The impact of intracellular generation of nitric oxide on the radiation response of human tumor cells.

Nitric oxide (NO) is a potent radiosensitizer of hypoxic mammalian cells. There have been many reports demonstrating radiosensitization in vitro and in vivo by the use of NO donors to generate NO by chemical means or by producing agents that mimic the free radical mechanism(s) of NO for potentiating radiosensitivity. However, much of this work has been done without taking account of the endogenous NO that is generated in tumor cells by NO synthase (NOS) in vitro or in tumor cells and host cells in solid tumors in vivo. To evaluate the contribution of intracellular generated NO to cellular radiosensitivity, we exposed human HT1080 and MDA231 tumor cells to a cytokine cocktail that results in an increase in cellular NOS expression to a level that is seen in many human solid tumors. We also carried out parallel studies to determine the radiosensitivity of HT1080 and MDA231 cells engineered to constitutively overexpress the iNOS gene. When cells are treated with cytokines under anoxic conditions (<0.01% O(2)), there is up to a 9-15-fold increase in NOS expression, but no detectable NO is generated (since O(2) is required for the generation of NO via the NOS-mediated conversion of arginine to citrulline). As a consequence, when these cells are irradiated under hypoxic conditions, no radiosensitization is observed. However, as the oxygen tension was increased, the amount of NO generated also increased, and we show that this NO then contributes to an overall increase in the radiosensitivity of cells. For example, at 1% O(2) in control HT1080 cells, with little measurable NOS activity, the dose of radiation required to reduce survival by 90% was 6 Gy compared to 10 Gy in anoxic conditions. After cytokine treatment, the level of NO generated at 1% O(2) was significantly increased and the dose of radiation needed for 90% cell killing was reduced further to 4 Gy. The presence of the NOS inhibitor N(G)-methyl-l-arginine (NMLA) shortly before and during irradiation ablated this increase in radiosensitivity, confirming that the effect was due to the generation of NO. We conclude that cytokine-mediated up-regulation of the NOS expression in tumor cells can produce sufficient NO to significantly increase the cytotoxic effect of radiation and that this is particularly apparent at intermediate oxygen concentrations.