MCF-7 breast carcinoma cells do not express caspase-3

Abstract

Apoptosis, a fundamental process essential for normal tissue homeostasis and development, is closely associated with the activation of a class of aspartate-specific cysteinedependent proteases, called caspases, that lead to the demise of the cell via limited proteolysis of a multitude of cellular substrates [1, 2]. Caspases are expressed as inactive zymogens that become activated upon cleavage by other caspases in a so-called caspase activation cascade, or by mere oligomerization instigated by the formation of large multi-protein complexes such as the death-inducing signaling complex (DISC) or the apoptosome [3, 4]. Whereas DISC formation occurs via the so-called extrinsic death pathway that is instigated by activation of members of the death receptor family such as CD95, tumor necrosis factor receptor (TNF-R) or the receptors of the TNF-Rrelated apoptosis-inducing ligand (TRAIL), the apoptosome is formed following activation of mitochondria and is hence termed the intrinsic or mitochondrial death pathway. Based on their order in cell death pathways, caspases can be divided into initiator (caspase-2, -8, -9, and -10) and effector (caspase-3, -6, and -7) caspases. Among them, caspase-3, a member of the latter group, is absolutely crucial for apoptosis induction, as this enzyme is not only activated downstream of both the extrinsic and intrinsic death pathway, it is also responsible for the cleavage of the majority of substrates known so far [1, 5]. More importantly, with the proteolysis of discrete substrates, caspase-3 evokes some of the typical morphological and biochemical alterations associated with apoptosis. For instance, whereas the caspase-3-mediated cleavages of a-fodrin, gelsolin, rho-associated kinase-1 (ROCK-1) and p21-activated kinase 2 (PAK2) contribute to membrane blebbing, cleavage of the inhibitor of the caspase-activated DNase (ICAD) leads to the typical DNA fragmentation pattern observed in apoptosis [1]. Furthermore, with the cleavagemediated activation of the calcium-independent phospholipase A2 and subsequent production of the chemotactic phospholipid lysophosphatidylcholine, caspase-3 appears to be also responsible for the generation of so-called ‘‘eatme’’ signals that induce migration of phagocytes to the site of apoptotic cell death [6]. Thus, caspase-3 not only instigates and pursues the demise of a cell, but, in addition, makes sure that the corpse is properly disposed, a function crucial for avoiding inflammatory processes. Hence, determination of the processing and activation of caspase-3 are common means to assess apoptotic signal transduction pathways in numerous cell lines of varying origin. Curiously, several reports still claim the presence of caspase-3 in the breast carcinoma cell line MCF-7 in which this enzyme is supposed to contribute to apoptosis signaling [7–15]. This was not only demonstrated indirectly via fluorometric assay systems measuring caspase-3-like activities—that might be also elicited by the closely related caspase-7—but by Western blotting analyses demonstrating directly the presence of this protease in MCF-7 cells. However, in addition to the lack of caspase-10 [16], an initiator caspase in the extrinsic death pathway, MCF-7 cells do also not express caspase-3 [17]. Ten years ago, we demonstrated unambiguously that the lack of caspase-3 in these cells is caused by a 47-base pair deletion within exon 3 of the CASP3 gene resulting in the skipping of this exon during premRNA splicing and introduction of a premature stop codon at position 42 that completely abrogates translation of the CASP-3 mRNA [17]. Although caspase-3-deficient MCF-7 R. U. Janicke (&) Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, University of Dusseldorf, Universitatsstrasse 1, 40225 Dusseldorf, Germany e-mail: Janicke@uni-duesseldorf.de