The p53 protein directly regulates the expression of the WAFl (wild-type p53activated fragment 1) protein which is a cyclin-dependent kinase inhibitor (CDKI). DNA damaging agents such as ionizing or UV radiation, and some chemical agents induce WAF1 in wild-type p53 containing cells, thereby halting cell cycle progression. WAF1 expression is also induced through a p53-independent pathway. Tumor necrosis factor a (TNFa) is a cytotoxic/cytostatic compound for some human cancer cells. We examined a series of myeloid leukemic cell lines that expressed either no p53 (HL-60, K562) or mutant inactive p53 (KG-1, KCL22, THP-1, U937). The KG-1, HL-60, K562, and KCL22 myeloid leukemic cells increased their levels of WAF1 mRNA in the presence of TNFa. We focused on KG-1 cells to determine how TNFa modulated WAF1 expression. WAFl mRNA increased in a dose-dependent manner in the cells after exposure to increasing concentrations of TNFa, and this increase occurred in the absence of new protein synthesis. An increase of WAF1 protein and a concominant decrease of cyclindependent kinase 2 activity also was found in KG-1 cells. Flow cytometry using 5-bromo-2'-deoxyuridine showed an increase in the proportion of TNFa-treated KG-1 cells in the GdG, phase of the cell cycle. TNFa enhanced the rate of WAFl transcription only 1.4-fold in TNFa-treated KG-1 cells as compared to untreated cells. Notably, however, the half-life (ti) of WAF1 mRNA in TNFa-treated cells was 2.5 hours as compared to 0.5 hours in untreated cells. These results indicate that TNFa increases WAFl levels at least in part via a posttranscriptional stabilization of the mRNA; and T N F a may mediate its cytostatic effects through WAFl in some cell types. o 1996 WiIey-Liss, Inc. 0 1996 WILEY-LISS, INC their growth, suggesting that WAF1 is a tumor suppressor gene (El-Deiry et al., 1993). However, we have not identified any alterations of WAFl in a variety of cultured or uncultured cancers (Shiohara et al., 19941, suggesting that wild-type WAFl may play an essential role in cell viability. Expression of WAFl is induced in wild-type p53-containing cells by exposure to DNA damaging agents like doxorubicin or ionizing radiation, but this induction does not occur in mutant p53-containing cells (El-Deiry et al., 1994). Studies showed that induction of expression of WAFl protein occurred in cells undergoing either p53-associated GI arrest or apoptosis, but not in cells induced either to arrest in GI or to undergo apoptosis through p53-independent mechanisms.
Recently, p53-independent induction of WAF1 was described in embryonic fibroblasts from p53 "knock-