EXCLI Journal 2008;7:163-168 – ISSN 1611-2156 Received: July 30, 2008, accepted: September 05, 2008, published: October 16, 2008 163 Original article: Mitochondrial DNA might be influenced in calprotectin-induced cell death Sayed-Amir Marashi 1,†, *, Mostafa Rezaei-Tavirani 2,4, *, Hakimeh Zali 3,5 , Mohammad Ali Shokrgozar 5 1 Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran 2 Clinical Proteomics Research Center, Faculty of Paramedical Sciences, Shahid Beheshti University (M.C), Tehran, Iran 3 Department of Molecular Biology, Khatam Universiy, Tehran, Iran 4 Skin Research Center, Shahid Beheshti University (M.C), Tehran, Iran 5 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran †: Present address: IMPRS-CBSC, Max Planck Institute for Molecular Genetics, Ihnestr. 63-73, D-14195, Berlin, Germany. * Corresponding authors: e-mail address: rezaei.tavirani@ibb.ut.ac.ir (M. Rezaei-Tavirani); marashi@molgen.mpg.de (S.-A. Marashi) ABSTRACT It is generally believed that calprotectin acts via exclusion of extracellular zinc, and/or bind- ing of calprotectin to a cell membrane receptor, and consequently, activation of a signaling pathway for apoptosis. Recently, we suggested that calprotectin may have an “internal target” within cells. Here, using target theory, we provided evidence that this internal target is DNA. Trypan blue (TB) and dimythylthiazol diphenyl tetrazolium bromide (MTT) assays were used to estimate survival of calprotectin-treated cells. TB assay relies on the viability of cell mem- brane, while MTT assay relies on the functionality of mitochondria. We demonstrated that MTT-based survival values fit to the “single-hit, single-target” model, while TB-based sur- vival values are best matched to the “single-hit, multi-target” model with N=2. Assumption of DNA as the target of calprotectin is fully consistent with the models, since each mitochon- drion contains one chromosome and each “cell” is diploid and contains two chromosome sets. To the best of our knowledge this is the first report that suggests mitochondrial DNA is af- fected during calprotectin-induced cell death. Furthermore, our results explain why toxicity measures (like LD 50 ), when estimated based on TB assay, are sometimes significantly greater than toxicity measures based on MTT assay. Keywords: S100A8/A9; MRP8/14; Apoptosis; DNA damage; mtDNA; MTT assay; TB assay INTRODUCTION Apoptosis is a type of programmed cell death, in which cells die without releasing their contents to the environment. Some characteristics of apoptosis are ruffling and appearance of smooth-surfaced protuber- ances of the plasma membrane with its pre- served integrity, cellular shrinking, cyto- plasmic condensation, cytoskeletal disinte- gration and chromatin clumping and margi- nation (Fietta, 2006). In addition, a distinc- tive internucleosome DNA cleavage hap- pens during apoptosis (van Cruchten and van den Broeck, 2002). Calprotectin is a calcium- and zinc- binding protein heterodimer of S100 protein family (Hunter et al., 2002; Yui et al., 1995) which has apoptosis-inducing activ- ity against various tumor cells and normal cells (Yui et al., 1997). While calprotectin (S100A8/A9) has a central role in apop- tosis, calgranulin A (S100A8) and cal-