Down-regulation of PERK enhances resistance to ionizing radiation

doi:10.1016/j.bbrc.2013.09.129

Biochemical and Biophysical Research Communications

Volume 441, Issue 1, 8 November 2013, Pages 31-35

https://doi.org/10.1016/j.bbrc.2013.09.129 Get rights and content

Highlights

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PERK enhances the sensitivity of cancer cells to ionizing radiation.
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Down-regulation of PERK results in enhanced DNA repair.
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Ionizing radiation-induced apoptosis is inhibited in PERK-down regulated cancer cells.

Abstract

Although, ionizing radiation (IR) has been implicated to cause stress in endoplasmic reticulum (ER), how ER stress signaling and major ER stress sensors modulate cellular response to IR is unclear. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is an ER transmembrane protein which initiates unfolded protein response (UPR) or ER stress signaling when ER homeostasis is disturbed. Here, we report that down-regulation of PERK resulted in increased clonogenic survival, enhanced DNA repair and reduced apoptosis in irradiated cancer cells. Our study demonstrated that PERK has a role in sensitizing cancer cells to IR.

Introduction

Radiation therapy is an extensively used mode of treatment for a wide range of cancers. However, like many other anti-cancer agents, ionizing radiation (IR) induces stress response pathways [1], [2], [3]. Cancer cells have been known to utilise stress response mechanisms in order to evade death as well as to enhance survival [4], [5]. Proteins targeted for secretion, along with membrane proteins, are synthesized and modified in the endoplasmic reticulum (ER). Apart from the expression of mutant proteins, changes in ATP, calcium and redox status that impede correct protein folding activates the unfolded protein response (UPR) or ER-stress signaling [6], [7]. Initiation of UPR is mediated by PKR like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol requiring enzyme 1 (IRE1) [6], [7]. These three ER transmembrane proteins sense perturbations in ER and activate corresponding arm of UPR pathways. Though, activation of UPR is directed to resolve the disturbed ER homeostasis, it can lead to the activation of cell death pathways if the stress persists and damage is irreversible [6], [7].

ER stress induces homodimerization and autophosphorylation of PERK and thereby its activation. Activated PERK phosphorylates eukaryotic initiation factor 2-alpha (eIF2α) and inhibits cap dependent translation of proteins to reduce further stress in ER [8]. Nevertheless, PERK mediated translational block is not absolute since this in turn activates the cap-independent translation of activating transcription factor 4 (ATF4). ATF4 aids in resolving ER-stress by inducing the transcription of ER chaperones and proteins involved in amino acid synthesis and transport [9]. However, in addition, ATF4 also induces the expression of C/EBP homologues protein (CHOP) [10], [11], a transcriptional factor, which activates the expression of Bim, a pro-apoptotic member of Bcl-2 family [12] and represses the expression of anti-apoptotic protein, Bcl-2 [13]. Although, the activation of PERK is a part of survival UPR, its persistent activation is a switch to apoptosis via regulation of CHOP [14]. Interestingly, PERK has been previously implicated to play a significant role in the activation of autophagy in irradiated caspase-3/7 null cells [15]. However, it remains largely unknown whether PERK modulates cellular response to IR in cancer cells that harbour functional caspase-3. In this study, we examined the role of PERK in determining the sensitivity of human cancer cells that express functional caspase-3 to IR.

Section snippets

Chemicals and antibodies

All chemicals were purchased from Sigma–Aldrich, Dorset, UK unless indicated otherwise. Rabbit polyclonal antibodies against PARP, Phospho-eIF2α, PERK, Grp78 / Bip and CHOP were obtained from Cell Signaling Technology, Danvers, MA. Rabbit polyclonal antibody against 53BP1 and Alexa Fluor 488-labelled anti-rabbit IgG secondary antibody were purchased from Novus Biologicals, Cambridge, UK and Molecular Probes, Paisley, UK respectively.

Cell culture and treatments

MDA-MB-231 cells and T98G cells were maintained in DMEM (PAA,

Down-regulation of PERK results in increased resistance to IR

In order to understand the role of PERK in modulating cellular response to IR, its expression was down-regulated in human cancer cell lines MDA-MB-231 and T98G cells by using siRNA methodology. Both cell lines express functional caspase-3 [19], [20]. Cell survival was determined by clonogenic cell survival assay at doses of 2, 4 and 6 Gy. Cells treated with PERK siRNA exhibited increased clonogenic cell survival compared to scrambled controls (Fig. 1A and Supplementary Fig. 1A). In PERK

Discussion

Activation of ER stress by IR has been reported in various cell types [2], [3], [15]. However, the relative impact of stress caused by IR is considered moderate compare to agents that severely affect ER-homeostasis [3]. In our study, it was apparent that IR does not cause an acute ER stress as there was no major induction of Grp78 compared to thapsigargin treated cells. However, PERK–eIF2α pathway was activated by IR. The absence of phosphorylated form of eIF2α in PERK-silenced cells suggested

Acknowledgment

The authors acknowledge funding to K.M.P. from CR-UK (Grant No. C1513/A7047).

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View full text

Down-regulation of PERK enhances resistance to ionizing radiation

Highlights

Abstract

Introduction

Section snippets

Chemicals and antibodies

Cell culture and treatments

Down-regulation of PERK results in increased resistance to IR

Discussion

Acknowledgment

Toxicol. In Vitro

Pharmacol. Ther.

Mol. Cell

Mol. Cell

J. Mol. Biol.

Cell

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells

Int. J. Radiat. Biol.

ER stress induced by ionising radiation in IEC-6 cells

Int. J. Radiat. Biol.

ER stress-regulated translation increases tolerance to extreme hypoxia and promotes tumor growth

EMBO J.

Perk-dependent translational regulation promotes tumor cell adaptation and angiogenesis in response to hypoxic stress

Mol. Cell. Biol.

Mediators of endoplasmic reticulum stress-induced apoptosis

EMBO Rep.

Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase

Nature

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state

Mol. Cell. Biol.