Tumor Suppressor p53 as a Component of the Tumor Necrosis Factor-induced, Protein Kinase PKR-mediated Apoptotic Pathway in Human Promonocytic U937 Cells

Abstract

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.

Double-stranded RNA-dependent Protein Kinase Phosphorylation of the α-Subunit of Eukaryotic Translation Initiation Factor 2 Mediates Apoptosis

“…It has been proposed that PKR mediates apoptosis at the transcriptional level. For example, PKR signals through STAT1 and STAT3 (36,43,44), interferon regulatory factor 1 (IRF1) (16,45), IRF3 (46), p53 (17,47,48), and the IKK complex to regulate transcription during proinflammatory (16,17,40,45) and/or proapoptotic responses (12,40,45,49). PKR-dependent apoptosis was also associated with Fas-associated death domain (FADD)-mediated activation of caspase 8 (50,51) and up-regulation of Fas and Bax (18,52,53).…”

Genetic deletion of PKR abrogates TNF-induced activation of IκBα kinase, JNK, Akt and cell proliferation but potentiates p44/p42 MAPK and p38 MAPK activation

“…Indeed, we found that TNF-induced cell proliferation was significantly suppressed by PKR deletion. Although PKR is known to mediate TNFinduced apoptosis in certain cells (Yeung and Lau, 1998), its role in TNF-induced proliferation of cells is not known. The difference in results may be owing to cell types used; human myeloid U-937 cells used by Yeung and Lau (1998) may have multiple genetic defects, as compared to PKR-deleted MEF used in our studies.…”

“…Although PKR is known to mediate TNFinduced apoptosis in certain cells (Yeung and Lau, 1998), its role in TNF-induced proliferation of cells is not known. The difference in results may be owing to cell types used; human myeloid U-937 cells used by Yeung and Lau (1998) may have multiple genetic defects, as compared to PKR-deleted MEF used in our studies. Consistent with our results, Donze et al (2004) showed that PKR activates a cell survival pathway through the expression of NF-kB-regulated cIAP and A20 gene products.…”

p53 and Tumor Necrosis Factor α Regulate the Expression of a Mitochondrial Chloride Channel Protein

“…Previously, interactions of these two pathways have been documented. p53 was induced during apoptosis mediated by TNF-␣ in U987 cells (43) and in MCF-7 cells (44); in both cases, the presence of wild-type p53 was shown to be essential for the cytotoxic action of TNF-␣. In ME-180 cells, TNF-␣ induced apoptosis leads to a rapid accumulation of p53 protein with no induction of p21/WAF1 (45).…”