NSAID-INDUCED APOPTOSIS IN ROUS-SARCOMA VIRUS-TRANSFORMED CHICKEN-EMBRYO FIBROBLASTS IS DEPENDENT ON V-SRC AND C-MYC AND IS INHIBITED BY BCL-2

Mounting epidemiological and experimental evidence implicates nonstero idal antiinflammatory drugs as anti-tumorigenic agents. Our previous w ork showed that nonsteroidal antiinflammatory drug treatment of src-tr ansformed chicken embryo fibroblasts caused apoptosis - a mechanism by which these drugs might exert their anti-tumorigenic effeet. The pres ent studies employ a sensitive technique for detecting single-and doub le-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60(v-src), which antagonizes apoptosis in many cell sys tems, was found to induce apoptosis in 11-23 % of serum-starved fibrob lasts. However, treatment with diclofenac following pp60(v-src) activa tion produced a much stronger response beginning within 6 hours of tre atment that resulted in 100% lethality. During cell death, cyclooxygen ase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increase d by all apoptotic drugs tested. Examination of the expression of apop tosis-associated genes showed that c-rel and p53 (found in normal or v -src-transformed chicken embryo fibroblasts at moderate levels), and b cl-2 (present at an extremely low level) were largely unchanged by tre atment with eight different nonsteroidal antiinflammatory drugs. Howev er, over-expression of human bcl-2 inhibited diclofenac-mediated apopt osis by 90%, demonstrating directly that bcI-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto -oncogene c-myc is known to cause apoptosis in chicken embryo fibrobla sts when artificially overexpressed in cells deprived of trophic facto rs. We found that nonsteroidal antiinflammatory drug treatment followi ng pp60(v-src) activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60(v-src) activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced dru g-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammat ory drug-induced apoptosis proceeds through a src/myc dependent pathwa y which is negatively regulated by bcI-2.