The effects of three bioreductive drugs (mitomycin C, RSU-1069 and SR4233) on cell lines selected for their sensitivity to mitomycin C or ionising radiation

Abstract

Summary We have investigated the cross-sensitivity of a number of cell lines to three different classes of bioreductive drugs under both aerobic and hypoxic conditions. The cell lines used were selected for their sensitivity to DNA-damaging agents and fall into two groups. One group, MMC cells derived from CHO-KI cells (Robson et al., 1985), show a range of sensitivities to mitomycin C in air. The second group, irs cells were cloned from V79 Chinese hamster fibroblasts (Jones et al., 1987) and exhibit sensitivity to ionising radiation. The sensitivity of both groups of cells to mitomycin C (MMC), RSU-1069 Hypoxic cells within tumours are known to limit the efficacy of radiotherapy. Bioreductive and radiosensitising drugs are under development in order to improve tumour therapy by sensitising the hypoxic tumour cells to either radiation or cytotoxic chemotherapy. One process by which hypoxic cells can be selectively killed is to utilise the reductive metabolic pathways which can be exploited readily under poorly oxygenated conditions.The mechanism of action of three different classes of bioreductive compounds have been investigated in vitro, using cell lines selected for their sensitivity to DNA damaging agents. The drugs investigated were the bioreductive quinone antibiotic mitomycin C (Kennedy et al., 1980); a monofunctional alkylating 2-nitroimidazole RSU-1069 (Adams et al., 1984;Stratford et al., 1986); and the benzotriazine-di-N-oxide SR4233 (Zeman et al., 1986(Zeman et al., , 1989.The cell lines used fall into two groups, both of which were cloned from Chinese hamster cell lines. The first, MMC cells, were isolated from Chinese hamster ovary cells, CHO-K1 (Robson et al., 1985), and were either hypersensitive (MMC-2 and MMC-3), or resistant (MMCl) to mitomycin C. The concentrations of MMC required to reduce cell survival to 0.37 of the control following 24 hour aerobic exposure (D37 value) have been determined by Robson et al. (1985) and Hoban et al. (1990) for each cell line. These are: CHO-KI, 0.32; MMC-2, 0.05; MMC-3, 0.07; MMCr, 6.07,UM respectively. The second group of cells, irs cells were originally cloned from V79 Chinese hamster fibroblasts (Jones et al., 1987) and exhibit a range of sensitivities to ionising radiation in air. For these cell lines D37 values (Gy) have been determined as; V79, 4.2; irs 1, 1.34; irs 2, 1.41; irs 3, 2.06 (Jones et al., 1987).The cross-sensitivity of these cell lines to MMC, RSU-1069 and SR4233 has been assessed under both hypoxic and aerobic conditions. This type of study may help to determine the mechanism of action of these agents and the factors influencing cellular sensitivity to bioreductive cytotoxic drugs, in particular the enzymes involved in drug activation and cellular repair pathways. gassed with N2 + 5% CO2 at 37°C for 3 h. The drug-containing medium was then removed from the dishes, replaced with fresh medium and cells assayed 7-10 days later for colony formation.

SR 4233 cytotoxicity and metabolism in DNA repair-competent and repair-deficient cell cultures

“…This may be related to the fact that AT cells have not shown a gross defect in DNA double strand break rejoining, although they do show reduced rejoining of chromosome breaks compared to normal cells (Cornforth & Bedford, 1985), and lack the ability to repair poterftially lethal damage as judged by delayed plating experiments (Biedermann & Brown, 1989). This lack of correlation between X-ray and SR4233 sensitivity is also seen in the data of Keohane et al (1990). These investigators showed that irs cells, which are radiationsensitive, but not deficient in the repair of DNA double strand breaks (Jones et al, 1990), display similar hypoxic cytotoxicities by SR 4233 to the parental V79 vells.…”

“…We have proposed that SR 4233 is reduced via a single-electron reduction pathway to form a free radical intermediate, the structure of which has been recently confirmed by ESR (Lloyd & Mason, personal communication). Under aerobic conditions, the radical reacts with oxygen in a futile one-electron reduction cycle, generating superoxide in the process (Laderoute et al, 1988). In the absence of oxygen, the SR 4233 radical may abstract hydrogen from DNA and other macromolecules, forming strand breaks which ultimately result in cell death.…”

“…The drug has a high selective toxicity for hypoxic cells in vitro (Zeman et al, 1986) and is effective as an antitumour agent in vivo when combined either with radiation Brown & Lemmon, 1990) or with an agent which specifically induces tumour hypoxia (Brown 1987; Sun & Brown, 1989). SR 4233 is reduced in hypoxic cells to form the two-and four-electron reduction products, SR 4317 and SR 4330, as measured by HPLC (Baker et al, 1988;Laderoute & Rauth, 1986;Walton et al, 1989). Both of these products are nontoxic to cells under hypoxic or aerobic conditions (Baker et al, 1988;Zeman et al, 1986).…”

SR 4233 (Tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours

“…Despite the variability in differential hypoxic cytotoxicity for different cell lines shown for SR4233, values obtained from different investigators are consistently higher than those obtained for the quinone antibiotic mitomycin C, for which values of only 1 to 5 are found (Fracasso & Sartorelli, 1986;Keohane et al, 1990;Stratford & Stephens, 1989), and for the 2-nitroimidazole misonidazole for which values of five to 15 are typical (Stratford & Stephens, 1989;Taylor & Rauth, 1978). However, comparable high values are often, though not universally, reported for the dual function nitroimidazole, RSU 1069 (Adams et al, 1992;Roizin-Towle et al, 1990;Stratford & Stephens, 1989).…”

“…Second, the nitroimidazoles, although developed as radiation sensitisers, show preferential toxicity to hypoxic cells which is high in the case of the dual function agent, RSU 1069 (Stratford & Stephens, 1989;Stratford et al, 1986). The third class of bioreductive cytotoxic drugs are the benzotriazine di-N-oxides of which SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide, WIN 59075, tirapazamine) is the prototype compound (Zeman et al, 1986 (Adams et al, 1992;Costa et al, 1989;Stratford & Stephens, 1989), although it has also been reported that certain cell lines with repair deficiencies have somewhat lower differential cytotoxicities (Keohane et al, 1990).…”