TEMPERATURE-DEPENDENCE OF RADIATION-INDUCED DNA-PROTEIN CROSS-LINKS FORMED UNDER HYPOXIC CONDITIONS

Recently, we demonstrated that the oxygen dependence of the formation of DNA-protein crosslinks (DPCs) in irradiated mammalian cells measure d by the alkaline elution technique is the mirror image of the oxygen dependence of radiation-induced cell killing. Consequently, these radi ation-induced DPCs could be used to detect hypoxic cells or estimate t he hypoxic fraction of cells in solid tumors. Although several techniq ues, including alkaline elution, gas chromatography/mass spectrometry (GC/MS) and nitrocellulose filter binding, have been used to measure r adiation-induced DPCs, the published reports suggest that the characte ristics of these DPCs may depend on both the type of sample irradiated (cellular compared to model systems, oxygenated compared to hypoxic, etc.) and the technique used to measure these radiation-induced DPCs. In the present study, the radiation-induced DPCs measured by our alkal ine elution technique with and without proteinase K in the lysis solut ion were characterized by studying the dependence of their formation o n temperature in hypoxic rat 9L brain tumor cells. Exponentially growi ng 9L rat brain tumor cells were rendered hypoxic at 4 degrees C or at 37 degrees C and then irradiated with either 7.5 Gy or 15 Gy. The cel ls were trypsinized at 4 degrees C, either immediately after the irrad iation or after one half-time of strand break repair at 37 degrees C. The results demonstrated that the radiation-induced DPCs produced in 9 L cells under hypoxic conditions, measured by our alkaline elution tec hnique after low to moderate radiation doses, required metabolism for their formation, unlike the radiation-induced DPCs reported by others using the GC/MS or nitrocellulose filter binding technique. (C) 1997 b y Radiation Research Society.