Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells I. Tsoneva a, * , B. Nikolova a , M. Georgieva b , M. Guenova c , T. Tomov a , M.-P. Rols d , M.R. Berger e a Institute of Biophysics, Bulg. Acad. Sci., Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria b Department of Oncogenesis, National Oncological Center, Unit of Toxicology and Chemotherapy, Plovdivsko Pole 6, 1756 Sofia, Bulgaria c National Centre of Clinical and Transfusion Hematology, Lab. of Cytopathology and Flow Cytometry, Plovdivsko Pole 6, 1756 Sofia, Bulgaria d Institute of Pharmacologie, CNRS (UNRS5089), 205, Route de Narbonne, 31077 Toulouse Cedex 4, France e German Cancer Research Center, Unit of Toxicology and Chemotherapy, Im Neunheimer Feld 280, 69 120 Heidelberg, Germany Received 20 April 2004; received in revised form 21 July 2004; accepted 7 October 2004 Available online 22 October 2004 Abstract Cytochrome C (Cyt. C) is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. The linker Histone H1 is another basic protein with highly conservative structure, composition, and equal molecular weight, not changed during the evolution. An attempt was made to understand better the apoptotic processes by electroloading of leukemic cells, such as K562, HL-60, and SKW3, and human lymphocytes with positively charged proteins, such as Cyt. C, Histone H1, and methylated BSA albumin (mBSA). The triggering apoptotic processes followed by MTT test, FACS analysis, and DNA fragmentation after the electrotransfer of these proteins into the cells were observed. Histone H1 and mBSA induce the release of Cyt. C from rat liver mitochondria. Cytochrome C release was higher when mitochondria were in bhigh-energyQ state. It is supposed that release of Cyt. C from mitochondria is due to the mechanical rupture of the outer mitochondrial membrane, rich in negatively charged groups, predominately due to cardiolipin. The reason for the morphological rupture of the outer mitochondial membrane could be the rigidification and segregation of the membrane and the destroyed membrane asymmetries of both monolayers in the presence of positively charged proteins at higher linear charges such as Histone H1. We suggested that Histone H1, at a given moment of activated signaling for apoptosis, could be not transported to the nucleus and could lead to the release of Cyt. C from the mitochondria in the cytoplasm. It is temping to speculate that Histone H1 has other physiological extranuclear functions involved in apoptosis. D 2004 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Cytochrome C; Histone H1; methylated BSA (mBSA); Electroporation; Mitochondria 1. Introduction Apoptosis (programmed cell death) is an important process for maintaining homeostasis in multicellular organ- isms. All cells in such organisms have the capacity to undergo this form of death. The dysregulation of apoptosis is implicated in a number of human diseases, including cancer, autoimmune diseases, viral infections, neurodege- nerative diseases, AIDS, and cardiovascular diseases [1]. Cyt. C is a basic protein composed of 104 amino acids, including 19 lysine residues. Cyt. C is a mitochondrial protein inducing apoptosis when it is accumulated in the cytosol by a currently unknown mechanism, but regulated by the bcl-2 family of proteins. Recent reports demonstrated that the electroloaded or microinjected Cyt. C into the cells could activate apoptosis [2–5]. There are data that apoptosis induction could be observed without accumulation of Cyt. C in the cytosol and the above process is a cell type- and inducer-dependent phenomenon [6]. On the other hand, the linker Histone H1 is another basic protein with highly conservative structure, composition, and 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.10.002 * Corresponding author. Tel.: +35 92 9792622; fax: +35 92 9712493. E-mail address: itsoneva@obzor.bio21.bas.bg (I. Tsoneva). Biochimica et Biophysica Acta 1721 (2005) 55 – 64 http://www.elsevier.com/locate/bba