Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells: Modulation of cellular signals regulating cell cycle and survival

Sanli, Toran; Storozhuk, Yaryna; Linher-Melville, Katja; Bristow, Robert G.; Laderout, Keith; Viollet, Benoit; Wright, James; Singh, Gurmit; Tsakiridis, Theodoros

doi:10.1016/j.radonc.2011.11.014

« Previous Next »Radiotherapy & Oncology
Volume 102, Issue 3 , Pages 459-465, March 2012

Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells:

Modulation of cellular signals regulating cell cycle and survival

Toran Sanli
- Translational Radiation Biology Laboratory, Juravinski Cancer Center, McMaster University, Hamilton, Canada
- Department of Pathology, McMaster University, Hamilton, Canada
- These authors have contributed equally to this work.
Yaryna Storozhuk
- Translational Radiation Biology Laboratory, Juravinski Cancer Center, McMaster University, Hamilton, Canada
- Department of Oncology, McMaster University, Hamilton, Canada
- These authors have contributed equally to this work.
Katja Linher-Melville
- Department of Pathology, McMaster University, Hamilton, Canada
Robert G. Bristow
- Department of Radiation Oncology, University of Toronto, Canada
Keith Laderout
- SRI International, Menlo Park, CA, USA
Benoit Viollet
- INSERM U567, CNRS UMR 8104, Université Paris Descartes, France
James Wright
- Department of Oncology, McMaster University, Hamilton, Canada
Gurmit Singh
- Department of Pathology, McMaster University, Hamilton, Canada
Theodoros Tsakiridis
- Translational Radiation Biology Laboratory, Juravinski Cancer Center, McMaster University, Hamilton, Canada
- Department of Oncology, McMaster University, Hamilton, Canada
- Corresponding author. Address: Department of Oncology, McMaster University, Radiation Oncology, Juravinski Cancer Center, 699 Concession Street, Hamilton, Ontario, Canada L8V 5C2.

Received 3 August 2011; received in revised form 8 November 2011; accepted 29 November 2011. published online 09 January 2012.

Abstract

Purpose

To analyze the (i) expression of AMPK in a variety of epithelial cancer cells, (ii) regulation of AMPK subunit expression by ionizing radiation (IR) and (iii) impact of AMPK on signaling pathways regulating cell cycle and survival.

Methods and materials

Human lung, prostate, and breast normal and cancer cells were treated with 0 or 8Gy IR and mRNA and protein levels of AMPK were evaluated by RT-PCR and immunoblotting 24 or 48h later. Untreated and radiated wild type (WT) and AMPKα^−/− mouse embryonic fibroblasts (MEFs) were analyzed by immunoblotting using total- and phosphorylation-specific antibodies. Histone H2Ax was examined by fluorescence microscopy. The cell cycle and survival of WT and AMPKα^−/− MEFs was also evaluated following 8Gy by IR.

Results

AMPK subunits were found widely expressed in normal and cancer epithelial cells. IR increased subunit protein levels and stimulated gene transcription in cancer cells. AMPKα^−/−-MEFs showed enhanced basal total levels of ATM and phosphorylation of its substrates histone H2Ax, but inhibited response of these markers and of checkpoint kinase Chk2 phosphorylation to IR. AMPKα^−/−-MEFs showed increased basal levels of p53 and cyclin-dependent kinase inhibitors p21^cip1, but lack of response of both genes to IR. These cells had increased basal levels and activation of the Akt-mTOR-p70^S6K/4-EBP1 signalling pathway. IR increased Akt, p70^S6K and 4-EBP1 phosphorylation in WT-MEFs, but this was reduced in AMPKα^−/−-MEFs. AMPKα^−/−-MEFs failed to arrest at the G2-M checkpoint after IR and showed a trend for radio-resistance in proliferation assays.

Conclusions

AMPK is widely expressed in human normal and cancer epithelial cells and its gene transcription, protein levels, and enzymatic activity is stimulated by IR. Work with AMPKα knockout cells suggests that AMPK (i) may mediate a suppressive regulation on basal expression and activity of ATM and its downstream effector pathways Chk2/p53-p21^cip1 and Akt-mTOR, (ii) facilitates the normal response of these pathways to IR and, (iii) mediates the IR-induced G2-M checkpoint.

Keywords: AMPK, Ionizing radiation, Cell cycle, ATM