Flow cytometric detection of p53 protein after incubation of a pre-B cell line with antitumor agents

Background: The study of new substances capable of counteracting tumor deve lopment has focused, in recent years, on several of the steps in a cell's i nitiation of the process of apoptosis. One of the crucial events is the act ivation of p53, leading to a cell cycle G1/S block or to programmed cell de ath. Methods: We report here a parallel flow cytometric method for semiquantitat ive detection of p53 protein and apoptosis (percent of apoptotic cells) in a pre-B leukemic cell line (NALM-6) exposed to various antitumor agents (2. 35 mu g/ml etoposide; 0.175 mu g/ml FCE296; 0.4 mu g/ml FCE624; and 1.5 mu g/ml L-PAM). Results: hll of the substances proved to be capable of inducing an increase of p53 after 16 or 24 h of incubation. in all experiments with antitumor a gents we also found an onset of apoptosis after 24 h of incubation with the substance, as determined by the annexin V flow cytometric assay and by DNA fragmentation. Conclusions: This technique, based on now cytometric data of both p53 intra cellular content and percentage of apoptotic cells, is suitable to determin e the amount of antitumor agent needed to induce p53, and thus to dose the drug in relation to the sensitivity of a defined tumor as well as choose th e more efficacious drug, depending on cell responsiveness. The study of ant itumor substances that induce apoptosis, bypassing p53, could also be evalu ated by this method, in view of the development of substances for the treat ment of p53-mutated tumors. (C) 1999 Wiley-Liss, Inc.