Olga Greco

Gene directed enzyme/prodrug therapy of cancer : historical appraisal and future prospectives

Gene therapy of cancer is a novel approach with the potential to selectively eradicate tumour cells, whilst sparing normal tissue from damage. In particular, gene‐directed enzyme prodrug therapy (GDEPT) is based on the delivery of a gene that encodes an enzyme which is non‐toxic per se, but is able to convert a prodrug into a potent cytotoxin. Several GDEPT systems have been investigated so far, demonstrating effectiveness in both tissue culture and animal models. Based on these encouraging results, phase I/II clinical trials have been performed and are still ongoing. The aim of this review is to summarise the progress made in the design and application of GDEPT strategies. The most widely used enzyme/prodrug combinations already in clinical trials (e.g., herpes simplex 1 virus thymidine kinase/ganciclovir and cytosine deaminase/5‐fluorocytosine), as well as novel approaches (carboxypeptidase G2/CMDA, horseradish peroxidase/indole‐3‐acetic acid) are described, with a particular attention to translational research and early clinical results. J. Cell. Physiol. 187:22–35, 2001.

Rejoining and Misrejoining of Radiation-Induced Chromatin Breaks. IV. Charged Particles

We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation.

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

Chromosome aberration dosimetry in cosmonauts after single or multiple space flights

Background and aims: Cosmic radiation is one of the main hazards for manned space exploration. Uncertainty in radiation risk estimates for crews of long-term missions are very high, and direct biological measurements are necessary. We measured chromosomal aberrations in peripheral blood lymphocytes from 33 cosmonauts involved in space missions during the past 11 years. Methods: Blood lymphocytes from the cosmonauts were stimulated to grow in vitro and were harvested at their first mitosis. Slides were either stained with Giemsa stain for dicentrics analysis, or painted with whole-chromosome DNA probes for translocation analysis (FISH). Results: A statistically significant increase in the yield of chromosomal aberrations was measured following long-term space missions in lymphocytes from cosmonauts at their first flight. No significant changes in aberration frequencies were observed for short-term taxi flights. The increase in long-term missions was consistent with the values calculated from physical dosimetry data. However, for cosmonauts involved in two or more space flights, the yield of interchromosomal exchanges was not related to the total duration of space sojourn or integral absorbed dose. Indeed, the yield of aberrations at the end of the last mission was generally in the range of background frequencies measured before the first mission. Conclusions: Chromosome aberration dosimetry can detect radiation damage during space flight, and biological measurements support the current risk estimates for space radiation exposure. However, for cosmonauts involved in multiple space missions the frequency of chromosomal aberrations is lower than expected, suggesting that the effects of repeated space flights on this particular endpoint are not simply additive. Changes in the immune system in microgravity and/or adaptive response to space radiation may explain the apparent increase in radioresistance after multiple space flights.

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells. Cancer Gene Therapy (2000) 7, 1414–1420

How to overcome (and exploit) tumor hypoxia for targeted gene therapy

Tumor hypoxia has long been recognized as a critical issue in oncology. Resistance of hypoxic areas has been shown to affect treatment outcome after radiation, chemotherapy, and surgery in a number of tumor sites. Two main strategies to overcome tumor hypoxia are to increase the delivery of oxygen (or oxygen‐mimetic drugs), and exploiting this unique environmental condition of solid tumors for targeted therapy. The first strategy includes hyperbaric oxygen breathing, the administration of carbogen and nicotinamide, and the delivery of chemical radiosensitizers. In contrast, bioreductive drugs and hypoxia‐targeted suicide gene therapy aim at activating cytotoxic agents at the tumor site, while sparing normal tissue from damage. The cellular machinery responds to hypoxia by activating the expression of genes involved in angiogenesis, anaerobic metabolism, vascular permeability, and inflammation. In most cases, transcription is initiated by the binding of the transcription factor hypoxia‐inducible factor (HIF) to hypoxia responsive elements (HREs). Hypoxia‐targeting for gene therapy has been achieved by utilizing promoters containing HREs, to induce selective and efficient transgene activation at the tumor site. Hypoxia‐targeted delivery and prodrug activation may add additional levels of selectivity to the treatment. In this article, the latest developments of cancer gene therapy of the hypoxic environment are discussed, with particular attention to combined protocols with ionizing radiation. Ultimately, it is proposed that by adopting specific transgene activation and molecular amplification systems, resistant hypoxic tumor tissues may be effectively targeted with gene therapy. J. Cell. Physiol. 197: 312–325, 2003© 2003 Wiley‐Liss, Inc.

Prodrugs in genetic chemoradiotherapy.

Improvements in the radiotherapeutic management of solid tumors through the concurrent use of gene therapy is a realistic possibility. Of the broad array of candidate genes that have been evaluated, those encoding prodrug-activating enzymes are particularly appealing since they directly complement ongoing clinical chemoradiation regimes. Gene-Directed Enzyme-Prodrug Therapy (GDEPT) only requires a fraction of the target cells to be genetically modified, providing that the resultant cytotoxic prodrug metabolites redistribute efficiently (the bystander effect). This transfer of cytotoxicity to neighboring non-targeted cancer cells is central to the success of any gene therapy strategy, irrespective of the therapeutic gene employed. In the context of genetic chemoradiotherapy, efficient prodrug metabolite diffusion will be a prerequisite for efficient radiosensitization. Some, but not all GDEPT approaches have been analysed in combination with radiotherapy. Examples of prodrugs of clinically established chemotherapeutic agents currently used in conjunction with radiotherapy include: 5-fluorocytosine (5FC), cyclophosphamide (CPA), irinotecan (CPT-11), gemcitabine (dFdC), capecitabine, mitomycin C (MMC) and AQ4N. Other GDEPT paradigms, such as ganciclovir (GCV) and Herpes Simplex thymidine kinase (HSV-tk), dinitrobenzamide (DNB) mustard or aziridinyl analogs and the E. coli nitroreductase (NTR), CMDA or ZP2767P with Pseudomonas aeruginosa carboxypeptidase G2 (CPG2), and indole-3-acetic acid (IAA) activated by horseradish peroxidase (HRP) have no clinically established chemotherapeutic counterpart. Each prodrug is discussed in this review in the context of GDEPT, with a particular attention to translational research and clinical utility in combination with radiotherapy.

5-Fluoroindole-3-acetic acid: a prodrug activated by a peroxidase with potential for use in targeted cancer therapy

Indole-3-acetic acid and some derivatives are oxidized by horseradish peroxidase, forming a radical-cation that rapidly fragments (eliminating CO(2)) to form cytotoxic products. No toxicity is seen when either indole-3-acetic acid or horseradish peroxidase is incubated alone at concentrations that together form potent cytotoxins. Unexpectedly, 5-fluoroindole-3-acetic acid, which is oxidized by horseradish peroxidase compound I 10-fold more slowly than indole-3-acetic acid, is much more cytotoxic towards V79 hamster fibroblasts in the presence of peroxidase than the unsubstituted indole. The fluorinated prodrug/peroxidase combination also shows potent cytotoxic activity in human and rodent tumor cell lines. Cytotoxicity is thought to arise in part from the formation of 3-methylene-2-oxindole (or analogues) that can conjugate with thiols and probably DNA or other biological nucleophiles. Levels of the fluorinated prodrug in the murine carcinoma NT after intraperitoneal administration of 50 mg/kg were about 200 microM. Although these were 4-5-fold lower than plasma levels (which reached 1mM), the integrated area under the concentration/time curve in tumors over 2 hr was approximately 20 mM min, almost double the exposure needed to achieve approximately 90-99% cell kill in human MCF7 breast or HT29 colon tumor cell lines and CaNT murine cells in vitro, although the human bladder T24 carcinoma cell line was more resistant. The high cytotoxicity of 5-fluoroindole-3-acetic acid after oxidative activation suggests its further evaluation as a prodrug for targeted cancer therapy involving antibody-, polymer-, or gene-directed delivery of horseradish peroxidase or similar activating enzymes.

Molecular approaches to chemo-radiotherapy

Although radiotherapy is used to treat many solid tumours, normal tissue tolerance and inherent tumour radioresistance can hinder successful outcome. Cancer gene therapy is one approach being developed to address this problem. However, the potential of many strategies are not realised owing to poor gene delivery and a lack of tumour specificity. The use of treatment-, condition- or tumour-specific promoters to control gene-directed enzyme prodrug therapy (GDEPT) is one such method for targeting gene expression to the tumour. Here, we describe two systems that make use of GDEPT, regulated by radiation or hypoxic-responsive promoters. To ensure that the radiation-responsive promoter is be activated by clinically relevant doses of radiation, we have designed synthetic promoters based on radiation responsive CArG elements derived from the Early Growth Response 1 (Egr1) gene. Use of these promoters in several tumour cell lines resulted in a 2-3-fold activation after a single dose of 3 Gy. Furthermore, use of these CArG promoters to control the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene in combination with the prodrug ganciclovir (GCV) resulted in substantially more cytotoxicity than seen with radiation or GCV treatment alone. Effectiveness was further improved by incorporating the GDEPT strategy into a novel molecular switch system using the Cre/loxP recombinase system of bacteriophage P1. The level of GDEPT bystander cell killing was notably increased by the use of a fusion protein of the HSVtk enzyme and the HSV intercellular transport protein vp22. Since hypoxia is also a common feature of many tumours, promoters containing hypoxic-responsive elements (HREs) for use with GDEPT are described. The development of such strategies that achieve tumour targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy.