ANALYSIS OF HUMAN BREAST ADENOCARCINOMA MCF7 RESISTANCE TO TUMOR NECROSIS FACTOR-INDUCED CELL-DEATH - LACK OF CORRELATION BETWEEN JNK ACTIVATION AND CERAMIDE PATHWAY

Considerable progress has been made in the understanding of tumor necr osis factor (TNF) signaling; however, the molecular and biochemical ba sis of tumor resistance to the cytotoxic action of TNF are still not d efinitively identified yet. Although a role of c-Jun N-terminal kinase (JNK) pathway has been suggested as an effector in TNF signaling, its exact relative contribution and its interaction with ceramide pathway and tumor resistance to TNF remain unknown. The relationship between JNK activation and human breast adenocarcinoma MCF7 resistance acquisi tion to the cytotoxic action of TNF was therefore investigated. We dem onstrate that TNF triggers JNK activation in both TNF-sensitive MCF7 c ells and its resistant derivative, RA1/1001, In addition, when MCF7 ce lls were stably transfected with mitogen-activated protein kinase kina se 4 (MKK4) dominant-negative cDNA or transiently transfected with a d ominant-negative c-Jun mutant (TAM: 67), their susceptibility to the c ytotoxic action of TNF remains comparable with control cells. We also demonstrated that JNK activation does not require ceramide generation since in MCF7 cells transfected with a dominant-negative derivative of FADD (FADD-DN), which are resistant to the cytotoxic action of TNF, T NF induced JNK activation in the absence of ceramide generation. Furth ermore, our data indicate that exogenous permeable synthetic ceramide C-6 induced the killing of MCF7 cells transfected with MKK4 dominant-n egative cDNA. These results provide strong evidence indicating that tu mor acquisition of resistance to the cytotoxic action of TNF may occur either independently or at a level downstream of JNK activation and s uggest that JNK activation is not linked to ceramide pathway in TNF-me diated apoptosis.