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  • Original Paper
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Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation

Oncogene volume 23, pages 3915–3931 (2004)Cite this article

Abstract

Bcl-2 family members either negatively or positively regulate the apoptotic threshold of cells. Bcl-xES (extra short), a novel Bcl-x member, possesses a unique combination of BH4 and BH2 domains as well as a COOH-terminal hydrophobic transmembrane anchor domain. Bcl-xES contains sequences of hydrophobic α-6 helices but lacks sequences of α-5 helices, suggesting that it does not have pore channel-forming activity but functions uniquely as a trapping protein. mRNA expression analysis by reverse transcriptase–polymerase chain reaction and RNase protection assay reveal that Bcl-xES is expressed in a variety of human cancer cell lines and human tumors, including bone marrow from patients with acute lymphoblastic leukemia. Bcl-xES expression is much less pronounced in some specimens of normal human tissues, including the breast, ovary, testis and lung. Stable, transfected human B lymphoma Namalwa variant cells expressing Bcl-xES were derived to investigate its role in apoptosis. Bcl-xES had a preventive effect on cell death induced by tumor necrosis factor-alpha and various concentrations of anticancer drugs, including camptothecin, etoposide and cisplatin. Its protective action on cell death was correlated with the inhibition of mitochondrial cytochrome c release and caspase activation. In a yeast two-hybrid system, Bcl-xES interacted with most Bcl-2 family members, including those containing only a BH3 domain, and with the Ced-4 homolog Apaf-1. Co-immunoprecipitation and gel filtration chromatography experiments suggest that Bcl-xES delays drug-induced apoptosis by disturbing the formation of Bax oligomers and preventing cytochrome c release, but also by interacting with Apaf-1 and inhibiting procaspase-9 activation, thus averting the apoptogenic proteolytic caspase cascade and cell death.

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Abbreviations

Ac-DEVD-AMC:

Ac-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin

Ac-LEHD-AFC:

Ac-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin

ANT:

adenine nucleotide translocator

ATP-γS:

adenosine 5′-O-(-3-thiotriphosphate)

BH:

Bcl-2 homology domain

cisPT:

cis-platinum(II) diammine dichloride

CPT:

20-S-camptothecin lactone

DSP:

dithiobis[succinimidylpropionate]

HA-tag:

hemagglutinin epitope tag sequences

HSP:

heat shock protein

LOOP:

long flexible loop domain

mPTP:

mitochondrial permeability transition pore

RT–PCR:

reverse transcriptase–polymerase chain reaction

SDS–PAGE:

sodium dodecyl sulphate–polyacrylamide gel electrophoresis

TM:

transmembrane domain

TNF-α:

tumor necrosis factor alpha

VDAC:

voltage-dependent anion channel

VP-16:

etoposide

Δψm:

mitochondrial inner transmembrane potential

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Acknowledgements

This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society to RB. RB is a scholar of the Fonds de la recherche en santé du Québec. CP obtained a studentship from the Canadian Institutes of Health Research. We thank Dr Yves Théoret (Hôpital Sainte Justine, Montreal, Que.) for providing bone marrow samples from acute lymphoblastic leukemia patients and Mr Ovid Da Silva (Research Support Office, Centre de recherche, CHUM) for editing this manuscript.

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  1. Centre de recherche, Centre hospitalier de l' Université of Montréal (CHUM), Hôpital Notre-Dame and Institut du cancer de Montréal, Montreal, Quebec, Canada

    Estelle Schmitt, Claudie Paquet, Myriam Beauchemin & Richard Bertrand

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Schmitt, E., Paquet, C., Beauchemin, M. et al. Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation. Oncogene 23, 3915–3931 (2004). https://doi.org/10.1038/sj.onc.1207554

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