Apoptose et cytometrie La Grande Motte, 12-13 octobre 2000. Resumes

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : Y 32962 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Apoptosis is antagonized by large T antigens in the pathway to immortalization by polyomaviruses

International audienceThe viability of rat embryo cells immortalized by thermosensitive mutants of SV40 or polyoma Large T antigen is impaired at the non-permissive temperature thus demonstrating that the immortal phenotype is dominantly maintained by Large T antigens. We have observed that exposing these cells to the restrictive temperature not only induces growth arrest but also causes apoptotic cell death. We present evidence supporting the model that polyomaviruses may indeed establish immortality by antagonizing the lethal effects of tumor suppressor genes via physical interactions between their products and Large T antigens. In the case of SV40-immortalized cells REtsAF, shift-up to 39.5 degrees C dissociates Large T antigen/p53 complexes releasing wild-type p53 molecules capable of inducing apoptotic cell death. In polyomavirus-immortalized cells, apoptosis may result from an alternative pathway mediated by other unidentified negatively acting molecules

A cytofluorometric assay of nuclear apoptosis induced in a cell-free system: Application to ceramide-induced apoptosis

Purified nuclei exposed to apoptogenic factors in vitro undergo morphological and biochemical changes in chromatin organization. Most cell- free models of nuclear apoptosis are based on the quantitation of endonuclease-mediated DNA fragmentation on agarose gels or on the changes of nuclear morphology revealed by the DNA-intercalating fluorochrome 4'-6- diamidino-2-phenylindole dihydrochloride. In this work we develop a cytofluorometric system for the accurate quantitation of nuclear DNA loss. This system has been used to determine the conditions of nuclear apoptosis induced by apoptosis-inducing factor (AIF) contained in the supernatant of mitochondria induced to undergo permeability transition. AIF can provoke significant nuclear DNA loss in ≤ 5 min, acts over a wide pH range (pH 6 to 9), and resists cysteine protease inhibitors such as iodoacetamide and N- ethylmaleimide. Moreover, we applied this system to the question of how the proapoptotic second messenger ceramide would induce apoptosis in vitro: via a direct effect on nuclei, a direct effect on mitochondria, or via indirect mechanisms? Our data indicate that ceramide has to activate yet unknown cytosolic effectors that, in the presence of mitochondria, can induce nuclear apoptosis in vitro.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Bcl-2 and Bax modulate adenine nucleotide translocase activity

Bcl-2 is a prosurvival factor that reportedly prevents the nonspecific permeabilization of mitochondrial membranes, yet enhances specific ADP/ATP exchange by these organelles. Here, we show that Bcl-2 enhances the ADP/ATP exchange in proteoliposomes containing the purified adenine nucleotide translocase (ANT) in isolated mitochondria and mitoplasts, as well as in intact cells in which mitochondrial matrix ATP was monitored continuously using a specific luciferase-based assay system. Conversely, Bax, which displaces Bcl-2 from ANT in apoptotic cells, inhibits ADP/ATP exchange through a direct action on ANT. The Bax-mediated inhibition of ADP/ATP exchange can be separated from Bax-stimulated formation of nonspecific pores by ANT. Chemotherapy-induced apoptosis caused an inhibition of ANT activity, which preceded the loss of the mitochondrial transmembrane potential and could be prevented by overexpression of Bcl-2. These data are compatible with a model of mitochondrial apoptosis regulation in which ANT interacts with either Bax or Bcl-2, which both influence ANT function in opposing manners. Bcl-2 would maintain the translocase activity at high levels, whereas Bax would inhibit the translocase function of ANT