Apoptotic cell death 351 Scanning Microscopy Vol. 12, No. 2, 1998 (Pages 351-360) 0891-7035/98$5.00+.25 Scanning Microscopy International, Chicago (AMF O’Hare), IL 60666 USA APOPTOTIC CELL DEATH INDUCED BY DIFERENT TRIGGERING AGENTS MAY FOLLOW A COMMON ENZYMATIC PATHWAY Abstract Molt-4 human leukemia cells were triggered to undergo apoptosis by various agents with different mechanisms of action. Staurosporine [a protein kinase C (PKC) inhibitor], camptothecin (a topoisomerase I blocking drug), and tiazofurin [an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH)], were used. Ultrastructural analysis showed morphological changes characteristic for apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in all staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotis inducing agents with different mechanisms of action, induced the formation of large genomic DNA fragments and very similar ultrastructural changes. This could mean that these phenomena follow a pathway that is common to the three apoptosis-triggering agents, despite their different mode of action. Key words: Apoptosis, Molt-4 cells, ultrastructure, DNA electrophoresis, nick translation. *Address for correspondence: Elisabetta Falcieri Istituto di Anatomia Umana Normale Via Irnerio 48 40126 Bologna, Italy Telephone number: 39 51 243369 FAX number: 39 51 251735 E-mail: pietro@biocfarm.unibo.it F. Luchetti 1 , A. Di Baldassarre 2 , A.R. Mariani 3 , M. Columbaro 4 , C. Cinti 5 and E. Falcieri 5,6 * 1 Ist. Science Morfologiche, Univ. Urbino; 2 Ist. Morfologia Umana Normale, Univ. Chieti; 3 Ist. Anatomia Umana Normale, Univ. Bologna; 4 Lab. Biol. Cell. Microsc. Elettr., Ist. Ortop. Rizzoli, Bologna; 5 Ist. Citomorfologia Norm. Patol., CNR, Bologna; 6 Ist. Anatomia e Fisiologia, Univ. Urbino, Italy (Received for publication July 8, 1996 and in revised form October 25, 1996) Introduction Apoptosis is a physiologic phenomenon of cell death involved in the regulation of embryonic organ devel- opment and cell proliferation in normal and neoplastic tissues (Wyllie et al., 1980; Ellis et al., 1991; Kouri et al., 1992; Barr et al., 1994; Schwartz, 1995). Apoptosis appears in different cell types. It can also be experimentally induced in various cell types, and is accompanied by generally comparable structural changes (Falcieri et al., 1994c). The molecular events underlying the apoptotic be- haviour are mostly correlated to the activation of Ca 2+ /Mg 2+ dependent endonucleases, causing DNA cleavage in nucleosomic or oligonucleosomic fragments (Arends et al., 1990; Peitsch et al., 1993a,b). This pattern of DNA fragmentation, clearly identifiable by DNA agarose gel electrophoresis (Boe et al., 1991), has long been considered a hallmark of apoptosis. Recently, a number of cell types has been described in which, despite the presence of other apoptotic signs, DNA oligonucleosomic fragmentation does not take place (Collins et al., 1992; Falcieri et al., 1993; Fady et al., 1994). Larger DNA fragments, prior to or in the absence of internucleosomic cleavage, were reported (Oberhammer et al., 1993a,b; Walker et al., 1995; Falcieri et al., 1996; Weaver et al, 1996). Apoptosis thus can proceed via different pathways and it is therefore advantageous to use a multiple technical approach for the study of this process. In the present study, apoptosis was induced in human leukemia Molt-4 cells, and studied by different techniques. Three different agents, staurosporine, camp- tothecin and tiazofurin were used and compared. The role of cell metabolism and mechanism triggering the start and progression of apoptosis is discussed. Materials and Methods Cell culture and drug treatment Molt-4 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and were main- tained in RPMI1640 medium supplemented with 10% fetal