Effects of Hypoxia on Detoxicating Enzyme Activity and Expression in HT29 Colon

Resistance of hypoxic tumor cells to ionizing radiation and cytotoxic drugs has been attributed to changes in the reacUvity and/or the half times of reactive species in the altered redox environment. Exposure of eukaryotic cells to such hypoxic conditions results in the induction of the synthesis of several unrelated proteins. To Investigate further the phe nomenon of hypoxic cell resistance to cytotoxic drugs, we examined the effects of hypoxia on the expression of a group of enzymes involved in drug metabolism. Exposure of HT29 colon carcinoma cells to hypoxia resulted in a marked increase in the activity of DT-diaphorase and in glutathione content. The activity of glutathione transferase was not in creased by this treatment. The response was proportional to the duration of hypoxia. After the cells were exposed to hypoxic conditions for 8 h, followed by restoration of an oxic environment, the elevation in enzyme activity and glutathione content reached a peak at 48 h (40 h after the restoration of an oxic environment) and returned to baseline at 72 h. Elevation of steady-state levels of DT-diaphorase and y-glutamylcysteine synthetase mRNA followed a similar time course, with >10-fold increases over oxic cells at 24 h. The elevation ofDT-diaphorase mRNA content was found to result both from transcriptional induction and from increased message stability. The magnitude and persistance ofelevated detoxicating enzyme activity following a relatively short hypoxic exposure followed by reoxygenation suggest a novel potential mechanism of resistance to cyto toxic drugs in hypoxic tumors.