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Vol. 19, Issue 1, 41-50, January 2008

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Interferon Induces Nucleus-independent Apoptosis by Activating Extracellular Signal-regulated Kinase 1/2 and c-Jun NH₂-Terminal Kinase Downstream of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin

Theocharis Panaretakis^*,, Linn Hjortsberg^*,, Katja Pokrovskaja Tamm^*, Ann-Charlotte Björklund^*, Bertrand Joseph, and Dan Grandér^*

*Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Hospital and Institute, S-171 76 Stockholm, Sweden; and Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden

Submitted April 19, 2007; Revised September 10, 2007; Accepted October 10, 2007
Monitoring Editor: Gerard Evan

Interferon (IFN) induces apoptosis via Bak and Bax and the mitochondrial pathway. Here, we investigated the role of known IFN-induced signaling cascades upstream of Bak activation. By pharmacological and genetic inhibition of the kinases protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH₂-terminal kinase (JNK) in U266-1984 and RHEK-1 cells, we could demonstrate that all three enzymes are critical for the apoptosis-associated mitochondrial events and apoptotic cell death induced by IFN, at a step downstream of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR). Furthermore, the activation of JNK was found to occur in a PKC/ERK-dependent manner. Inhibition of these kinases did not affect the canonical IFN-stimulated Janus tyrosine kinase-signal transducer and activator of transcription signaling or expression of IFN-responsive genes. Therefore, enucleated cells (cytoplasts) were examined for IFN-induced apoptosis, to test directly whether this process depends on gene transcription. Cytoplasts were found to undergo apoptosis after IFN treatment, as analyzed by several apoptosis markers by using flow cytometry, live cell imaging, and biochemical analysis of flow-sorted cytoplasts. Furthermore, inhibition of mTOR, ERK, and JNK blocked IFN-induced apoptosis in cytoplasts. In conclusion, IFN-induced apoptosis requires activation of ERK1/2, PKC, and JNK downstream of PI3K and mTOR, and it can occur in a nucleus-independent manner, thus demonstrating for the first time that IFN induces apoptosis in the absence of de novo transcription.

These authors have contributed equally to this work.

Address correspondence to: Dan Grandér (dan.grander{at}cck.ki.se)

Abbreviations used: BOP, BH3-only protein; cyt c, cytochrome c; ERK, extracellular signal-regulated kinase; IFN, interferon; IRS, insulin receptor substrate; ISG, interferon-stimulated gene; JAK, Janus tyrosine kinase; MEK, mitogen-activated protein kinase kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PS, phosphatidylserine; STAT, signal transducer and activator of transcription; TMRE, tetramethylrhodamine ethyl ester.