INVOLVEMENT OF DT-DIAPHORASE (EC-1.6.99.2) IN THE DNA CROSS-LINKING AND SEQUENCE SELECTIVITY OF THE BIOREDUCTIVE ANTITUMOR AGENT EO9

The chemistry of the mitomycin C-related drug indoloquinone EO9 would suggest that its mechanism of action is likely to involve DNA damage a fter reductive activation. The ability of this agent to induce DNA dam age in intact cells has been examined using alkaline filter elution. A fter treatment with pharmacologically relevant concentrations of EO9, both DNA strand breaks and interstrand cross-links were detected in ra t Walker tumour cells and human HT29 colon carcinoma cells. These cell lines express relatively high levels of DT-diaphorase (NAD(P)H: quino ne acceptor oxidoreductase), which is believed to be involved in EO9 a ctivation. The extent of DNA damage was increased by approximately 30- fold under hypoxia in BE colon carcinoma cells that express non-functi onal DT-diaphorase, but this dramatic hypoxia enhancement was not seen in HT-29 cells. These data are consistent with cytotoxicity studies t hat indicate that DT-diaphorase appears to be important in EO9 activat ion under aerobic conditions, but other enzymes may be more relevant u nder hypoxia. The involvement of DT-diaphorase in DNA damage induction was further investigated using cell-free assays. DNA cross-links were detectable in plasmid DNA coincubated with EO9, cofactor and DT-diaph orase but not in the absence of this enzyme. In contrast, using a Tao polymerase stop assay, monofunctional alkylation was detected in plasm id DNA without metabolic activation, although the sequence selectivity was altered after reduction catalysed by DT-diaphorase.