PARENT SESSION
1:30 PM to 3:30 PM
Saturday, April 20, 2002
Poster Session 2 Heat Shock Response and Mechanisms of Heat Damage
Room: Nevada 4-5

(MP02-22) Nitric Oxide-mediated Inhibition of NF-kB Regulates Hyperthermia-induced Apoptosis.

Natarajan, Mohan^*,1, Aravindan, Natarajan¹, Herman, Terence¹, ¹ Molecular & Therapeutic Radiation oncology, San Antonio, Texas

ABSTRACT-
Hyperthermia is a modality used to treat cancer, mostly combined with chemotherapy and/or radiation. Although considerable effort has been expended towards understanding the cellular and biochemical responses to heat, the underlying processes of cellular injuries at the molecular level are still not well understood. In this study, using human adenocarcinoma MCF-7 breast cancer cell line, we have investigated whether (i) hyperthermia-induced apoptosis is mediated through the nitric oxide signaling pathway and (ii) inhibition of post-translational modification of IB- and the down regulation of NF-B DNA-binding activity is an intermediate step in NO-dependent apoptosis. For hyperthermia treatment the cells were exposed to 43oC in a T-25 flasks for 1 h. Intracellular nitric oxide levels measured by the fluorescent intensity of DAF-2A and iNOS expression examined by immunobloting revealed an increased level of iNOS dependent NO production after 43oC. Apoptotic cell death measured by Annexin V expression and cell survival determined by clonogenic assay showed a 20% increase in cell death after 43oC treatment compared to 37oC. EMSA analysis on the other hand showed a time dependent inhibition of NF-B DNA binding activity in those cells after 43oC treatment. The hyperthermia-mediated inhibition of NF-kB was persistent even after 48 h. Immunobloting analysis with phospho IB- showed a decreased site-specific phosphorylation of IB- at Ser 32. Inhibition of NO by either L-NAME or L-NMMA rescued the NF-B DNA-binding activity and inhibits heat-induced apoptosis. Similarly, overexpression of NF-B by transient transfection inhibits heat-induced apoptosis. These results suggest that apoptosis upon hyperthermia exposure of human adenocarcinoma cells is regulated by NO-mediated suppression of NF-B.

KEYWORDS: Heat response, I-kappa B, MCF-7 cells, signaling