Reported here are representative samples (samples 1, 3, 4, and 6) showing a variable induction of p53 and MDM-2, and one sample (sample 5) showing a lack of induction of p53 and MDM-2. and 4) a RING finger and an E3 ligase website that are responsible for the ubiquitination of p53 [2,3]. Recently, Nutlin-3, a small-molecule inhibitor of MDM-2, has been developed . Nutlin-3 binds to MDM-2, liberating p53 from bad control and leading to effective p53 stabilization and activation in cells with wild-type, but not mutant or erased, [4,5]. Of notice, recent studies possess shown that Nutlin-3, used alone or in combination with chemotherapeutic medicines, effectively increases the degree of apoptosis in acute myeloid leukemia (AML), as well as in additional hematologic malignancies characterized by wild-type [6C9]. Even though therapeutic effect of most standard anticancer medicines has been attributed for years to their ability to induce apoptosis, it has been identified that growth arrest with morphologic features reminiscent of SRPKIN-1 terminal maturation constitutes an alternative drug-induced response system controlled, at least in part, from the p53 pathway . Therefore, the experiments illustrated with this study were designed to investigate whether Nutlin-3, used only or in combination with the death-inducing ligand TNF-related apoptosis-inducing ligand (TRAIL), was able to modulate the maturation of main blasts acquired by AML individuals. Moreover, we have performed a series of experiments in the wild-type and treatments. The AML individuals’ cells, as well as the wild-type SKW6.4 cell lines, were cultured in RPMI 1640 (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco) and WAF1 seeded SRPKIN-1 at an optimal cell density of 0.8 x 106 to 1 1.0 x 106 cells/ml before treatments. Table 1 Clinical and Laboratory Features of AML Individuals. Bad Control siRNA were used to demonstrate the transfection did not induce nonspecific effects on gene manifestation. A cocktail of two different bad control siRNA, each composed of a 19-bp scrambled sequence with 3 dT overhangs, was used. The sequences have no significant homology to any known gene sequence from humans and have been previously tested for lack of nonspecific effects on gene manifestation (Ambion). Statistical Analysis The results were evaluated using analysis of variance, with subsequent comparisons by Student’s test and Mann-Whitney rank-sum test. Statistical significance was defined as .05. Results Nutlin-3 Induces Variable Levels of Cytotoxicity in Main AML Blasts To investigate the effect of Nutlin-3 on cell viability, AML samples freshly isolated from individuals free SRPKIN-1 of therapy were incubated with predetermined ideal concentrations of Nutlin-3 (10 M) and were assayed for viability by trypan blue dye exclusion at 72 hours of treatment (Table 1). During this time frame, the number of viable cells in untreated ethnicities remained relatively constant, never shedding below 70% compared to the cell number seeded at time 0 (1 x 106 cells/ml). On Nutlin-3 treatment, a variable decrease in cell viability (61 18.5; mean SD of the percentage of cell viability compared with untreated controls; Table 1) was observed, with all patient samples being susceptible to Nutlin-3 cytotoxicity, with the exception of patient SRPKIN-1 SRPKIN-1 5. The decrease in cell viability induced by Nutlin-3 was paralleled by a concomitant increase in the percentage of apoptosis, evaluated in terms of Annexin V-positive cells (data not demonstrated) and/or PARP cleavage (Number 1) with respect to control cultures. Open in a separate window Number 1 p53 and MDM-2 induction in response to Nutlin-3 in main AML cells. AML cells were left untreated or were incubated with Nutlin-3 (10 M) before Western blot analysis. Reported here are representative samples (samples 1, 3,.