SummaryIn a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (Aqtm). This A~ m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system ofapoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis: glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic A~,n disruption, BA prevents a number of phenomena linked to apoptosis: depletion ofnonoxidized glutathione, generation of reactive oxygen species, translocation of NFKB, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic Aqr,, disruption is secondary to transcriptional events. These data connect the PT control point to the p53-and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.

Apoptosis and particularly Fas-mediated apoptosis has been proposed to play a key role in controlling monocyte homeostasis. We and others have documented the regulatory function of human growth hormone (hGH) on monocytic cells, which prompted us to investigate the role of hGH on their response to Fas antigen cross-linking. Using human promonocytic U937 cells constitutively producing hGH upon gene transfer and human primary monocytes cultured in the presence of recombinant hGH, we demonstrated that hGH diminished Fas-mediated cell death by enhancing the expression of the antiapoptotic oncoprotein Bcl-2 as well as the level of bcl-2alpha mRNA. In parallel, we established that overexpression of Bcl-2 through gene transfer into normal U937 cells also diminished Fas-induced apoptosis. Further, as a result of Bcl-2 overexpression, we found that hGH greatly depressed Fas-induced activation of the cysteine protease caspase-3 (CPP32), which in turn affected the cleavage of poly(ADP-ribose) polymerase. Altogether, these data provide evidence that hGH mediates its protective effect through a Bcl-2-dependent pathway, clearly a crucial step in enhanced survival of monocytic cells exposed to Fas-induced death.

Chemotherapy remains the main tool for the treatment of cancers, but is often hampered by tumor cell resistance. In this context, the transfer of genes able to accentuate the effect of anticancer drugs may constitute a useful approach, as exemplified by inactivation of nuclear factor ( NF ) -B via direct transfer of a gene encoding a negative dominant of its natural inhibitor IB, leading to improved response to cancer chemotherapy. Following our previous report that transfection of human growth hormone ( hGH ) gene into human monocytic cell lines may also inactivate NF -B in another situation, we decided to test the consequences of hGH gene transfer on cancer treatments. We demonstrated that hGH -transfected human myeloid leukemia U937 cells were sensitized to an apoptotic signal mediated by the anticancer drugs. In parallel, we found that, by inhibiting degradation of IB, hGH gene transfer diminished NF -B entry into the nuclei of U937 cells exposed to daunorubicin. Finally, we report that hGH -transfected tumor cells engrafted in nude mice responded in vivo to chemotherapy with nontoxic doses of daunorubicin whereas, under the same conditions, control tumor cells remained insensitive. Overall, this study therefore suggests that hGH gene transfer may offer new therapeutic prospects in cancer therapy.