Aminoflavone (AF, NSC 686288) is beginning clinical trials. It induces replication-mediated histone H2AX phosphorylation, DNA-protein crosslinks and activates p53. Here, we studied p21 CIP1/WAF1 and Mdm2 responses to AF. Although p53 stabilization and phosphorylation at serine 15 increased with dose and time of exposure, Mdm2 and p21 CIP1/WAF1 protein levels displayed a biphasic response, as they accumulated at submicromolar doses and then decreased with increasing AF. As both Mdm2 and p21 CIP1/WAF1 mRNA levels increased with AF concentration without reduction at higher concentrations, we measured the half-lives of Mdm2 and p21proteins. Mdm2 and p21 CIP1/WAF1 half-lives were shortened with increasing AF concentrations. Proteasomal degradation appears responsible for the decrease of both Mdm2 and p21 CIP1/WAF1, as MG-132 prevented their degradation and revealed AF-induced Mdm2 polyubiquitylation. AF also induced protein kinase B (Akt) activation, which was reduced with increasing AF concentrations. Suppression of Akt by small interfering RNA was associated with downregulation of Mdm2 and p21 CIP1/WAF1 and with enhanced apoptosis. These results suggest that the cellular responses to AF are determined at least in part by Mdm2 and p21 CIP1/WAF1 protein levels, as well as by Akt activity, leading either to cell cycle arrest when Mdm2 and p21 CIP1/WAF1 are elevated, or to apoptosis when Mdm2 and p21 CIP1/WAF1 are degraded by the proteasome and Akt insufficiently activated to protect against apoptosis.

The effects of monocytic/macrophage and granulocytic differentiation induced by phorbol myristate acetate (TPA) and all-trans retinoic acid, respectively, were tested on the induction of apoptosis in human promyelocytic leukemia HL-60 cells treated with topoisomerase I and II inhibitors. Using a filter-binding assay, we observed a strong inhibition of DNA fragmentation induced by 3- and 24-hour continuous exposure to camptothecin, VP-16, VM-26, and m-AMSA in TPA- differentiated cells. The inhibition of the typical internucleosomal DNA fragmentation was confirmed by agarose gel electrophoresis. By contrast, drug-induced DNA fragmentation was not inhibited in retinoic acid-differentiated cells, and apoptosis occurred in these cells after 4 to 5 days in the absence of drug treatment. The TPA inhibitory effect was maximal after 24 hours of treatment and was correlated with differentiation, because phorbol dibutyrate ester was active, whereas 4- alpha-TPA, a nontumor promoter that does not induce differentiation, was not active. Using alkaline elution, we observed that TPA and retinoic acid differentiation were associated with changes in topoisomerase-mediated DNA breaks that were not correlated with their differential effects on drug-induced DNA fragmentation. Moreover, TPA also inhibited DNA fragmentation induced by vinblastine, cycloheximide, calphostin C, and x-rays. Using a cell-free system, we observed that DNA fragmentation was not inhibited in nuclei from TPA-differentiated cells. Rather, inhibition of apoptosis seemed to take place in the cytoplasm. We conclude that phenotypic changes associated with TPA- induced differentiation include inactivation of a cytoplasmic activity that can induce DNA fragmentation associated with apoptosis.