A Short Caspase-3 Isoform Inhibits Chemotherapy- Induced Apoptosis by Blocking Apoptosome Assembly Fre´ de´ rique Ve´ gran1,2,3, Romain Boidot1,2, Eric Solary2,3, Sarab Lizard-Nacol1,2* 1 Unit of Molecular Biology - Centre Georges Franc¸ois Leclerc, Dijon, France, 2 Federative Institute of Research IFR ‘‘Sante´-STIC’’ - University of Burgundy, Dijon, France, 3 UMR-INSERM U-866, Dijon, France Abstract Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and a-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments. Citation: Ve´gran F, Boidot R, Solary E, Lizard-Nacol S (2011) A Short Caspase-3 Isoform Inhibits Chemotherapy-Induced Apoptosis by Blocking Apoptosome Assembly. PLoS ONE 6(12): e29058. doi:10.1371/journal.pone.0029058 Editor: Nai Sum Wong, University of Hong Kong, Hong Kong Received April 27, 2011; Accepted November 20, 2011; Published December 22, 2011 Copyright: 2011 Ve´gran et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Slizard@cgfl.fr Introduction Caspases are a family of evolutionary conserved cysteine proteases that play a central role in a majority of apoptotic cell death pathways. Death signals activate the proteolytic cascade of caspases through two main pathways, i.e. an extrinsic pathway that starts at the level of plasma membrane death receptors and an intrinsic pathway that activation is a response to irreversible cellular damage [1]. Both pathways converge to the activation of caspase-3, the closer homolog of the in Caenorhabditis elegans CED-3 [2]. Procaspase-3 is a 32 kDa caspase-3 zymogen (also known as CPP32). CPP32 exists in the cells as inactive dimmers. Its proteolytic cleavage on C-terminal side of aspartate residues eliminates the pro-domain, separates the remaining protein into a large and a small subunit and generates an active tetramer constituted by two large and two small subunits. In turn, caspase-3 activates downstream enzymes of the caspase family and contributes with them to generate the characteristic apoptotic cell death phenotype. Activation of caspase-3 is required for membrane bleeding and internucleosomal DNA fragmentation that occur during apoptosis. Caspase activity is regulated at several levels, including gene transcription and post-translational modifications. The alternative splicing of caspase genes generates full-length and truncated proteins whose functions can be antagonistic [3]. This character- istic maintains the threshold of response to certain levels of stimuli [4]. The human Caspase-3 gene is located on 4q33-q35.1 and possesses 2635 base pairs leading to 7 exons. Its alternative splicing generates two transcripts, caspase-3 and caspase-3s that were detected in all the studied tissues [5] and may have different apoptotic activities [5,6]. The principal mRNA variant, caspase-3, is 834 base-long and the short transcript, caspase-3s, has lost the sixth exon leading to a loss of 122 bases (representing 95 amino- acids initially present in the procaspase-3 protein). The lost sequence includes the short subunit and the C-terminal part of the long subunit in which is located the QACRG motif that participates in the formation of the catalytic site. The caspase-3s protein has around a 20 KDa molecular weight and expression of the short isoform of caspase-3 in 293T cells prevents DNA fragmentation and poly(ADP-ribose) polymerase 1 (PARP1) cleavage in response to an apoptotic stimulus [5]. The role of caspase-3 in the response of breast cancer cells to chemotherapeutic drugs remains a controversial issue. The lost of caspase-3 expression as well as defaults in cytochrome c release from the mitochondria, which is requested in most apoptotic pathways to activate caspase-3 through caspase-9 activation, are associated with multidrug resistance. Accordingly, expression of caspase-3 in the human MCF-7 breast tumor cell line (which is deficient for caspase-3) restores the apoptotic response to the topoisomerase II inhibitor, etoposide [7–9]. Caspase-3 was also involved in breast cancer cell apoptosis upon exposure to anthracyclines [9–12] and cisplatin [13–16]. Its role in tumor cell response to paclitaxel has been challenged [17–24]. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29058