Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.

Abstract

Abstract. In this paper we used a multiparametric approach to analyze extensively the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure and function are occurring in early stages of apoptosis. A multiparametric quantitative analysis was performed on normal or apoptotic thymocytes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scattering properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apoptotic cells) allowed a precise analysis of the preapoptotic and apoptotic stages. Moreover a thorough study of mitochondrial transmembrane potential (Aq~m) assessed following in a time course study the uptake by apoptotic cells of the cationic lipophilic dye DiOC6(3) or the J-aggregate-forming cation JC-1, indicates that a drop in Atem occurs very early in thymocyte apoptosis, before DNA fragmentation. This is associated with alteration in mitochondrial structure assessed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function occurring in early stages of apoptosis were confirmed by confocal and electron microscopy analysis. p ROGRAMMED Cell Death (PCD) 1 of thymocytes is a crucial event that is involved in the negative intrathymic selection of the T cell repertoire, leading to the clonal deletion of autoreactive T cells and to the establishment of self-tolerance (10, 4). This death of physiological significance, called apoptosis, is an active process of self-destruction associated to profound structural changes including a nuclear collapse characterized by the condensation of chromatin and fragmentation of DNA into single and multiple oligonucleosomes leading to a final and irreversible cell destruction (2,20,46).It was stated that no marked changes in energy metabo-

A novel indole ethyl isothiocyanate (7Me-IEITC) with anti-proliferative and pro-apoptotic effects on platinum-resistant human ovarian cancer cells

“…The ADP:ATP ratio and ΔΨm can be used as an indicator of apoptosis [22,23]. Moreover, the loss of ΔΨm due to chemical agents for other drug-treated cell types has been reported to be indicator of early apoptosis and as the first irreversible step in the induction of apoptosis [24]. Accordingly, loss of the ΔΨm within 3hrs in SKOV-3 following 7-MeIEITC treatment may be the first irreversible step in the induction of apoptosis by this agent.…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…Accordingly, the ratio of red-to-green JC-1 fluorescence can be used as a sensitive measure of the m , that is relatively independent of putative interfering factors, including mitochondrial mass, shape or density [85][86][87]. DiOC 6 (3) has become the most commonly used carbocyanine fluorochrome for the cytofluorometric measurement of m [88], due to its rapid mitochondrial equilibration and negligible quenching effects, at least at low concentrations (recommended dose 10-20 nM) [77,89,90]. However, higher concentrations of DiOC 6 (3) result in relevant quenching and in the staining of non-mitochondrial compartments, such as the endoplasmic reticulum.…”

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

“…For example, strong evidences suggest that CL participates in the interaction of cytochrome c with the outer face of the inner mitochondrial membrane, thereby ensuring the presence of a pool with limited solubility close to respiratory complexes III and IV, increasing the efficiency of electron transport. 10 During the past decade, it has become unequivocally clear that most proapoptotic stimuli require a mitochondria-dependent step involving the disruption of mitochondrial bioenergetics 11,12,13,14 and outer membrane permeabilization, 15,16 leading to the release of apoptogenic factors, including cytochrome c.…”