The role of reduced nicotinamide adenine dinucleotide phosphate in glucose- and temperature-dependent doxorubicin cytotoxicity.

The mechanism of doxorubicin resistance induced by glucose deprivation was examined using an L929 cell system. Resistance developed even when the synthesis of glucose-regulated proteins was suppressed by supplementing glucose-deprived cultures with uridine. Resistance was also not correlated with pyruvate availability, with DNA strand breaks, or with intracellular drug or nucleotide levels. However, intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate (NADPH) decreased to undetectable levels in glucose-deprived cells with or without uridine supplementation. NADPH depletion induced by treating glucose-fed cells with low concentrations of methylene blue afforded the same degree of protection as glucose deprivation, and normal sensitivity could be restored to glucose-deprived cells by adding NADPH to the culture medium. These results suggest that decreased NADPH availability is responsible for the doxorubicin resistance induced by glucose deprivation. Although drug uptake and NADPH production increased with temperature, these effects could not fully account for the > 1000-fold decrease in clonogenic survival observed over the 25 degrees-37 degrees C temperature range. Similarly, manipulation of NADPH levels confirmed a role for drug bioreduction in the cytotoxic mechanism but did not suggest that NADPH availability was rate-limiting for this process at any temperature employed.