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The fourth isoform of the adenine nucleotide translocator inhibits mitochondrial apoptosis in cancer cells

https://doi.org/10.1016/j.biocel.2009.12.024Get rights and content

Abstract

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1–4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANT1 and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondrial network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli.

Introduction

Mitochondria, the cells’ powerhouse of energy, are also an important checkpoint in the signaling pathways of cell death. In response to a wide range of lethal stimuli, mitochondria undergo changes in their membrane permeability (MMP) that triggers the cytosolic translocation of pro-apoptotic proteins such as cytochrome c (Cyt c), apoptosis inducing factor (AIF), endonuclease G (EndoG) and Smac/Diablo that normally reside in the inter-membranous space (for reviews: Green and Kroemer, 2004, Kroemer et al., 2007). Thus, MMP activates cytosolic and nuclear caspase-dependent and caspase-independent signaling cascades that coordinate the final steps of apoptosis. It is now well established that MMP could be induced by diverse stimuli and involve either outer membrane permeabilization (OMP) or inner membrane permeabilization (IMP); both processes are under the control of Bcl-2 family proteins. IMP is often related to the so-called “permeability transition”, a process involving prolonged opening of the mitochondrial polyprotein complex, the permeability transition pore complex (PTPC), and manifests as the early loss of integrity of the IM permeability barrier associated with a loss of the transmembrane potential (ΔΨm) and arrest of mitochondrial bioenergetic functions. Despite intense research and presumably due to tissue specificity, the PTPC composition is still a matter of debate. In a variety of human cancers, such as cervical carcinoma, breast cancer, lung and liver carcinomas, the components of the PTPC might involve the outer membrane voltage-dependent anion channel (VDAC), the inner membrane ANT, and the matrix protein cyclophilin D (CypD) (Grimm and Brenner, 2006, Faustin et al., in press), notwithstanding the association of other mitochondrial proteins, such as the phosphate carrier (Leung et al., 2008).

The ANTs form a sub-family of mitochondrial ADP/ATP transporters (Pfaff and Klingenberg, 1968). The human genome contains at least four genes encoding four protein isoforms, namely ANT1–4. The four isoforms are highly homologous (68–88%), expressed in a tissue-, hormone-, and development-specific manner and conserved through evolution, although ANT3 is not present in mice (Dorner et al., 1997, Stepien et al., 1992). Wan-1, the C. elegans ortholog of mammalian ANT is an important cell death regulator, which cooperates with the core cell death machinery (Shen et al., 2009). In human, rat, mouse, and partially in yeast, pro-apoptotic stimuli (e.g. calcium, ROS, and carboxyatractyloside) favoring a non-specific lethal pore conformation of ANT1 and 3 (at least in human) lead to the classical hallmarks of mitochondrial apoptosis (Beutner et al., 1996, Bauer et al., 1999, Marzo et al., 1998a, Marzo et al., 1998b, Zamarin et al., 2005, Zamora et al., 2004). The pro-apoptotic ANT isoforms cooperate with a panel of proteins, in particular members of the Bcl-2 family (e.g. Bax, Bid, Bcl-2) to form a new class of lethal channels. These dynamic interactions are notable during apoptotic signaling in response to chemotherapeutic drugs, ischemia/reperfusion, and viral infection (Belzacq et al., 2001, Cao et al., 2001, Zamarin et al., 2005, Zamora et al., 2004, Jacotot et al., 2001). In contrast, the ANT2 isoform, which is overexpressed in proliferating cells and some human cancers, has been reported to play an anti-apoptotic role (Stepien et al., 1992). As a consequence, ANT2 inactivation by RNA interference favors chemotherapy-induced cell death in vitro and in vivo (Le Bras et al., 2006, Jang et al., 2008). Whether ANT isoforms could have a direct effect on cell proliferation has not yet been established. Interestingly, the double knock-out of ANT1 and 2 in mouse liver did not have any major effect on the calcium-induced permeability transition in liver mitochondria (i.e. permeability transition requires more calcium), but abolished the sensitivity of ANT ligands, suggesting that, at least in mice, ANT1 and 2 are dispensable for permeability transition (Kokoszka et al., 2004). However, this scenario might not be entirely true based on two possibilities: (1) compensatory phenomenon in conventional knock-out mice cannot be excluded, and (2) the existence of previously unknown ANT isoforms, like the recently discovered ANT4 (Dolce et al., 2005). As a corollary, ADP/ATP carriers were shown to be required for efficient MMP induction, Cyt c release and apoptosis in yeast, independent of the translocase activity (Pereira et al., 2007).

Whereas the ADP/ATP antiport activity of ANT4 has been extensively characterized (Dolce et al., 2005), not much is known regarding its involvement in PTPC and mitochondria-dependent apoptosis. ANT4 is highly conserved through evolution, and notably, human ANT4 mRNA is present in liver, brain and testis. In mice, although ANT4 is expressed in pluripotent stem cells and germ cells, its physiological expression is restricted to developing gametes in adults (Rodic et al., 2005). Ant4-deficient mice are infertile thus strongly suggesting that ANT4 could serve as the sole ADP/ATP translocase isoform during spermatogenesis, precisely where the sex chromosome-linked Ant2 gene is inactivated. In addition, Ant4-deficient male mice exhibit enhanced levels of apoptosis within the testis suggesting an anti-apoptotic role of this isoform (Brower et al., 2009).

To determine the role of human ANT4 in apoptosis, we investigated the effect of its stable overexpression in tumoral cell lines in comparison with the pro-apoptotic ANT3 isoform. Here we report that ANT4-overexpressing cells exhibited increased growth rate without a significant change in cell morphology or mitochondrial respiration. Similarly to ANT3, ANT4 overexpression resulted in a decrease in intracellular hydrogen peroxide (H2O2) without affecting the levels of superoxide anion (O2). Of note, in contrast to the apoptosis sensitizing activity of ANT3, ANT4 expression had an inhibitory effect on apoptosis. Altogether, these data suggest a new cytoprotective function of the human ANT4 isoform.

Section snippets

Materials and methods

All chemicals were purchased from Sigma–Aldrich unless otherwise indicated.

Contrasting effects of ANT3 and 4 overexpression on cell proliferation, cell morphology and mitochondrial topology

To investigate the role of human ANT4 in mitochondrial apoptosis, we first generated cell lines expressing various isoforms of ANT. HeLa cells were used as a model system as these cells constitutively express ANT1, 2, and 3, but not the ANT4 isoform (data not shown). We generated the following HeLa cell variants: (a) cells overexpressing ANT4 (Q9H0C2, 315aa) and (b) cells overexpressing ANT3 isoform (P12236, 298aa; positive control for pro-apoptotic isoform). The integration of the ant gene

Acknowledgments

CB is supported by grants funded by Association pour la Recherche sur le Cancer (ARC), Egide, l’Agence Nationale pour la Recherche (ANR, ANR-08PCVI-0008-01) and l’Institut National pour le Cancer (INCa, 2008-1-PL BIO-04-CNRS ON1). CD, CM, and EM received fellowships from the Ministère de l’Enseignement et de la Recherche (MENR) and OS received a postdoctoral fellowship from the DIM Sent, Région Ile de France. E. Maillier is acknowledged for his technical help.

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    Contributed equally to the work.

    2

    Present address: INSERM U769, Université Paris-Sud 11, Faculté de Pharmacie, 5, Rue J.-B. Clément, Châtenay-Malabry cedex, France.

    3

    Present address: Mitologics S.A.S. Hôpital Robert Debré, 48, Boulevard Sérurier, 75019 Paris, France.

    4

    Present address: Imperial College London, Department of Reproductive Biology, Cancer division, Hammersmith Hospital, DuCane Rd., London, W120NN, United Kingdom.

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