EXPRESSION OF THYMIDINE KINASE MESSENGER-RNA AND A RELATED TRANSCRIPT IS MODULATED BY RADIOPROTECTOR WR1065

Previous studies have shown that the radioprotector WR1065 protects against mutagenesis across a wide concentration range (i.e., 40 mu M to 4 mM) but protects against cell killing by ionizing radiation at concentrations greater than 1 mM. Other work has demonstrated that many genes are induced or repressed after exposure of cells in culture to ionizing radiation, but the actual inducing agents for this gene modulation response are unknown. In these experiments, we set out to identify genes that would be modulated in response to two different concentrations of WR1065 (i.e., a lower dose that is incapable of protecting against cell killing but effective in protecting against mutation induction, and a high dose that is effective in protecting against both end points). Using differential display reverse transcription-PCR, we compared genes expressed in untreated cells to those expressed in cells treated with different concentrations of WR1065 (4 mM or 40 mu M) with or without radiation exposure (7.5 Gy). One band, which showed a differential response, was sequenced and found to have homology in the 3'-untranslated region of the mouse thymidine kinase (tk) gene but not identity to the Chinese hamster ovary tk gene. Dot blot and Northern blot analyses confirmed the differential display results and also determined that regulation of the tk-like gene is similar to that of tk itself. These experiments established that in Chinese hamster ovary cells, radiation causes a repression in accumulation of tie mRNA and a related tk-like transcript. This repression Is made less dramatic by the presence of 40 mu M WR1065, and, in fact, expression becomes enhanced when cells are pretreated with 4 mM WR1065. This suggests a role for regulation of tk and its related gene in the survival response of cells after exposure to ionizing radiation.