Interaction of heat and hypoxia in modulating transcription of DT diaphorase in human colon adenocarcinoma cells.

To study the regulation of the human detoxicating enzyme DT diaphorase (DTD) under hypoxic conditions, we examined the effects of heat and hypoxia, and their interaction, on the steady-state levels of mRNA and DTD enzyme activity in human colon adenocarcinoma HT29 cells. We found that a 1-h heat treatment (42.5 degrees C) markedly increased the specific activity of DTD. Elevated enzyme activity was observed within 30 min, peaked at 6 h, and had almost returned to baseline by 36 h. The effect of hypoxia alone on DTD enzyme activity developed more slowly, with a maximal response at 24 h, and return to baseline at 72 h. The effect of a 1-h heat treatment was not inhibited by subsequent hypoxic exposure for 8 h. The effect of hypoxia was also not inhibited by heat in any schedule. However, a 1-h exposure to heat during 8 h hypoxic exposure induced the transcriptional effects of heat treatment much earlier. Heat shock followed by hypoxic stress resulted in prolonged elevation of DTD activity similar to that observed with hypoxia alone. We found that DTD mRNA content was elevated with a time course concordant with that of the enzyme activity. These data suggest that hypoxia and heat shock induce expression of the DTD gene independently. The mechanism of heat effect on DTD gene expression was investigated. Gel retardation assays demonstrated the induction of a binding activity of heat-induced transcription factor(s) to heat shock elements following both heat and hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)