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 non-steroidal antiinflammatory drugs as anti-tumorigenic agents. Our previous work showed that nonsteroidal antiinflammatory drug treatment of src-transformed chicken embryo fibroblasts caused apoptosis--a mechanism by which these drugs might exert their anti-tumorigenic effect. The present studies employ a sensitive technique for detecting single- and double-stranded DNA cleavage (the comet assay) to quantitate apoptosis. By this method pp60v-src, which antagonizes apoptosis in many cell systems, was found to induce apoptosis in 11-23% of serum-starved fibroblasts. However, treatment with diclofenac following pp60v-src activation produced a much stronger response beginning within 6 hours of treatment that resulted in 100% lethality. During cell death, cyclooxygenase-2 but not cyclooxygenase-1 mRNA was found to be uniformly increased by all apoptotic drugs tested. Examination of the expression of apoptosis-associated genes showed that c-rel and p53 (found in normal or v-src-transformed chicken embryo fibroblasts at moderate levels), and bcl-2 (present at an extremely low level) were largely unchanged by treatment with eight different nonsteroidal antiinflammatory drugs. However, overexpression of human bcl-2 inhibited diclofenac-mediated apoptosis by 90%, demonstrating directly that bcl-2 expression can regulate nonsteroidal antiinflammatory drug induction of cell death. The proto-oncogene c-myc is known to cause apoptosis in chicken embryo fibroblasts when artificially overexpressed in cells deprived of trophic factors. We found that nonsteroidal antiinflammatory drug treatment following pp60v-src activation persistently induced myc protein and mRNA by more than 20-fold above that evoked by pp60v-src activation alone. Moreover, transfection of antisense c-myc oligonucleotides reduced drug-induced myc expression by 80% and caused a concomitant 50% reduction in cell death. These findings suggest that nonsteroidal antiinflammatory drug-induced apoptosis proceeds through a src/myc dependent pathway which is negatively regulated by bcl-2.
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Department of Zoology, Brigham Young University, Provo, UT 84602, USA.
Fairbairn
D W
DW
Bradshaw
W S
WS
O'Neill
K L
KL
Ewert
D L
DL
Simmons
D L
DL
D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001358:Avian Sarcoma Viruses; D002461:Cell Line, Transformed; D002642:Chick Embryo; D053938:DNA Fragmentation; D005347:Fibroblasts / Q000502:physiology*; D015870:Gene Expression; D016259:Genes, myc / Q000235:genetics; D006801:Humans; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000494:pharmacology*; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000494:pharmacology*
D000894:Anti-Inflammatory Agents, Non-Steroidal; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D015688:Oncogene Protein pp60(v-src)