Unit: Apoptosis and Immune System - CNRS URA 1961

Director: SUSIN, Santos A.

Programmed cell death (PCD) or apoptosis is part of the normal physiology of an organism. PCD deregulation may give rise to several pathologies (cancer, neurodegenerative and autoimmune diseases). In several models, this highly controlled process is linked to the activation of a family of cysteine proteases called caspases. However, there is a considerable amount of data pointing out the existence of several caspase-independent cell death pathways. The Apoptosis and Immune System team is devoted to research on caspase-independent cell death mechanisms in the immune system. The main objectives of our scientific project are to: 1) identify the mechanisms that regulate the necrosis-like cell death mediated by CD47 in B-chronic lymphocytic leukaemia (B-CLL) cells; 2) expand the knowledge of the regulation mechanism involved in apoptosis-like cell death by seeking to define the molecular mechanisms that determine the functions of AIF (one of the essential agents of this type of caspase-independent apoptosis).

Identification of mechanisms that regulate necrosis-like cell death mediated by CD47 in B-chronic lymphocytic leukaemia (B-CLL) cells (Marlène Bras, Nadine Robert & Gaël Roué).

The involvement of antigen CD47 (IAP) via its physiological ligand, thrombospondin, or via an anti-CD47 agonist antibody, induces cell adhesion and motility through the ß3 integrins. It also induces a form of caspase-independent cell death whose mechanism is yet to be defined. Our objective is to identify, through a multi-parametrical cell analysis, the actors involved in this pathway. Special attention should be paid to the importance of this type of apoptosis in the context of B-CLL. Indeed, the functionality of this way to death is preserved even in B-CLL cells offering resistance to apoptosis mediated by the intrinsic mitochondrial pathway (activated by most of the agents used in chemotherapy) and/or by the extrinsic death receptors.

Upon inducing cell death in B-CLL cells, via anti-CD47, we do not observe any nuclear change (hypodiploidy or DNA fragmentation) despite the reduction in the mitochondrial membrane potential, the production of oxygen free radicals and the aberrant exposure of phosphatidylserine residues on the outer plasma membrane leaflet. Furthermore, downstream of mitochondria, we neither observe cytochrome c or AIF release nor caspase-3 activation. These parameters as a whole enable us to identify the cell death mediated by CD47 as a necrosis-like PCD. In contrast, when treating these B-CLL cells with hydrocortisone, cell death is accompanied by all the typical hallmarks of apoptosis, including the alteration in mitochondrial potential and nuclear DNA fragmentation. These phenomena induced by hydrocortisone treatment depend on caspase activity and can no longer be observed when the general caspase inhibitor z-VAD.fmk is present. All these results indicate that at least two cell death pathways coexist in most B-CLL cells.

In order to identify the elements involved in the CD47 cell death pathway, we first sought to determine, through a flow cytometry and a immunofluorescence analysis, the activation level of two proapoptotic proteins of the Bcl-2 family: Bax and Bak. Using these two techniques we noticed that Bax and Bak are activated in the CD47 death pathway, and in a much stronger way than under the caspase-dependent hydrocortisone treatment. These results were confirmed in the T Jurkat leukemic cells using etoposide - a topoisomerase II inhibitor - as a positive control.

We currently study the role of Bax and Bak activation in this death pathway. At the same time, we analyse the possible role of the BH3-proteins such as Bid, Bim or Bmf, which could be potentially involved in Bax and Bak activation. Finally, through the use of specific inhibitors of the different signal transduction paths we seek to characterize the events that precede Bax and Bak activation.

Expansion of the knowledge of regulation mechanisms in apoptosis-like cell death (Cécile Delettre-Cribaillet, Rana Moubarak & Victor J. Yuste-Mateos).

One of the essential agents acting in apoptosis-like, caspase-independent PCD, is AIF (Apoptosis-Inducing Factor). AIF is a mitochondrial intermembrane flavoprotein that has both oxydoreductase and apoptotic functions. Although AIF functions have already been described, several questions about the mode of action of AIF still remain unanswered.

In order to expand our knowledge of apoptosis-like cell death, the aim of our project is to define the molecular mechanisms that determine the functions of AIF.

-Characterization of AIF isoforms: there are two AIF isoforms issued from the alternate splicing of exon 2 (variant 1) and exon 2b (variant 2). In order to better understand the role of these isoforms in the caspase-independent apoptotic process, we characterized their tissue distribution in human through RT-PCR and Northern blot analysis. The two variants show a different tissue expression. At present, we are working on a more detailed characterization of these two splice variants and of their functions in caspase-independent cell death.

-Identification of factors that interact with AIF before and after the apoptotic stimulus: Based on an AIF-FLAG construction and with the help of co-immunoprecipitation assays we obtained, in close collaboration with the Proteomic Platform at the Pasteur Institute, the sequences of several potential AIF partners. Studies of the links between these proteins and AIF will be performed in order to demonstrate their functional interaction. The resulting data will be completed by functional tests that will enable us to select the AIF cofactors.

-AIF elimination by RNAi technique, effects on apoptosis: The RNAi technique (RNA interference) makes it possible to reduce protein expression by introducing a small double-chained ARN into the cell. We outlined 19-nucleotide siRNAs (short interference RNA) that correspond to region 5' of AIF's ARN messenger. After the transfection of these oligonucleotides into HeLa cells, we were able to observe, by Western blot and by immunofluorescence, a reduction of 80% in AIF expression. Having confirmed these results by RT-PCR, we are currently working on the apoptosis signalling pathways that could be affected by a lower level of AIF expression.

-AIF anti-tumoral activity: In an effort to further analyse AIF's implication in different types of cancer, we are also studying AIF expression in a series of tumoral cell lines treated with apoptotic inducers such as oxidative stress inducers, chemotherapeutic agents or irradiation. We presently aim to demonstrate the cause and effect relationship between AIF and oncogenesis.

All of these studies will allow for a deeper knowledge of the molecular mechanisms that characterize apoptosis-like death. The consequent results will provide data that is needed in order to know AIF partners, thereby paving the way for evaluating the role of AIF in tumour development and determining whether it can be a diagnosis marker for the study of cancer.

Photos :

1. Subcellular distribution of cytochrome c and AIF in B-CLL B cells treated with hydrocortisone or with an immobilised anti-CD47 antibody.

2. Morphologic modifications induced by AIF in HeLa cell nuclei.

Keywords: Apoptosis, Apoptosis Inducing Factor (AIF), Cancer, B-CLL, Caspase-independent cell death

Activity Reports 2003 - Institut Pasteur

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