas in the Balb/c study, TNFerade was administered by subcutaneous injection. TNFerade was well tolerated. In the nude mice studies, no significant toxicity occurred in any dose group. In the Balb/c study, 6/40 mice at the top dose (4x10(11) pu) were sacrificed in moribund condition (5/20 in the TNFerade+radiation group, 1/20 in the TNFerade alone group). Necropsy showed local necrosis and ulceration at the site of the injection. No deaths or significant toxicity were observed at the lower dose levels (4x10(9) and 4x10(10) pu), indicating a large safety margin for initial studies in humans. The pharmacokinetic study demonstrated high sustained levels of TNFalpha in the tumor homogenate with no "spillover" to plasma, where TNFalpha levels were below the level of detection. Radiation increased intratumoral levels of TNFalpha by a factor of 12 (from 0.998 to 11.55 ng/g). In conclusion, a gene therapy approach with TNFerade, in combination with radiation, represents a potential way to utilize the potent anticancer activity of TNFalpha without systemic toxicity.

Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma.

The ability to infect tumor cells limits the antitumor effects of gene therapy. The addition of radiotherapy to treatment with Ad.Egr.TNF.11D, a replication-deficient adenovirus containing a radiation-inducible promoter, early growth response-1, and the tumor necrosis factor-alpha (TNFalpha) complementary DNA may enhance the therapeutic ratio. Seg-1 human esophageal adenocarcinoma cells were treated with Ad.Egr.TNF.11D with or without radiation. TNFalpha levels were quantified with enzyme-linked immunosorbent assay. Athymic nude mice bearing Seg-1 tumors were randomized to buffer, ionizing radiation, Ad.Egr.TNF.11D, and combination therapy. Tumor growth delay was used to compare treatment regimens. TNFalpha levels were measured in tumor homogenates and plasma. Seg-1 cells treated with Ad.Egr.TNF.11D and ionizing radiation demonstrated increased TNFalpha levels at 72 hours compared with cells exposed to vector alone (124 +/- 0 pg/mL vs. 31.11 +/- 22 pg/mL; P =.008). In vivo, Ad.Egr.TNF.11D-treated tumors expressed low TNFalpha levels (151.5 +/- 107.11 pg/mg protein) compared with tumors receiving combined treatment (793.92 +/- 489.13 pg/mg protein; P =.067). Increased TNFalpha levels were associated with increased tumor growth delay after combined treatment (P <.05). Radiotherapy enables focal stimulation of TNFalpha expression in Ad.Egr.TNF.11D-infected cells and thus improves local tumor control.

420. A Search for Radiation-Inducible Promoters by Gene Expression Profiling of Irradiated Microvascular Endothelial Cells

420. A Search for Radiation-Inducible Promoters by Gene Expression Profiling of Irradiated Microvascular Endothelial Cells

A new perspective of carcinogenesis from protracted high-let radiation arises from the two-stage clonal expansion model

When applied to the Colorado Plateau miner population, the two-stage clonal expansion (TSCE) model of radiation carcinogenesis predicts that radiation-induced promotion dominates radiation-induced initiation. Thus, according to the model, at least for alpha-particle radiation from inhaled radon daughters, lung cancer induction over long periods of protracted irradiation appears to be dominated by radiation-induced modification of the proliferation kinetics of already-initiated cells rather than by direct radiation-induced initiation (i.e., mutation) of normal cells. We explore the possible consequences of this result for radiation exposures to space travelers on long missions. Still unknown is the LET dependence of this effect. Speculations of the cause of this phenomenon include the suggestion that modification of cell kinetics is caused by a “bystander” effect, i.e., the traversal of normal cells by alpha particles, followed by the signaling of these cells to nearby initiated cells which then modify their proliferation kinetics.

Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro.

The promoter of the early growth response gene (Egr-1) has been described to be activated by ionizing radiation, and it seems to be clear that this process involves different mitogen activated protein (MAP) kinases, dependent on the specific cell type examined. However, early steps leading to activation of the corresponding pathways and thus to overexpression of Egr-1 are not well understood. In this study, deletion mutants of the 5' upstream region of the Egr-1 gene were generated which allowed us to correlate the radiation-induction of the Egr-1 promoter in U87 glioma cells to five serum response elements. Based on the data shown, a possible role of two cAMP responsive elements for radiation-dependent promoter regulation could be ruled out. On the basis of activator/inhibitor studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and potentially SAPK/JNK, but not the p38MAPK/SAPK2, pathway contribute to the radiation-induction of Egr-1 promoter. In addition, it was observed that irradiated cells secrete a diffusible factor into the culture media which accounts for the radiation-induced promoter upregulation. By blocking growth factor receptor activation with suramin, this effect could be completely abolished.

Defining a future role for radiogenic therapy

The goal of cancer therapy is to eliminate the cancer and/or to arrest further growth while decreasing normal tissue toxicity, i.e. to increase the therapeutic ratio. This review focuses on a group of therapeutics that are either (1) directly stimulated by radiation to produce either directly or indirectly cytotoxic agents (i.e. genes under the control of a radiation inducible promoter that produce a cytotoxic protein or an enzyme that converts a prodrug to an active form, respectively); (2) auger-electron emitting radiolabelled oligonucleotides, antibodies, nucleotide analogues, or other small molecules that are internalized; (3) radiation inducible genes that produce a ligand or transporter (or the like) which then can be targeted by cytotoxic agents (e.g. radiolabelled substance). We have termed this group of therapeutics radiogenic therapy.

Prospects for viral-based strategies enhancing the anti-tumor effects of ionizing radiation

Ionizing radiation (IR) has been extensively used to treat a variety of solid tumors to improve local control and overall survival in patients. Gene therapy strategies represent one experimental direction to improve radiocurability. These gene therapy strategies include (1) replacement of mutated or deleted tumor-suppressor genes, (2) delivery of prodrugs, (3) transduction of genes under the control of radiation-inducible promoters, and (4) genetically engineered viruses that replicate preferentially in tumor cells after IR. Although any one of these viral-based gene therapy approaches is unlikely to succeed independently, experimental results suggest that clinically important antitumor can be achieved when these strategies are combined with IR. Several of these strategies are currently being or soon will be evaluated in clinical trials. This review focuses on molecular mechanisms and potential clinical application of these viral-based gene therapy strategies to improve the therapeutic index of IR.

Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation.

One of the major problems with gene therapy today is the lack of tumour specificity. The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour. These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour. As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter. A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy. A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05). These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter. Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression. Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

The use of radiation-induced bacterial promoters in anaerobic conditions: a means to control gene expression in clostridium-mediated therapy for cancer.

Nuyts, S., Van Mellaert, L., Theys, J., Landuyt, W., Lambin, P. and Anné, J. The Use of Radiation-Induced Bacterial Promoters in Anaerobic Conditions: A Means to Control Gene Expression in Clostridium-Mediated Therapy for Cancer. Radiat. Res. 155, 716-723 (2001). Apathogenic clostridia, which have been genetically engineered to express therapeutic genes, will specifically target hypoxic and necrotic regions in tumors. This specificity can be improved further if the expression of these genes is controlled by a radiation-induced promoter, leading to spatial and temporal control of gene expression. We isolated two radiation-inducible genes of the SOS repair system of Clostridium. Northern blot experiments confirmed radiation activation of the recA and recN genes at a dose of 2 Gy. The promoter region of these genes was isolated and used to regulate expression of the lacZ gene under anaerobic conditions. For the recA promoter, a significant increase of beta-galactosidase activity of 20-30% was seen after 2 Gy irradiation. The recN promoter did not show a significant induction and had a 50-100 times lower basal expression. Treatment of the recombinant clostridial cultures with the cytostatic agent mitomycin C also resulted in a significant increase of beta-galactosidase activity that was under the control of recA or recN promoter. Oxygen does not appear to be necessary in the activation of the SOS repair system by irradiation as tested with Escherichia coli since recA-deficient and recA-containing strains showed similar survival after treatment with UV and ionizing radiation in the presence or absence of oxygen.

Gene expression under control of the radiation-inducible Egr-1 promoter in an adenoviral vector: vector optimization for reduction of unspecific gene expression in the absence of irradiation

Gene expression under control of the radiation-inducible Egr-1 promoter in an adenoviral vector: vector optimization for reduction of unspecific gene expression in the absence of irradiation

The nitroxide Tempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culture model of cutaneous photoaging.

The generation of reactive oxygen species is among the various mechanisms by which ultraviolet radiation damages skin. Tempol, a superoxide dismutase analogue which readily penetrates cell membranes when administered exogenously, has been shown to provide protection against some forms of oxygen-dependent damage. In this study, we measured the ability of Tempol to protect against ultraviolet A- and ultraviolet B-induced damage, using a previously described transgenic mouse model of cutaneous photoaging. The ability of Tempol to prevent ultraviolet radiation-induced elastin promoter activation was determined in vitro. Tempol provided over 50% protection against ultraviolet B and over 70% protection against ultraviolet A as measured in our in vitro system. These results demonstrate the ability of the superoxide dismutase mimic, Tempol, to protect against ultraviolet induced elastin promoter activation. This compound could be a useful pharmacological agent to prevent cutaneous photoaging.

Modification of vascular tone using iNOS under the control of a radiation-inducible promoter.

It may be therapeutically advantageous to alter tumour blood supply specifically. Nitric oxide is a potent vasodilator which is produced in many tissues by the enzyme nitric oxide synthase (NOS). We have transfected cDNA for the inducible isoform of this enzyme (iNOS), under the control of the radiation-inducible promoter WAF1. The activity of the promoter was initially assessed using green fluorescent protein (GFP) in both endothelial cells and rat tail artery segments. Induction of protein expression by 9.5- and 4.5-fold respectively, was observed after a radiation dose of 4 Gy. Artery sections were then transfected with the WAF1/iNOS construct; this gave five-fold induction of iNOS protein after a dose of 4 Gy. The transfected artery was also tested functionally for relaxation, indicative of NO production. One hour after exposure to 4 Gy there was a significant (65%) relaxation of artery segments that had been preconstricted with phenylephrine. This could be partially reversed by the NOS inhibitor nitro-L-arginine. This study demonstrates that we can regulate vascular tone using an X-ray inducible promoter.

NF-κB and AP-1 are responsible for inducibility of the IL-6 promoter by ionizing radiation in HeLa cells

Purpose : To investigate the mechanisms leading to initiation by ionizing radiation of IL-6 transcription in HeLa cells. Materials and methods : HeLa cells were irradiated with X-rays at a dose rate of approximately 1 Gy/min or treated with TPA (100 ng/ml). Transient transfection analysis with truncated IL-6 promoter CAT constructs was used to identify the radiation-sensitive region within the IL-6 promoter/enhancer. Results : For basal expression of the IL-6 gene in unirradiated control cells the presence of the binding site for the nuclear factor kappa B (NF- κB) and the multiple response elements (MRE) were necessary. After deletion of either the activator protein (AP)-1 or the MRE site, radiation-induced IL-6 promoter CAT activity was significantly reduced, whereas after deletion of the NF-kappaB site it was completely abolished. Maximal radiation-induced IL-6 promoter CAT activity was observed when the AP1, NF κB and MRE motifs were present. In electrophoretic mobility shift analyses (EMSA), X-ray-inducible activity was found for NF-kappaB and AP-1 at the MRE constitutive, but no inducible activities were detectable. The nuclear factor IL-6 (NFIL6) element showed no specific radiation-responsive activity. Conclusions : These results demonstrate that NF- κB plays a major role in X-ray-inducible IL-6 expression in HeLa cells. The fact that IL-6 promoter activity was dramatically enhanced in the presence of the MRE and distal AP-1 binding motif is indicative of a cooperative mode of transcriptional activation involving all three transcription factor systems. These data provide new insights into the prodromal events of radiation-induced inflammation of epithelial cells and putatively the cutaneous radiation syndrome.

Transgene expression in malignant glioma using a replication-defective adenoviral vector containing the Egr-1 promoter: activation by ionizing radiation or uptake of radioactive iododeoxyuridine.

One approach to improving the specificity of gene therapy involves using radiosensitive promoters to activate gene expression selectively in the radiation field. In this study, we evaluated the ability of irradiation to regulate the transcription of a recombinant replication-defective adenovirus vector, Ad.Egr-1/lacZ, containing the radiation-inducible Egr-1 promoter driving the beta-galactosidase reporter gene in glioma cells. Transcripts of the Egr-1 gene in human and rat glioma cells were induced following irradiation with as little as 2 Gy. This dose was 10-fold less than previously reported, and comparable to doses of irradiation used clinically in standard fractionated radiotherapy for brain tumors. When 9L rat gliosarcoma cells were infected with Ad.Egr-1/lacZ in vitro and exposed to 2 Gy of external beam irradiation, there was a threefold increase in beta-galactosidase expression. Irradiation of intracerebral 9L tumors infected with the Ad.Egr-1/lacZ virus, using either external beam radiotherapy (2 Gy) or the thymidine analog 5-iodo-2'-deoxyuridine radiolabeled with the Auger electron emitter iodine-125 ([125I]IdUrd), also resulted in increased beta-galactosidase activity of the tumor cells. These results indicate that the use of viral vectors containing radiation-inducible promoters represents a novel therapeutic approach that enables gene therapy to be spatially and temporally regulated by ionizing radiation. These findings also support a potential role for radiation-inducible promoters in the treatment of malignant brain tumors.

2020 Function of the [formula omitted] radiation inducible promoter in a minimal HSV-1 amplicon system

2020 Function of the [formula omitted] radiation inducible promoter in a minimal HSV-1 amplicon system