NUCLEAR C-MYC PLAYS AN IMPORTANT ROLE IN THE CYTOTOXICITY OF TUMOR-NECROSIS-FACTOR-ALPHA IN TUMOR-CELLS

The phosphoprotein c-Myc has the potential to kill cells by apoptosis. To investigate whether c-Myc is involved in tumor necrosis factor alp ha (TNF-alpha)-mediated cell killing, we have examined two HeLa cell l ines (D98 and H21) which show dramatic differences in their susceptibi lities to TNF-alpha cytotoxicity. Northern (RNA) blot analyses shelved that there were no significant differences between these cell lines i n basal or TNF-alpha-induced mRNA expression for a variety of proteins , including manganous superoxide dismutase, A20 zinc finger protein, p lasminogen activator inhibitor type 2, and hsp70, all of which are kno wn to influence the susceptibility of certain cells to TNF-alpha killi ng. On the other hand, there was a dramatic increase in c-Myc mRNA exp ression in TNF-alpha-sensitive D98 cells, but not in TNF-alpha-resista nt H21 cells, which was only observed when the cells were treated with cycloheximide. Western blot (immunoblot) analyses revealed that even in the absence of TNF-alpha or cycloheximide, c-Myc was detectable onl y in nuclear extracts of TNF-alpha-sensitive D98 cells, implying a rol e for preexisting c-Myc in TNF-alpha killing. In support of this inter pretation, a c-myc antisense oligonucleotide specifically inhibited th e TNF-alpha killing of D98 cells, provided that the oligonucleotide wa s added 6 h prior to TNF-alpha treatment. Either dexamethasone treatme nt or transient expression of c-myc antisense cDNA fragments decreased nuclear c-Myc in D98 cells and rendered the cells more resistant to T NF-alpha cytotoxicity. Nuclear c-Myc was also detectable in a TNF-alph a-sensitive human HT-1080 fibrosarcoma cell line, but it was undetecta ble in a derivative of HT-1080 (SS-HT-1080) known to be resistant to T NF-alpha killing because of overexpression of plasminogen activator in hibitor type 2. HT-1080 cells transfected with antisense c-myc cDNA ha d significantly less nuclear c-Myc and were resistant to TNF-alpha cyt otoxicity. Together, these data indicate that a nuclear transcription factor, c-Myc, plays an important role in sensitizing two different tu mor cell types to TNF-alpha cytotoxicity.