RAS-DEPENDENT APOPTOSIS CORRELATES WITH PERSISTENT ACTIVATION OF STRESS-ACTIVATED PROTEIN-KINASES AND INDUCTION OF ISOFORM(S) OF BCL-X

Has proteins are signal transducers for many cellular responses. Howev er, it is not well established whether Ras-signaling also contributes to apoptosis. We have constructed H-Ras(R12)-transformed Rat1 fibrobla sts using tetracycline operator/repressor (TetO/TetR)-based conditiona l vectors. Rat1/TetO-Ras(R12) (Rat1-Ras) cells produced high levels of H-Ras(R12) protein and exhibited oncogenic transformation. Treatment of Rat1-Ras cells with 0.1% serum triggered massive apoptosis. Rat1-Ra s cells expressed increased basal activities of extracellular response kinase (ERK) and p46/p54 stress-activated protein kinase/c-Jun NH2-te rminal kinase (SAPK/JNK). Interestingly, Ras-dependent apoptosis corre lated with further persistent activation of both p46 and p54 SAPK/JNK and concurrent inhibition of ERK. Differential modulation of SAPK/JNK and ERK was not detected in tetracycline-treated cells that did not co mmit apoptosis. Furthermore, two Bcl-x related proteins of 15 kDa and 18 kDa were highly induced in apoptotic Rat1-Ras cells. Our results es tablish a direct role for Ras in apoptosis, and suggest a functional r elationship between H-Ras, SAPK/JNK, ERK and Bcl-x in regulating apopt osis.