Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells

The present study explored the relationship between activation of c-Jun N-terminal kinase (JNK) and apoptosis following exposure of primary human kidney cells to hypoxia/reoxygenation. Apoptosis induction was apparent only after prolonged exposure of cells to hypoxia (>48 hr), when a 2-fold increase in DNA fragmentation was observed. In contrast, 15 hr of reoxygenation following either 4 or 8 hr of hypoxia was associated with a pronounced (>17-fold) increase in DNA fragmentation. Fluorescence microscopy, using DNA binding dyes, demonstrated that cell death following hypoxia/reoxygenation was due predominantly to apoptosis and not necrosis. Furthermore, reoxygenation, but not hypoxia alone, caused a time-dependent increase in the activation of JNK as monitored by western blot analysis using a phospho-specific JNK antibody. In contrast, p38 mitogen-activated protein kinase was activated following hypoxia, but this activation was not augmented during reoxygenation. Exposure of human kidney cells to a 2'-methoxyethyl mixed backbone antisense oligonucleotide directed against human JNK1 (JNK1 AS) resulted in a potent suppression of JNK mRNA and protein expression, whereas a 6-base mismatch control oligonucleotide was without effect. Moreover, a significant diminution of reoxygenation-induced apoptosis was observed in cells exposed to JNK1 AS but nor: to the mismatch control oligonucleotide. Taken together, these results strongly indicate that activation of the JNK signaling cascade is a major mechanism whereby hypoxia/reoxygenation induces apoptosis. (C) 2000 Elsevier Science Inc.