The subject of this thesis was to study the sensitivity of mitochondrial dysfunction harbouring cells to staurosporine-induced apoptosis. The work is divided into two chapters, each one concerning a particular mitochondrial dysfunction. The first part is devoted to the sensitivity of 143B human osteosarcoma cells compared with the mtDNA-depleted 143B ρ0 cells to staurosporine-induced apoptosis. Treating these two cell lines with staurosporine, we first showed that the mtDNA-depleted 143B ρ0 are more sensitive to staurosporine than the parental cell line. Trying to unravel the origin of this hypersensitivity, we first looked for staurosporine-induced cell death mechanisms. Staurosporine induces cytochrome c release and caspases activation in both cell lines, initiating cell death by the well-described intrinsic pathway of apoptosis. However, in 143B ρ0 cells, the anti-apoptotic proteins Bcl-2, Bcl-XL and Mcl-1, known to inhibit the mitochondrial outer membrane permeabilization, are less abundant than in 143B cells, sensitizing the mtDNA-depleted cells. Moreover, we show that in 143B ρ0 cells, staurosporine induces the permeabilization of the lysosomal membrane and the release of cathepsin B in the cytosol, where it participates to the initiation of cell death. The second part of this work is dedicated to the sensitivity of cybrid cells derived from 143B human osteosarcoma cells harbouring or not the A8344G mutation in the mtDNA, responsible for the MERRF syndrome (Myoclonic Epilepsy with Ragged Red Fibers). Apoptosis was induced in the wild type (WT) and in the MERRF mutated cybrid cells with staurosporine, and as for the mtDNA-depleted 143B cells, the cybrid cells harbouring the A8344G mutation in the mtDNA are more sensitive to staurosporine than their WT counterpart. However, the mechanisms of cell death are different. Indeed, in WT and MERRF mutated cybrid cell lines, staurosporine induces caspases activation in the absence of cytochrome c release. In these cells, caspases are activated, at least partially, by the calcium-activated proteases calpaines, through the initial activation of caspase-4. Following staurosporine treatment, the cytosolic calcium concentration increases in both cell lines, although more rapidly in the mutated ones. It thus seems that the hypersensitivity of A8344G harbouring cybrid cells to staurosporine-induced apoptosis is due to an altered calcium homeostasis. However, our results show that the calcium-calpain-caspase-4 pathway is only partially involved in staurosporine cell death, and is not the only pathway activated by staurosporine to induce cell death. Preliminary results suggest that Reactive Oxygen Species, mitochondrial membrane potential and maybe autophagy may also be involved in staurosporine-induced cell death and participate to the hypersensitivity of MERRF mutated cybrid cells to staurosporine-induced apoptosis.
Etude de l’impact d’un dysfonctionnement mitochondrial sur la réponse apoptotique de cellules humaines en culture
Rommelaere, G. (Author). 14 Jun 2012
Student thesis: Doc types › Doctor of Sciences