Pergamon Lcwkemia Research Vol. 21, No. 2, pp. 163-172, 1497 Copyright 8 1997 Published by Elsevier Science Ltd. All rights reserved Printed m Great Brxain “1452126,97 $17.00 + 0.00 PII: SO145-2126(96)00102-6 STUDY OF THE APOPTOSIS INDUCED IN VITRO BY ANTITUMORAL DRUGS ON LEUKAEMIC CELLS Jean-Philippe Vial*, Francis Belloc’f, Patrice Dumaint, Sabine Besnard*, Francis Lacombef, Michel R. Boisseaut, Josy ReiffersS and Philippe Bernard* *Laboratoire Universitaire d’Hematologie, Universite de Bordeaux II, 33000 Bordeaux, France, TLaboratoire d’Hematologie, Hopital Ham Leveque, 33604 Pessac, France; and $Laboratoire de Greffe de Moelle, URA CNRS 1456, Universite de Bordeaux II, 33000 Bordeaux, France (Received 14 March 1996. Revi.rion accepted 23 September 1996) Abstract-A flow cytometric method for simultaneous apoptotic cell detection and cell cycle analysis was applied on the U937 cell line. Four antitumoral drugs currently used in the treatment of acute myeloid leukaemia were studied in vitro: DNR, IDR, MIT0 and Ara-C. Our results show a dissociation between the cytostatic effect (the block in the cell cycle observed for low drug concentrations) and the cytotoxic effect (the induction of apoptosis induced by higher concentrations) for all the tested molecules. Low concentrations of Ara-C induced a block in the S phase while higher concentrations (>lO-’ M) induced apoptosis at the Gl-S boundary. Low concentrations of anthracyclines (~40 nM DNR and ~20 nM IDR) induced a block in G2 without apoptosis. Apoptosis was induced in Gl and/or early S phases by higher concentrations of anthracyclines. The concentration inducing 50% apoptosis (ICY,-,) was found to be, respectively, 200 and 40 nM for DNR and IDR. Analysis of MITO-treated cells showed a parallel increase in the percentages of S phase and apoptotic cells. However, the bivariate analysis showed that apoptosis did occur in a population with Gl DNA content. For two other drugs (CAM and COLC), apoptosis occurred for the same concentrations and in the same phase as the block (in S and G2M, respectively). The 1c5a of MIT0 was found to be 100 nM. Cotreatment of the cells with colchicin and either Ara-C or IDR showed that the passage through mitosis was not necessary for the completion of apoptosis at the Gl-S boundary. Short incubations of U937 cells with high concentrations of anthracyclines were found to be efficient in inducing further apoptosis. We conclude that, for all the assayed molecules, the cytotoxic and/or cytostatic effects of the antitumoral drugs tested greatly depend on the concentrations used and that, depending on their in viva pharmacokinetics, the induction of apoptosis could be an important mechanism of action for some of these drugs. c 1997 Published by Elsevier Science Ltd. All rights reserved. Key words: Leukaemic cells, apoptosis, cell cycle, flow cytometry, antitumoral agents. Introduction Apoptosis, or active cell death, is a specific mode of cell death, which is characterized by morphological changes such as chromatin condensation, fragmentation of the nucleus, cytoplasmic retraction and emission of ‘apop- totic bodies’ containing apparently intact organelles [l-5]. Indeed, apoptosis governs a mechanism of cell death that involves the active participation of the affected cell in carrying out the program of its own demise. The morphological and biochemical events of apoptosis have been known for a long time, and, in some respects, differ from those leading to necrosis [6, 71. Apoptosis occurs in physiological conditions as a regulatory mechanism of tissue growth, where it balances cell proliferation [8]. Moreover, several studies Abbreviations: DNR, daunorubicin; IDR, idarubicin; MITO, mitoxantrone; Ara-C, aracytine; CAM, camptothecin; COLC, colchicin; ,cSar 50% inhibiting concentration; FCS, fetal calf serum; H342, Hoechst 33342; PI, propidium iodide. Correspondence to: F. Belloc, Laboratoire d’Htmatologie, Hopital Haut LCv&que, Avenue Magellan, 33604 Pessac, in experimental hematology strongly suggest that programmed cell death plays a role in normal hemato- poiesis in relation with hematopoietic growth factors [9]. Apoptosis has become a focus of interest in oncology owing to its dysregulation, which could help to explain France (Tel: (33) 56 55 64 64; Fax: (33) 56 55 68 09). the pathogenesis of some malignant tumors [lo-121. 163