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AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts

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Abstract

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 / /α2 / MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

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Abbreviations

ACC:

Acetyl-CoA carboxylase

AICAR:

5-Aminoimidazole-4-carboxamide (AICA) riboside or acadesine

AMPK:

AMP-activated protein kinase

CLL:

Chronic lymphocytic leukemia

DKO:

Double knockout

MEFs:

Mouse embryonic fibroblasts

MOMP:

Mitochondrial outer membrane permeabilization

PI:

Propidium iodide

RT-MLPA:

Reverse transcriptase multiplex ligation-dependent probe amplification

RT-qPCR:

Real time quantitative PCR

SEM:

Standard error of the mean

WT:

Wild type

ZMP:

AICA ribotide

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Acknowledgments

We thank Dr. Lozano (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA) and Dr. Viollet (Inserm, Institut Cochin, Paris, France) for providing MEFs. We also thank Scientific-Technical Services (CCiTUB) of the Unitat de Bellvitge at the Universitat de Barcelona for helpful discussions and suggestions. Moreover, we would like to thank the Unitat de Genòmica from CCiTUB at the Universitat de Barcelona for their technical support. This study was supported by grants from the Ministerio de Ciencia e Innovación and European Regional Development Fund (ERDF, SAF2010-20519), the Instituto de Salud Carlos III (RTICC RD12/0036/0029) and the AGAUR-Generalitat de Catalunya (AGAUR- 2009SGR395). DMG-G, CM-M, DI-S, AP-P, and CR-P are recipients of research fellowships from the Ministerio de Economía y Competitividad.

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The authors declare no conflicts of interest and no financial disclosure.

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Correspondence to Joan Gil.

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Diana M. González-Gironès and Cristina Moncunill-Massaguer contributed equally to this study.

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González-Gironès, D.M., Moncunill-Massaguer, C., Iglesias-Serret, D. et al. AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts. Apoptosis 18, 1008–1016 (2013). https://doi.org/10.1007/s10495-013-0850-6

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