Tumor necrosis factor a (TNF-a) is wellcharacterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNFca-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-a to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-ai and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-a. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-ai-induced apoptosis. Further, TNF-a initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.

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Despite what is known about the early signaling events in tumor necrosis factor (TNF) ␣-induced apoptosis, characterization of the downstream events remains largely undefined. It is now known that a crosstalk exists between the interferon and TNF-␣ pathways. This linkage allows recruitment of the cell proliferation suppressor PKR (dsRNA-dependent protein kinase) from the interferon pathway to play a pivotal role in TNF-␣-induced apoptosis. In this study, we took advantage of the differential TNF-␣ susceptibilities of human promonocytic U937 subclones, deficient in or overexpressing PKR, to further characterize the role of PKR in apoptosis. By reverse transcription-polymerase chain reaction, we demonstrated that TNF-␣ transiently induces the tumor suppressor p53 in U937 cells. This p53 induction lags behind the TNF-␣ induction of PKR by 1 h. By cell viability determination, ultrastructural studies, apoptotic DNA laddering, and antisense techniques, it was shown that inhibition of p53 expression in PKRoverexpressing U937 cells abrogates the TNF-␣-induced apoptosis in these cells. Conversely, overexpressing wild type p53 in PKR-deficient U937 cells confers the susceptibility of these cells to TNF-␣-induced apoptosis. This latter result indicates that p53 induction is an event downstream of TNF-␣-induced up-regulation of PKR, thereby further establishing the critical role of p53 in TNF-␣-induced apoptosis in U937 cells. PKR-overexpressing U937 cells were found to possess a constitutively higher level of p53, which partly explains why these cells spontaneously undergo apoptosis even without TNF-␣ treatment. Finally, a model is presented on the interplay between PKR and p53 in effecting TNF-␣-induced apoptosis in U937 cells.

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