Treatment of different human cancer cell lines with 2-deoxyglucose (2dG), or thapsigargin (TG) or tunicamycin (TM) results in up-regulation/induction of GRP78. Previously we have demonstrated an excellent association between up-regulation of GRP78 and hypersensitivity to alkylating/platinating agents. However, the mechanisms of this association are still not clear. GRP78 is a Ca++ binding protein. Thus it is possible that over-expression of GRP78 can cause an alteration in Ca++ homeostasis which can have a regulatory role in Ca++-dependent signaling pathways. DNA damage after GRP78 induction can adversely affect these signaling pathways resulting in cell cycle alteration and augmented apoptosis. To examine the validity of our hypothesis, microarray assays were performed using apoptosis, cell cycle and MAP kinase pathway specific cDNA microarrays containing 100-280 genes (SuperArray Bioscience Corporation, Frederick, MD). A549 human lung cancer cells were exposed to 2dG (10 mM) or TG (25 nM) or TM (250 nM) for 48 h, followed by cisplatin (60 μM for 2 h) treatment. The results demonstrate that all Caspases, BAK1, BIK and p53 are significantly upregulated which may be the cause of observed increase in apoptosis. However, up-regulation of these genes occurs without the activation of upstream ATM. In contrast, anti-apoptotic protein Bcl-2 and E2F transcription factors that regulate cyclin-E levels are strongly up-regulated. These results suggest an intriguing possibility of existence of alternative pathways in which p53/Bcl-2 levels are regulated independent of ATM. Further studies are in progress to confirm transcript levels by quantitative RT-PCR, protein levels by western immunoblotting and cell cycle analysis by flowcytometry. Our study will provide better understanding of the pathways involved in DNA damage after GRP78 over-expression that can serve as the basis for developing new rationale for cancer chemotherapy. Acknowledgements: These studies were supported, in part, by grants RO1CA65920 from the NCI, 00A1 from the American Institute for Cancer Research, and EPSCoR from NDSU to SC.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]