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 alternative caspase-independent cell death pathways. The Apoptosis and Immune System team is devoted to research on these new cell death mechanisms in the immune system. The main objectives of our scientific project are to: 1) Unravel the biochemical mechanisms that regulate cell death mediated by CD47 in B-chronic lymphocytic leukaemia (B-CLL); 2) Identify the molecular mechanisms of AIF mitochondrial protein (one of the essential agents of caspase-independent apoptosis).
I. Mechanisms regulating cell death mediated by CD47 in B-chronic lymphocytic leukemia (Marlène Bras, Nadine Robert & Clémence Virely).
The involvement of antigen CD47 (IAP) via its physiological ligand, thrombospondin, or via an anti-CD47 agonist antibody, induces a form of caspase-independent cell death. Using a multi-parametrical cell analysis we have shown that, in B-chronic lymphocytic leukaemia (B-CLL) cells, the programmed cell death (PCD) mechanism induced by the stimulation of CD47 receptor has several biochemical characteristics which are typical of apoptosis: reduction in mitochondrial transmembrane potential (DYm), production of reactive oxygen species (ROS), aberrant outlet exposure of phosphatidylserine in the plasma membrane, and lysosomal permeabilization. An ultrastructure cell analysis by means of an electronic microscope has also revealed that these cytosolic alterations are accompanied by swelling of the endoplasmic reticulum and Golgi apparatus, as well as by a change in mitochondrial inner membrane morphology. These data have allowed us to identify the cytosolic programmed cell death mediated by CD47 as type III PCD or necrosis-like PCD. Importantly, the functionality of this death pathway is preserved even in B-CLL cells which are often resistant to other types of apoptosis.
PCD mediated by CD47 seems to be independent from the main Bcl-2 family members. Indeed, after inducing cell death via anti-CD47 antibody, we observe neither the active forms of Bax, Bak proteins, nor the mitochondrial relocation of Bim, a "BH3-only" protein. Furthermore, while overexpression of anti-apoptotic members of this family (Bcl-XL, Bcl-2, and Mcl-1) efficiently protects leukemic cells from death induced by agents affecting mitochondria, such as etoposide (a topoisomerase II inhibitor), no protective effect has been evidenced in death mediated by CD47. Similarly, downregulation of the main anti-apoptotic members of the Bcl-2 family (Bax, Bak and Bim) through siRNA technique does not modulate death induced by CD47 mAb. Downstream of mitochondria, we do not observe release of the pro-apoptotic mitochondrial proteins AIF, cytochrome c, EndoG, Omi/HtrA2 or Smac/Diablo. In contrast, treatment of B-lymphocytes with hydrocortisone induces a cell death that is characterized by DYm loss, release of cytochrome c and AIF, and caspase-3 activation. These events, which depend on this protease activity, are no longer observed when z-DEVD.fmk, a caspase-3 specific inhibitor, is used. In contrast, death induced by CD47 ligation is inhibited neither by the pan caspase inhibitor z-VAD.fmk nor by specific caspase 2, 3, 6, 7, 8, 9, and 10 inhibitors.
On the other hand, ultrastructural alterations observed in B-CLL cells treated with CD47 mAb suggest a total cytoskeleton disorganization. A more detailed immunofluorescence study of its different components (actin microfilaments, microtubules, and intermediate filaments) has enabled us to detect a specific disorganization of the actin microfilament network.
We currently analyse the effectors that could play a role in the mitochondrial alterations and cytoskeleton disorganization that was observed when CD47 was involved. Simultaneously, we seek to identify the elements involved in the death signal transduction characterizing necrosis-like PCD.
II. Expansion of the knowledge of cell regulation in AIF mitochondrial protein (Jeanne Lesbordes-Brion, Sophie Laine, Rana Moubarak & Victor J. Yuste-Mateos)
AIF is a mitochondrial protein that has both oxidoreductase and apoptotic functions. Although AIF functions have already been described, several questions about the mode of action of AIF still remain unanswered. The aim of our study is to define the molecular mechanisms that determine AIF functions, and to expand our knowledge of the cell regulation mechanism in caspase-independent "apoptosis-like" death, as well as of the role AIF plays in this regulation.
- Characterization of AIF isoforms: there are two AIF isoforms issued from the alternate splicing of exon 2 (variant 1) and exon 2b (variant 2). Using RT-PCR analysis, we identified two new forms of AIF resulting from the alternate splicing of a new identified exon. In order to better understand the role of these new variants in the apoptotic process, we characterized their tissue distribution in human as well as their cell location. One of these variants is the consequence of an alternative transcriptional start site which results in the loss of the N-terminal part of the AIF protein. This new form of AIF, which lacks the oxidoreductase region of the protein, triggers caspase-independent death and induces DNA fragmentation into 50kb fragments. We presently seek to expand our analysis of the function of these new variants inside the cell, as well as their role 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 immunoprecipitation assays, we obtained the sequences of several proteins that could be potential AIF partners in close collaboration with the Proteomic Platform at Pasteur Institute. Studies of the links between these proteins and AIF are being performed in order to demonstrate their functional interaction.
Furthermore, throughout 2004 our group was able to determine the minimal AIF domain that is responsible for inducing apoptosis. In this context, we have used a yeast double-hybrid approach in order to identify the molecules that are involved in the caspase-independent, AIF-dependent, cell death pathway. The resulting data will be completed by functional tests that will enable us to select the AIF partners.
- Description of cell death involving AIF: First, we determined the role of effector caspases along with Bid and other "BH-3-only"proteins in AIF release from mitochondria. In this way, we showed that a large spectrum of pro-apoptotic drugs induced AIF release from mitochondria of caspase -3 and -9 deficient mouse embryonic fibroblasts (MEFs). Therefore, AIF release from mitochondria to cytosol is indeed a caspase-independent process. On the other hand, we are studying the implication of Bid in AIF release within the context of a Pasteur-Wiezmann project prepared with Dr. Atan Gross's team at Weizmann Institute. We currently study the role of Bax and Bak in the AIF-related cell death pathway through Bax and/or Bak deficient MEF cells. Furthermore, stable cell lines expressing AIF under an inducible promoter have been generated in order to elucidate the biochemical consequences of AIF overexpression. In this way, we will establish the effect of AIF overexpression upon cell functions (oxidative/non-oxidative glucolysis, respiratory chain function, cell cycle and proliferation), as well as cell sensitivity to undergo apoptosis.
- AIF elimination by RNAi technique, effects on apoptosis: RNAi technique (RNA interference) makes it possible to reduce protein expression by introducing a small double-chained ARN specific to the ARN messenger to destroy. 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, an 80% reduction in AIF expression. Having confirmed these results by RT-PCR, we are now 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 analyze AIF's implication in different types of cancer, we are also studying AIF expression in different tumoral cell lines treated with apoptotic inducers such as oxidative stress inducers, chemotherapeutic agents, or irradiation. We currently seek to demonstrate the cause and effect relationship between AIF deficit and oncogenesis.
The results obtained will provide the necessary data 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 and prognosis marker for cancer.
Keywords: Apoptosis, Apoptosis Inducing Factor (AIF), Cancer, B-CLL, Caspase-independent cell death