VARIACIONES DE VOLTAJE PERMISIBLES EN ALIMENTADORES

Anticancer strategies, in order to be o f any significant advantage, should be characterised by high specificity as well as efficacy. On this basis, gene-directed enzyme/prodrug therapy (GDEPT) was introduced, whereby the target cells are genetically modified to synthesise an enzyme able to convert a prodrug into a cytotoxin. In the present work, a novel system consisting o f horseradish peroxidase (HRP) and indole-3-acetic acid (lAA) is proposed as an enzyme/prodrug combination for cancer gene therapy. In particular, this GDEPT approach was aimed at targeting the hypoxic areas o f solid tumours resistant to conventional treatment. The efficacy o f HRP/IAA gene therapy and the induction o f a bystander effect were demonstrated in vitro under normoxic as well as hypoxic conditions (Chapter 3). To date, the chemical agents and the cellular targets involved in HRP/IAA-induced toxicity are unknown, but the results presented here indicate that an apoptotic pathway may be activated (Chapter 4). An enhancement o f the therapeutic potential o f HRP-mediated GDEPT was demonstrated by adopting novel lAA analogues characterised by fast normoxic and hypoxic cytotoxic activation, high HRP^ cell kill or selectivity (Chapter 5). Moreover, with a view to combining GDEPT and radiotherapy protocols, the interaction with therapeutically significant doses o f ionising radiation (IR) was evaluated, and oxic and anoxic enhancement o f IR toxicity was observed (Chapter 6). Finally, selective transgene expression and prodrug activation in hypoxic and/or irradiated cells was demonstrated by the use o f synthetic promoters containing hypoxia regulatory elements (HREs) and IR-responsive CArG elements (Chapter 7).

For the choice o f effective enzyme/prodrug combinations for GDEPT, a number o f criteria have been listed (Connors, 1995; Knox, 1999). As also done in an ADEPT context (Wardman, 2002), these requirements, which are not fulfilled by most o f the current systems, are compared here with the properties o f the HRP/IAA combination:

Monomeric enzyme with no equivalent in humans. HRP is a monomeric glycoprotein, and glycosylation is not required for its activity (Welinder, 1979; Smith et ah, 1990). Human peroxidases are much less effective than HRP in oxidising lAA and analogues. lAA was moderately toxic to rodent neutrophils (-40% loss o f viability after 1 mM lAA for 24 h), while lymphocytes were not affected by lAA treatment (Fires de Melo et al., 1997, 1998). This effect was attributed to endogenous myeloperoxidase

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(MPO) activity, which in lymphocytes is 10-fold lower than in neutrophils (Pires de Melo et al., 1998). Analogously, human pro-myelocytic leukaemia lymphocytes (HL60) were moderately affected by lAA treatment, as 10 mM lAA for 1 h induced 25% cell kill only (Folkes et al., 1998). The formation o f the skatole radical was detected, but the MPO/IAA reaction was inhibited after a few minutes (Folkes et al., 1998). Some toxicity was measured in lAA-treated endothelial HMEC-1 cells, but it did not appear to depend on endogenous peroxidase levels (section 3.3.4). Therefore the pathways leading to cytotoxic prodrug activation appear to be specific for HRP, and not for endogenous peroxidases.

Active at physiological pH. Although HRP activity may be higher at pH -5 (Dunford, 1999), the experiments described in this work demonstrated significant cytotoxic prodrug activation at neutral pH.

Low Km and high kcat fo r the prodrug. lAA and derivatives are notably better substrates for HRP than for other biological indoles, such as tryptophan (Wardman, 2002). Comparison o f the Km. with other enzymes may be misleading, as there is no evidence o f typical Michaelis and Menten kinetics for the HRP/IAA reaction (Candeias et al., 1997). However, one study provided a value o f ATm = 19 pM for lA A with 23 nM HRP at pH 5 and 25°C (Smith et al., 1982). For therapeutic purposes, prodrug activation should occur rapidly and at low concentrations o f the substrate. Indeed, it was shown here that significant toxicity was induced in HRP-expressing cells after only 2 h- incubation, at levels o f prodrug in the 0.1-1 mM range.

No co-substrate requirements. To activate lAA and analogues neither H2O2 nor

other cofactors are required (Dunford, 1999).

Freely diffusible prodrug. At pH 7.4 lAA is hydrophilic and can cross cell membranes within a few minutes (Pires de Melo et al., 1997; Folkes et al., 1999).

Bystander effect. A significant bystander effect was demonstrated with lAA and analogues, under oxic as well as hypoxic conditions, not dependent on cell-to-cell contact (Chapter 3). The half-life o f the active drug will need to be investigated in vivo,

to ensure that no toxic species escape into the circulation to damage normal tissue.

High differential toxicity. After b rief incubation intervals (2 h) with up to 20 mM lAA, no toxicity could be detected in HRP cells, while 3-4 mM induced 1-2 log cell kill in HRP-transfectants. Longer exposure times induced differential toxicities o f 36-85 (IC50), depending on the tumour cell line. Higher differentials were measured using

some lAA derivatives, such as 1-Me-IAA (selectivity index = 740 in T24 cells). However, in microvascular endothelial HMEC-1 cells lower selectivity was observed, compared to the tumour cell lines analysed. This may limit therapeutic efficacy.

Active drug neither phase specific nor proliferation dependent. HRP/IAA induced a cytostatic effect that appeared to be independent on cell cycle phase at the time o f exposure (Chapter 4). Further studies may be necessary using cells synchronised in different phases. Also, cytotoxicity in quiescent cells may be specifically measured by blocking cell proliferation during treatment.

Thus HRP/IAA appears to fulfil most requirements, justifying further work and its evaluation in in vivo models. A number o f factors need to be taken into account, such as prodrug levels and kinetics, tumour selectivity, normal tissue toxicity, local and distant bystander effect and host immunicity. The compounds lAA, 1-Me-IAA and 5-Br-IAA will be tested in animal models, on their own and, based on the radiosensitisation observed in vitro, in combination with IR. When X-irradiation took place immediately before or after prodrug incubation, sensitivity enhancement ratios (SERs) o f 2.1-5.6 under normoxia, and o f 3.6 under anoxic conditions were observed (Chapter 6). Statistical evaluation o f combined radiation and GDEPT protocols has shown that in vitro SER values o f 1.2 can significantly increase local control after radiotherapy (Lambin et al., 2000). Therefore, it is possible that the HRP/IAA system may not only eradicate the hypoxic radioresistant tumour cells, but also directly enhance the cytotoxic effects o f IR. The scheduling o f a combination protocol should also be devised, in order to achieve maximum efficacy in vivo. Multiple dosing o f radiation and prodrug may be

needed to maximise gene expression, direct cytotoxicity and radiosensitisation in hypoxic and/or irradiated areas.

Future work is recommended to assess the chemical agents and the cellular mechanisms involved in HRP/IAA-induced cell death and radiosensitisation. In particular it would be o f interest to investigate if the DNA macromolecule is damaged in the exposed cells, as observed in a cell-free system (Folkes et al., 1999). High sensitivity in measuring DNA strand breaks within a cell population may be achieved with single-cell electrophoresis, or comet assay (Olive, 1999). Immunostaining the single cell preparations (Dachs et al., 1997) with anti-HR? antibodies may allow the identification o f HRP-expressing and non-expressing damaged cells, giving further insights into the bystander effect o f the HRP/IAA combination. The induction o f base damage and base loss may be evaluated by utilising purified repair enzymes such as AP- nucleases and DNA glycosylases (endonuclease III, endonuclease IV) and formamidopyrimidine-DNA glycosylase (Fpg protein), which are able to recognise specific lesions and convert them into strand breaks, detectable by comet assay (Chaudhry and Weinfeld, 1995; Banath et al., 1999).

HRP localisation within the transfected cell may play a role in lAA activation and cytotoxicity, due to proximity to the target molecules. The plasmids used in this work contained the HRP cDNA previously fused to the KDEL tetrapeptide, which caused its accumulation in the cytoplasm and the nuclear membrane. If DNA is the critical target, localisation o f the HRP in the nucleus via nuclear localisation sequences may result in enhanced toxicity. Nuclear accumulation and enhancement o f the bystander effect may also be achieved by fusing the HRP to the HSV virion protein VP22, which has been shown to export fusion transgene products from transfected to surrounding untransfected cells, where it specifically accumulates in the nucleus (Elliott and O ’Hare, 1999). These genetic manipulations should not affect the catalytic properties o f HRP, as it is a robust and efficient enzyme, able to retain at least 50% o f its peroxidase activity when conjugated to polymers and antibodies (Folkes and Wardman, 2001).

To minimise IR- and drug-mediated toxicity to normal tissues, synthesis o f the prodrug activating enzyme has to be targeted to the tumour. Selective gene expression at

the tumour site may be achieved by using promoters that respond to the tumour-specific stimulus o f hypoxia. HREs are DNA sequences found in the regulatory regions o f a number o f genes responsive to tissue hypoxia, such as erythropoietin (Epo), phosphoglycerate kinase (PGK)-l and vascular endothelial growth factor (VEGF), and they have been successfully utilised to target therapeutic genes to the hypoxic tumour environment (Dachs et ah, 1997). Moreover, IR itself may be exploited for selective transgene expression, by induction o f CArG elements from the early growth response (Egr)-1 gene. Chimeric promoters containing pentamers o f HREs from the human Epo

and the murine PGK-1 genes, and a novel sequence based on the human VEGF HRE (nVEGF) were constructed (Chapter 7). Surprisingly, the PGKl and nVEGF enhancers were responsive not only to hypoxia (0.1% O2) but also to IR (5 Gy). In order to clarify

this effect, future work will need to be carried out. For instance, to establish if the induction is due to transcriptional activation, reporter mRNA levels after IR will need to be analysed, and, to detect if an HIF-1-associated pathway is activated, HIF-1 a protein levels and HRE binding activity measured. In a similar fashion, when nine CArG elements controlled transgene expression, they were independently induced by IR as well as by hypoxia. Whether reactive oxygen species (ROS) generated during hypoxia/reoxygenation rather than a direct response to a decrease in oxygen levels are involved is yet to be elucidated.

In order to combine and enhance the response to the transcriptional stimuli o f hypoxia and IR, the HREs were inserted upstream to the CArG elements. These chimeric promoters containing combinations o f HREs and CArG elements retained the ability to respond to individual and dual trigger treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo and CArG enhancers, on their own or in combinations, could selectively sensitise hypoxic and/or irradiated cells to lAA, when regulating the HRP cDNA. Such chimeric promoters may therefore be an effective tool to control therapeutic gene expression within the tumour microenvironment in GDEPT approaches aimed at addressing the problem o f hypoxia in radiotherapy.

Although selective gene expression is o f paramount importance to minimise normal tissue toxicity, the use o f inducible promoters may result in reduced therapeutic

protein levels compared to those under the control o f strong constitutive promoters. Further improvements to the system may be achieved by optimising the spacing between the elements, their orientation and position from the transcription start site. Moreover, in order to amplify suicide gene expression further, a molecular switch (Scott et al., 2000) could be utilised. The resultant high-level, stimuli-controlled expression system may provide sufficient therapeutic product in the tumour environment for future clinical application.

For a successful GDEPT, the prodrug should be able to reach the transfected cells. This may pose a particular problem in targeting cells under chronic hypoxia, since lAA will need to diffuse efficiently in the extravascular compartment to reach the HRP- expressing cells, located 100-200 pm away from the blood vessels. It is likely that lAA would reach populations at intermediate oxygen concentrations, closer to functional vessels, which appear to be critical in tumour response to fractionated radiotherapy (Wouters and Brown, 1997). Also, dynamic changes in microregional perfusion have been related to the formation o f areas o f acute, intermittent hypoxia, which may initially allow the delivery o f the prodrug in the tumour mass, and, subsequently, activate a hypoxia-responsive promoter.

The diffusion properties o f lAA and analogues in tumour conditions will have to be analysed. Tumour penetration may be improved by encapsulating the drug in carrier molecules, such as liposomes, hydrogels or polymers (reviewed by Langer, 1998). After prodrug delivery, tumour hypoxia may be increased by concomitant use o f antivascular agents such as combretastatin A-4-P, which has been shown to induce a rapid shut-down o f tumour blood flow leading to significant hypoxia (Tozer et al., 1999, 2001).

Finally, as for all gene therapy strategies, gene delivery remains the main concern. To date, viral vectors are characterised by the highest transfection rates in vivo, and major clinical experience has been gained with adenoviruses. Modification o f the capsid proteins and fibre may allow increased selectivity via the recognition o f cell-specific receptors, but biosafety is at present still disputable. Non-viral systems, although remarkably safe and easy to produce, are limited by poor gene transfer. Hypoxia-

specific therapeutic strategies, such as bacteria and macrophages, may represent interesting approaches for hypoxia-targeted gene therapy. The analysis o f their pathogenesis and the induced inflammation in humans are the main problems that will need to be answered in future clinical studies.

The need for local control in the cure o f cancer is a matter o f crucial importance. Therapeutic strategies aimed at delivering high and localised concentrations o f cytotoxic agents to clinically resistant solid tumour populations may provide a fundamental clinical gain, improving not only the efficacy o f standard treatments, without concurrent systemic complications, but overall survival and patient quality o f life.

Gene therapy is a promising approach, and 12 years after the approval o f the first clinical trial, it is still in the early stages o f development. Some major problems remain to be solved before these new strategies become routinely adopted in the clinic. One o f the main challenges is the improvement o f gene delivery, and therefore therapeutic efficacy. Nevertheless, the results collected so far and their potential clinical application are encouraging, and illustrate both feasibility and future promise for cancer treatment. Clinical trials have already addressed the issues o f safety. As vector technology fulfils the requirement o f efficient delivery, it can be anticipated that the results observed in the pre-clinical studies will more quickly translate into clinical benefit.

These in vitro studies showed that the horseradish peroxidase/indole-3-acetic acid system has the potential for use in cancer gene therapy, particularly in cases currently refractory to treatment as a result o f inherent or hypoxia-mediated radioresistance. Taken collectively, these observations suggest that the use o f HRE- and CArG-mediated HRP-GDEPT may be an effective tool to target the microenvironment o f solid malignancies.