Graeme J. Dougherty

A sense of danger from radiation.

Abstract McBride, W. H., Chiang, C-S., Olson, J. L., Wang, C-C., Hong, J-H., Pajonk, F., Dougherty, G. J., Iwamoto, K. S., Pervan, M. and Liao, Y-P. A Sense of Danger from Radiation. Radiat. Res. 162, 1–19 (2004). Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic “danger” signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of “danger” signals in tissue responses to this agent. This review deals with the nature of putative “danger” signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of “danger” signaling in response to radiation exposure. “Danger” signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alter intrinsic cellular radiosensitivity or initiate radioadaptive responses to subsequent exposure. They may spread outside the locally damaged site and mediate bystander or “out-of-field” radiation effects. Finally, an important aspect of classical “danger” signals is that they link initial nonspecific immune responses in a pathological site to the development of specific adaptive immunity. Interestingly, in the case of radiation, there is little evidence that “danger” signals efficiently translate radiation-induced tumor cell death into the generation of tumor-specific immunity or normal tissue damage into autoimmunity. The suggestion is that radiation-induced “danger” signals may be inadequate in this respect or that radiation interferes with the generation of specific immunity. There are many issues that need to be resolved regarding “danger” signaling after exposure to ionizing radiation. Evidence of their importance is, in some areas, scant, but the issues are worthy of consideration, if for no other reason than that manipulation of these pathways has the potential to improve the therapeutic benefit of radiation therapy. This article focuses on how normal tissues and tumors sense and respond to danger from ionizing radiation, on the nature of the signals that are sent, and on the impact on the eventual consequences of exposure.

CD44 isoforms in normal and leukemic hematopoiesis

a Terry Fox Laboratory, British Columbia Cancer Agency and the University of British Columbia, Vancouver, BC, Canada; b Institut National de la Sante et de la Recherche Medicale U268, Hopital Paul Brousse, Villejuif, France; c GSF, Institute for Experimental Hematology, Munich, Germany; d Leukaemia Research Unit, Department of Haematology, Hanson Center for Cancer Research, Adelaide, Australia; e UCLA Medical Center, Department of Radiation Oncology, Experimental Division, Los Angeles, CA

Newly discovered anti-inflammatory properties of the benzamides and nicotinamides

Our laboratory has concentrated on the possible regulation the benzamides and nicotinamides may have on the processes of DNA repair and apoptosis. Recent reports [14-16] have suggested that both apoptosis and inflammation are regulated by the transcription factor NF-kB. We have initiated studies regarding the hypothesis that the benzamides and nicotinamides could inhibit the production of tumor necrosis factor alpha (TNFalpha) and the inflammatory response as well as induce apoptosis via inhibition of NF-kB. Our data have shown that nicotinamide and two N-substituted benzamides, metoclopramide (MCA) and 3-chloroprocainamide (3-CPA), gave dose dependent inhibition of lipopolysacharide induced TNFalpha in the mouse within the dose range of 10-500 mg/kg. Moreover, lung edema was prevented in the rat by 3 ï 50 mg/kg doses of 3-CPA or MCA, and 100-200 μM doses of MCA could also inhibit NF-kB in Hela cells. Taken together these data strongly support the notion that benzamides and nicotinamides have potent anti-inflammatory and antitumor properties, because their primary mechanism of action is regulated by inhibition at the gene transcription level of NF-kB, which in turn inhibits TNFalpha and induces apoptosis.

Diverse effects of anti‐CD44 antibodies on the stromal cell‐mediated support of normal but not leukaemic (CML) haemopoiesis in vitro

We have identified three non‐cross‐reacting anti‐human CD44 monoclonal antibodies that have significant positive or negative (or no) effects on normal human haemopoiesis in the long‐term culture (LTC) system. These effects manifested as increases or decreases in the number of LTC‐initiating cells (LTC‐IC), and the number of colony‐forming cells (CFC) recovered from cultures in which either unseparated or highly purified CD34+CD38− normal marrow cells were placed on pre‐established normal marrow feeder layers in the presence or absence of each antibody. The effects seen were rapid and sustained, and dependent on the presence of a preformed feeder layer. Interestingly, the same anti‐CD44 antibodies had no effect on the maintenance of leukaemic (Ph+) progenitors (from patients with chronic myeloid leukaemia) when these cells were cultured on preformed feeder layers established from normal marrow. CD44 appears to be part of a mechanism by which stromal elements can regulate primitive normal haemopoietic cells but not their leukaemic (Ph+) counterparts.

Combining radiation therapy with interleukin-3 gene immunotherapy

The goal of this study was to explore immunological strategies to increase local and systemic tumor control in patients receiving radiation therapy. In previous studies, interleukin-3 (IL-3) gene expression within murine tumors was shown to increase their response to irradiation through immune mechanisms. In this study, the efficacy of systemically administered IL-3 gene-transduced irradiated tumor cell vaccines was tested for their ability to augment radiation responses against established immunogenic (FSAR) and nonimmunogenic (FSAN) tumors. Vaccines of irradiated FSAR/FSAN or FSAN-JmIL-3/FSAR-JmIL-3 cells were given intraperitoneally just before and after local irradiation of parental tumors with diameters of 8 mm, as well as in two booster doses. The IL-3 gene-transduced tumor cell vaccines were more effective than the parental vaccines at delaying tumor growth after irradiation, although no complete cures resulted. Responses were largely specific to the tumor type, indicating that tumor-specific immunity was enhanced by IL-3 vaccine administration. When the experiment was repeated in the C3H/HeJ mice, which are deficient in tumor necrosis factor-α production, the vaccines were still effective, but less so than in C3H/HeN mice. Systemic IL-3 vaccine treatment increased intratumoral levels of intercellular adhesion molecule-1, Mac-1, EB22/5.3, tumor necrosis factor-α, and IL-1 mRNA in irradiated tumors, indicating that cellular infiltration was part of the response. The study demonstrates that local radiation therapy can enhance the efficacy of genetically altered vaccine-based immunotherapy for cancer by decreasing tumor burden. At the same time, tumor cell vaccines may improve the cure rate of local radiation therapy by eliminating residual cancer cells. Although less effective than intratumoral gene expression, administration of IL-3 gene-transduced tumor cell vaccines is clinically a more feasible strategy that may be useful in situations in which the tumor load is small.

Inhibitory effect of locally produced and exogenous interleukin-6 on tumor growth in vivo

In order to define the potential antitumor activity of the multifunctional cytokine interleukin-6 (IL-6), retrovirus-mediated gene transfer was used to introduce and express a cDNA encoding human IL-6 in the murine fibrosarcoma cell line Fsa-R. Although these genetically modified tumor cells appeared morphologically and phenotypically identical to control Fsa-R cells and had a similar plating efficiency in vitro, they were found to exhibit greatly reduced tumorigenicity in vivo following intravenous injection into syngeneic recipients. Exogenous IL-6 was shown to produce a similar inhibition of tumor growth in the lung if administered intraperitoneally. In contrast, tumor growth in subcutaneous sites was inhibited only if the tumor cells were engineered to express IL-6 locally, or if IL-6 was administered intratumorally. Intraperitoneal injection of IL-6 had no inhibitory effect. Tumors that did grow from IL-6-producing tumor cell inocula in subcutaneous sites were found to contain large numbers of macrophages. These results demonstrate that the antitumor activity of systemically administered IL-6 varies depending on the site of tumor growth and suggest an important role for IL-6 in the recruitment, proliferation and/or survival of tumor-associated macrophages.

Alternative splicing as a novel of means of regulating the expression of therapeutic genes.

In order to determine the potential of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo, a series of episomal plasmid-based “splice-activated gene expression” (pSAGE) vectors was generated, which contain minigene cassettes composed of various combinations of the three alternatively spliced exons present in the differentially expressed adhesion protein CD44R1 (v8, v9, and v10) with or without their corresponding intronic sequences, positioned in-frame between the CD44 leader sequence and a “leaderless” human liver/bone/kidney alkaline phosphatase (ALP) cDNA. Because both the v8–v9 and v9–v10 introns contain multiple in-frame stop codons, the expression and enzymatic activity of ALP are dependent upon the accurate removal of intronic sequences from the pre-mRNA transcripts encoded by these constructs. The various pSAGE constructs were introduced into CD44H-positive (T24) and CD44R1-positive (PC3) target cells by electroporation and transfectants selected in hygromycin B. ALP expression was determined by staining with the ALP substrate, BCIP/INT, and the transfected cells tested for their sensitivity to the inactive prodrug, etoposide phosphate. ALP-mediated dephosphorylation of etoposide phosphate generates the potent topoisomerase II inhibitor etoposide. The data obtained indicate that whereas the v8–v9 intron is spliced in both CD44H- and CD44R1-positive cells, the v9–v10 intron is efficiently and accurately removed only in CD44R1-positive cells. Furthermore, only CD44R1-positive cells were sensitized to etoposide phosphate when transfected with the v9–v10.ALP construct. These data emphasize the potential usefulness of alternative splicing as a novel means of targeting gene expression to tumor cells in vivo.

Modification of Tumor Microenvironment by Cytokine Gene Transfer

The tumor microenvironment is determined by the interactions between host and tumor cells, a process in which cytokines play a major role. We have used retroviral vectors to insert and express cytokine genes in tumor cells so as to induce predictable changes in the host cells that infiltrate tumors. This frequently caused changes in tumor cell phenotype through autocrine/intracrine pathways. We reasoned that cytokine-induced alterations in tumor cell phenotype and/or in infiltrating host cells might alter the in vitro and in vivo cellular response to irradiation. In the present paper we document some of the effects of expression of interleukin-6 (IL-6) and IL-7 genes in tumor cells in this regard. The studies support the hypothesis that cytokines may play a role in determining both intrinsic tumor radioresponsiveness and the tumor microenvironment and in these ways may influence in vivo tumor irradiation responses. Possible cytokine gene-mediated approaches to radiotherapy cancer are discussed.

Exploiting the differential production of angiogenic factors within the tumor microenvironment in the design of a novel vascular-targeted gene therapy-based approach to the treatment of cancer

PURPOSE The aim of this study is to explore a novel strategy through which the differential production of pro-angiogenic cytokines within the tumor microenvironment can be exploited as a means of selectively killing the vascular endothelial cells upon which the survival and growth of a tumor depend. METHODS AND MATERIALS Adenoviral vectors encoding a chimeric cell surface receptor composed of the extracellular domain of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR fused in frame to the membrane spanning and cytoplasmic domain of Fas were constructed and used to transduce primary human endothelial cells in vitro. The apoptotic response of these cells induced upon ligation of the chimeric receptor with VEGF was determined by measuring caspase-3 activation, AnnexinV-FITC binding, and the release of glucose-6-phosphate dehydrogenase. RESULTS The chimeric Flk-1/Fas protein is stable and expressed at high levels on the surface of adenovirally transduced cells. Upon the addition of exogenous VEGF, these cells undergo rapid apoptosis. CONCLUSIONS Receptor/Fas chimeras that recognize and bind pro-angiogenic cytokines represent a novel means by which the signal transduction events normally triggered in vascular endothelial cells upon the binding of angiogenic cytokines may be redirected toward the induction of apoptotic cell death. It is proposed that these constructs will prove of value in the further development of safe and effective vascular-targeted gene therapy-based approaches to the treatment of cancer.

Exploiting the tumor microenvironment in the development of targeted cancer gene therapy.

The future success of cancer gene therapy is critically dependent upon the development of safe, practical and effective targeting strategies. In this study, we describe a novel and broadly applicable targeting approach in which the induction of apoptotic tumor cell death is linked to the differential expression within the tumor microenvironment of elevated levels of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF). As VEGF is generally absent or produced at only low levels in most normal tissues, undesirable toxicity will not result even if the therapeutic gene in question is inadvertently expressed in non-targeted tissue sites. The basic approach makes use of a chimeric cell-surface protein in which the membrane-spanning and cytoplasmic ‘death domain’ of the pro-apoptotic protein Fas are fused in frame to the extracellular ligand-binding domain of the VEGF receptor Flk-1/KDR/VEGFR2 (Flk-1/Fas). The resultant chimeric Flk-1/Fas receptor was found to be stable and capable of inducing a rapid apoptotic response when expressed in tumor cells that produce endogenous VEGF. Importantly, in the absence of VEGF, transduced tumor cells remain viable although they can be triggered to die by the addition of recombinant VEGF. Given the key role played by VEGF in tumor development and progression, it is proposed that the Flk-1/Fas chimera may have great potential in the context of tumor cell-targeted cancer gene therapy.