INCREASED CYTOTOXICITY OF CHRONIC HYPOXIC CELLS BY MOLECULAR INHIBITION OF GRP78 INDUCTION

Purpose: To develop a molecular strategy of increasing cytotoxicity of chronically hypoxic cells by inhibiting Glucose Regulated Protein 78 kDal (GRP78) induction. Methods and Materials: A mutant nonGRP78 inducing cell line (78WO) was developed from its parent (DG44) by overexpressing antisense GRP78 mRNA. Following exposure to varying durations of hypoxia, Northern and Western blot analysis were used to characterize the amount of GRP78 expression both at the RNA and protein level. Hypoxia was achieved by placing cells in specially designed hypoxic chambers which were subjected to successive rounds of evacuation and flushing with 95% CO2/N2 to reduce the oxygen in the environment to 0.02% oxygen. After treatment with hypoxia, cells were assayed for colony forming ability. Results: GRP78 mRNA and protein induction following exposure to hypoxia was 3-4 fold lower in the 78WO cell line than in the parental DG44 cell line. Furthermore, it was observed that there was no difference in the cytotoxicity of 78WO and DG44 cells after 10 h of hypoxia. However, after 15 h of hypoxia, the survival of 78WO cells decreased by 1 log and after 20 h of hypoxia, the survival of 78WO decreased by another log. Conclusion: These results show that stress protein induction is important for cellular survival to chronic hypoxia and that inhibition of GRP78 induction may represent a novel therapeutic strategy by selectively sensitizing chronically hypoxic cells within solid tumors.