Regulación de la expresión de B-F1-ATPasa y función supresora de tumores de la H+-ATP sintasa

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 22-06-2004The mitochondrial H'-ATP synthase is required for the provision of cellular energy and for the efficient execution of apoptosis. In this work we have studied three main issues in relation with (i) the expression of the H+-ATP synthase in cancer. (ii) the role of the Ht-ATP synthase in cell death and (iii) the regulation of the expression of the Ht-ATP synthase in cells of mammals. We have analyzed the expression level of the P-catalytic subunit of the Ht-ATP synthase (P-F1- ATPase), as well as other mitochondrial and glycolytic markers, in paired normal and tumour biopsies of patients bearing colon and gastric adenocarcinomas. We show that carcinogenesis in these tissues promotes significant alterations of the mitochondrial proteome and, specifically, the repression of the expresion of the p-F1-ATPase when compared to the expression level in normal tissue biopsies. These findings suggest that the bioenergetic competente of mitochondria in human cancers of the colon and stomach is impaired. thus supporting the hypothesis put forward by Otto Warburg at the beginning of previous century. This alteration might be involved in the resistance and compromised apoptotic potential of cancer cells in these tumours. We have used staurosporine to trigger cell death in various rat liver cell lines (C9, FA0 and AS30D) that have different dependency on mitochondrial oxidative phosphorylation for the provision of cellular energy (FAO>C9>AS30D). Staurosporine induces apoptosis in C9 and FA0 cells through caspasedependent and independent mechanisms. The AS30D cells are resistant to staurosporine-induced apoptosis. Oligomycine, a specific inhibitor of the H'-ATP synthase, prevents staurosporine-induced apoptosis in C9 and FA0 cells suggesting a relevant role for the H'-ATP synthase in the death signalling cascade by the mitochondrial pathway. However, oligomycine is not able to prevent the rapid swelling of mitochondria and the release of apoptogenic molecules from the organelles after staurosporine treatment. Staurosporine-induced apoptosis in liver cells promotes the rapid generation of reactive oxygen species (ROS) by mitochondria as a function of its dependence on mitochondrial oxidative phosphorylation (FAOX9). Likewise, staurosporine-induced apoptosis promotes extensive covalent modification of mitochondrial proteins. 60th the production of ROS and the modification of mitochondrial proteins is prevented by oligomycine treatment of staurosporine primed cells. These results suggest that the mitochondrial Ht-ATP synthase controls the production of ROS and therefore, the extent of cellular damage. after a toxic cellular insult further providing a mechanistic explanation for the diminished apoptotic potential of cancer cells and for the contribution of the H'-ATP synthase in cancer progression. Finally, in previous studies we have shown that the assembly of the ribonucleoprotein complex containing the mRNA of the p-subunit of the mitochondrial Ht-ATP synthase requires the participation of two dista1 cis-acting elements (B1.2 and 3'PUTR) and a complex set of cellular trans-acting proteins. In this study we demonstrate that the p1.2 element acts as a repressor of P-mRNA translation by its ability to interact with the 5'-UTR of the mRNA. In addition, we show that the in vivo translational activity of spliced chimeric PmRNAs is much higher than the non-spliced variants, strongly supporting that the nuclear history of the transcript is involved in defining its further metabolic fate