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mTOR pp 125–169Cite as

Evaluation of Rapamycin-Induced Cell Death

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 821))

Abstract

Mammalian target of rapamycin (mTOR) is an evolutionarily conserved kinase that integrates signals from nutrients and growth factors for the coordinate regulation of many cellular processes, including proliferation and cell death. Constitutive mTOR signaling characterizes multiple human malignancies, and pharmacological inhibitors of mTOR such as the immunosuppressant rapamycin and some of its nonimmunosuppressive derivatives not only have been ascribed with promising anticancer properties in vitro and in vivo but are also being extensively evaluated in clinical trials. mTOR inhibition rapidly leads to the activation of autophagy, which most often exerts prosurvival effects, although in some cases it accompanies cell death. Thus, depending on the specific experimental setting (cell type, concentration, stimulation time, and presence of concurrent stimuli), rapamycin can activate/favor a wide spectrum of cellular responses/phenotypes, ranging from adaptation to stress and survival to cell death. The (at least partial) overlap among the biochemical and morphological responses triggered by rapamycin considerably complicates the study of cell death-associated variables. Moreover, rapamycin presumably triggers acute cell death mainly via off-target mechanisms. Here, we describe a set of assays that can be employed for the routine quantification of rapamycin-induced cell death in vitro, as well as a set of guidelines that should be applied for their correct interpretation.

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Abbreviations

AnnV:

Annexin V

ATCC:

American type culture collection

BSA:

Bovine serum albumin

CRTC1:

CREB-regulated transcription coactivator 1

CV:

Crystal violet

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

dH2O:

Deionized water

DiOC6(3):

3,3′-Dihexiloxalocarbocyanine iodide

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

EGFR:

Epidermal growth factor receptor

FITC:

Fluorescein isothiocyanate

FSC:

Forward scatter

HBSS:

Hank’s balanced salt solution

IARC:

International Agency for Research on Cancer

IC50 :

Half maximal inhibitory concentration

IM:

Inner mitochondrial membrane

IMS:

Mitochondrial intermembrane space

∆ψ m :

Mitochondrial transmembrane potential

MMP:

Mitochondrial membrane permeabilization

mTOR:

Mammalian target of rapamycin

NTP:

National Toxicology Program

OM:

Outer mitochondrial membrane

OSHA:

Occupational Safety and Health Administration

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

PI:

Propidium iodide

PI3K:

Phosphoinositide 3-kinase

PLSCR1:

Phospholipid scramblase 1

PMSF:

Phenylmethylsulfonyl fluoride

PS:

Phosphatidylserine

ROS:

Reactive oxygen species

RT:

Room temperature

SDS:

Sodium dodecylsulfate

SF:

Surviving fraction

siRNA:

Small interfering RNA

SSC:

Side scatter

TBS:

Tris-buffered saline

TMRM:

Tetramethylrhodamine methyl ester

TORC:

TOR complex

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Acknowledgments

GK is supported by the Ligue Nationale contre le Cancer (Equipe labellisée), Agence Nationale de la Recherche, Cancéropôle Ile-de-France, Fondation pour la Recherche Médicale, Institut National du Cancer, European Commission (Active p53, Apo-Sys, RIGHT, TransDeath, ChemoRes, ApopTrain), and Fondation pour la Recherche Médicale. LG, EM, and OK are supported by Apo-Sys, the ApopTrain Marie Curie training network of the European Union, and INSERM, respectively.

Author information

Authors and Affiliations

  1. INSERM, U848, Villejuif, France

    Lorenzo Galluzzi

  2. Institut Gustave Roussy, Villejuif, France

    Lorenzo Galluzzi, Eugenia Morselli, Oliver Kepp, Ilio Vitale, Aména Ben Younes & Maria Chiara Maiuri

  3. Université Paris-Sud, Le Kremlin-Bicêtre, France

    Lorenzo Galluzzi, Eugenia Morselli, Oliver Kepp, Ilio Vitale, Aména Ben Younes & Maria Chiara Maiuri

  4. INSERM Institut Gustave Roussy, U848, Villejuif, France

    Eugenia Morselli, Oliver Kepp, Ilio Vitale, Aména Ben Younes, Maria Chiara Maiuri & Guido Kroemer

  5. Dipartimento di Farmacologia Sperimentale, Università degli Studi di Napoli Federico II, Napoli, Italy

    Maria Chiara Maiuri

  6. Metabolomics Platform, Institut Gustave Roussy, Villejuif, France

    Guido Kroemer

  7. Centre de Recherche des Cordeliers, Paris, France

    Guido Kroemer

  8. Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France

    Guido Kroemer

  9. Sorbonne Paris Cité, Université Paris Descartes, Paris, France

    Guido Kroemer

Authors
  1. Lorenzo Galluzzi
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  2. Eugenia Morselli
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  3. Oliver Kepp
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  4. Ilio Vitale
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  5. Aména Ben Younes
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  6. Maria Chiara Maiuri
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  7. Guido Kroemer
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Corresponding author

Correspondence to Guido Kroemer .

Editor information

Editors and Affiliations

  1. Division of Nephrology and Dialysis, Medical University of Vienna, Währinger Gürtel 18-20, Vienna, 1090, Austria

    Thomas Weichhart

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© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Galluzzi, L. et al. (2012). Evaluation of Rapamycin-Induced Cell Death. In: Weichhart, T. (eds) mTOR. Methods in Molecular Biology, vol 821. Humana Press. https://doi.org/10.1007/978-1-61779-430-8_9

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  • DOI: https://doi.org/10.1007/978-1-61779-430-8_9

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-429-2

  • Online ISBN: 978-1-61779-430-8

  • eBook Packages: Springer Protocols

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