by HRP/IAA gene therapy
were 3.6-fold more sensitive to radiation than buffer-treated controls, and the OER was reduced from 2 to 1.2.
HRP* anoxia : CD4* anoxia 0.1 0.01 0.01 Dos. (Gy) 0.001 8 Radiation 0.001 0 2 4 6 8
Figure 6.5. Effect of HRP/IAA GDEPT on the radiosensitivity o f T24 cells under anoxia. CD4 (A) and HRP (B) cells were exposed to 0.1 mM lAA for 24 h prior to irradiation. Means of three independent experiments (triplicate samples) ± SE are shown. Lines are best-fit curves. Cell survival was normalised to exposure to lAA, to account for drug toxicity. Survival data for oxic cells without lAA is shown for comparative purposes. —V —; CD4 T24 cells, no prodrug, normoxia; A : CD4* cells, no prodrug, anoxia; - 0 - : CD4^ cells + 0.1 mM lAA, anoxia; -▼ —: HRP cells, no prodrug, normoxia; A : HRP cells, no prodrug, anoxia; HRP^ cells + 0.1 mM lAA, anoxia. Inset: survival of CD4 cells irradiated in normoxia and anoxia replotted for clarity.
Delivery and expression o f a foreign gene alone (HRP or the marker CD4) did not alter cellular radiosensitivity. Similar findings were reported by other investigators who
utilised retroviral (Kim et al., 1994; Khil et al., 1996) or adenoviral vectors (Hanna et al., 1997; Blackburn et al., 1999) for GDEPT. Moreover, Mansur et al. (2001) observed that neither retroviral nor liposome-mediated delivery o f the glutathione peroxidase gene resulted in an effect on radiosensitivity o f human T cells and Chinese hamster ovary (CHO) fibroblasts. On the other hand, an increase in sensitivity to IR was observed in cells transduced with Ads containing the CD gene even in the absence o f 5- FC (Pederson et al., 1997; Stackhouse et al., 2000).
True radiation sensitisers have no lethal effect o f their own. The prodrugs tested here reduced the survival o f HRP^ cells to -3 0 % at 0.1 mM and -0 .6 % at 0.5 mM after 24 h (Chapter 3). Further selective toxicity could be achieved with the addition o f IR. These concentrations o f prodrug were found to kill less than 20% o f HRP T24 and FaDu cells, they are likely to be achievable in vivo (tumour prodrug levels o f -1 mM can be obtained in mice, with no associated toxicity; J. Tupper, unpublished data), and therefore may represent a possible clinical scenario.
Limited transfection efficiency (10-14% in FaDu and 20-25% in T24 cells) compared with the levels o f cell kill measured (Figures 6.2-6.5) indicated the induction o f a bystander effect, whereby the activated prodrug induced radiosensitisation also in surrounding untransfected tumour cells.
Similar SERs were measured when prodrug incubation was performed prior to or after X-rays, suggesting that activated lAA may increase susceptibility to IR and also interfere with post-irradiation repair processes. To date, the activated drugs and the cellular targets involved in HRP/IAA-mediated cytotoxicity and radiosensitisation have not been identified. Exposure o f hamster fibroblast V79 cells to lAA or 5-F-IAA activated by purified HRP resulted in a decrease o f cellular glutathione (GSH) up to -70% o f control levels (Folkes et al., 2002). Moreover, the addition o f excess GSH to HRP/IAA-generated oxidation products significantly reduced cytotoxicity (Folkes et al., 1998), suggesting that potentially cytotoxic products had reacted with this thiol. Depletion o f intracellular thiols has been shown to have a radiosensitising effect in vitro
(Clark et al., 1984; Vames et al., 1984; Biaglow et al., 1986) and in vivo (Bump et al., 1982; Stevens et al., 1995). The effect has been observed under both oxic and anoxic conditions (Vames et al., 1984; Debieu et al., 1985), with preferential sensitisation o f cells at low or intermediate oxygen levels (Bump et al., 1982; Clark et al., 1984; Debieu et al., 1985; Scott et al., 1993; Stevens et al., 1995). GSH depletion did not induce any significant alteration o f the OER in normal mouse skin (Stevens et ah, 1995), V79 (Mitchell et ah, 1983) and A549 human lung carcinoma cells (Biaglow et ah, 1983 a; Mitchell et ah, 1983). A decrease in OER has been observed in cells that show no oxic response to thiol depletion (Biaglow et ah, 1983 b).
However, GSH depletion is unlikely to be solely responsible for the effects shown in this chapter. The reduction in GSH levels observed by Folkes et ah (2002) is probably insufficient to account for the SERs achieved here, as a reduction o f >90% was shown to be necessary to achieve full radiosensitisation (Clark et ah, 1984). Moreover, GSH depletion would only be expected to radiosensitise prior to irradiation, while HRP/IAA was shown to induce sensitisation also after IR. Additional biochemical alterations, probably involving the DNA macromolecule, are likely to take place. Further studies are warranted to shed light on the possible mechanisms associated with HRP/IAA-mediated enhancement o f IR toxicity.
With 50% o f all human cancer patients treated with radiation, improvement o f the efficacy o f a radiotherapy schedule remains a major issue in cancer research and treatment. Numerous clinical trials have focused on combining IR with conventional chemotherapeutic agents, such as doxorubicin, methotrexate, 5-FU and CDDP (Stewart and Saunders, 1997). However, normal tissue toxicity remains a major limiting factor with these protocols. The results presented here demonstrate that lAA and 1-Me-IAA selectively enhance radiation-induced lethality in human tumour cells expressing the enzyme HRP at doses o f prodrug achievable in vivo. In order to achieve a significant advantage by using combined gene therapy and radiotherapy protocols, prodrug activation should take place at the tumour site only. This can be obtained by selective HRP gene delivery and/or selective gene expression at the target, as suggested in Chapter 7.