TYROSINE PHOSPHORYLATION IN DNA-DAMAGE AND CELL-DEATH IN HYPOXIC INJURY TO LLC-PK1 CELLS

Hypoxia is classically considered to result in a necrotic form of cell injury. We have recently demonstrated a role of endonuclease activati on, considered a feature of apoptosis, in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells (LLC-PK1 ce lls). Tyrosine phosphorylation has been implicated to be involved in c ell signaling pathway leading to cell growth, proliferation, and apopt otic death. However, a role of tyrosine phosphorylation as a signal tr ansduction pathway involved in DNA damage and cell death has not been previously examined in hypoxic injury in any tissue. In the present st udy, we have demonstrated that chemical hypoxia with a combination of antimycin A, a mitochondrial respiration inhibitor, and substrate depr ivation resulted in rapid increase in protein tyrosine kinases activit y and protein tyrosine phosphorylation prior to any evidence of cell d eath in LLC-PK1 cells. The inhibitors of protein tyrosine kinases, gen istein, lavendustin A, tyrphostin, and herbimycin A provided a marked protection against chemical hypoxia-induced DNA damage (as measured by alkaline unwinding assay) and cell death (as measured by trypan blue exclusion assay). In a separate study, we confirmed the ability of the inhibitors, lavendustin A and herbimycin A to prevent chemical hypoxi a-induced increase in protein tyrosine kinases activity and protein ty rosine phosphorylation. In addition, the inhibitors used did not affec t ATP depletion induced by antimycin A, suggesting that the inhibitors do not alter cellular uptake of antimycin A. Taken together, our data provide a strong evidence that tyrosine phosphorylation plays an impo rtant role in DNA damage and cell death in chemical hypoxic injury to renal tubular epithelial cells.