Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans

https://doi.org/10.1016/j.jphotobiol.2012.08.005Get rights and content

Abstract

KillerRed, a red fluorescent protein, is a photosensitizer that efficiently generates reactive oxygen species (ROS) when irradiated with green light. Because KillerRed is genetically encoded, it can be expressed in a spatially and temporally regulated manner under control of a chosen promoter and thus is a powerful tool for studying the downstream cellular effects of ROS. However, information is still limited about the effects of KillerRed-mediated production of ROS inside the mitochondria (mtROS). Therefore, we investigated whether mtROS generated by KillerRed could trigger mitochondrial damage and cell death by generating human cell lines (HEK293T and HeLa cells) that stably expressed mitochondria-targeting KillerRed (mtKillerRed). We found that mtROS generated by mtKillerRed caused depolarization of the mitochondrial membrane and morphological changes, which were partly due to the mitochondrial permeability transition (MPT), as well as inducing both caspase-dependent cell death (apoptosis) and caspase-independent cell death. In order to study the pathological processes initiated by mtROS in animals, transgenic Caenorhabditis elegans expressing mtKillerRed in muscle tissue were generated. Transgenic larvae showed developmental delay following light irradiation, suggesting that mtROS influenced the development of C. elegans larvae. In conclusion, our studies demonstrated that the photosensitizer KillerRed is effective at inducing oxidative damage in the mitochondria, and indicated that our experimental systems may be useful for studying the downstream cellular effects of mtROS.

Highlights

► mtROS generated by KillerRed could trigger mitochondrial damage. ► mtROS induced caspase-dependent and caspase-independent cell death in human cells. ► mtKillerRed alone showed toxicity in C. elegans under unirradiated conditions. ► Photodynamic action of mtKillerRed induced developmental delay of C. elegans larvae.

Introduction

Photosensitizers are molecules that absorb and re-emit light at a specific wavelength unique to each molecule and produce reactive oxygen species (ROS), such as singlet oxygen 1O2 or other radicals (superoxide, hydroxyl radical) during the photoreaction [1], [2], [3], [4]. Photosensitizers are widely used in photodynamic therapy (PDT), which is a promising treatment for numerous cancers and certain noncancerous diseases. Cancer cells that absorb photosensitizers can be killed by the oxidative damage induced by ROS production following irradiation with light of an appropriate wavelength.

Bulina et al. developed a photosensitizer protein called KillerRed, which is a dimeric red fluorescent protein with excitation and emission maxima at 585 nm and 610 nm, respectively [5], [6], [7]. After irradiation with light at wavelengths of 520–590 nm, KillerRed generates high levels of ROS that can kill Escherichia coli and eukaryotic cells, exceeding the amounts of ROS produced by other green and red fluorescent proteins (e.g. GFP and DsRed) by at least 1000-fold. Because KillerRed is encoded by a gene, unlike other known photosensitizers, it can be expressed in a spatially and temporally regulated manner by choosing an appropriate promoter or by fusing with a protein of interest or a subcellular localization signal. Therefore, genetically encoded KillerRed provides a unique opportunity to not only investigate the downstream effects of ROS generated in subcellular organelles but also the functions of cells in various organs. For example, the effects of peroxisome-generated ROS on the cellular oxidation–reduction balance have been studied in mammalian cells by using peroxisome-targeting KillerRed [8]. More recently, membrane-targeting KillerRed was employed in zebrafish to disrupt neural circuits involving the habenula or to cause cardiac dysfunction following light irradiation [9], [10]. It has also been reported that light irradiation of HeLa or PC12 cells expressing mitochondria-targeting KillerRed induces mitochondrial translocation of mitophagy-related factors, including Parkin and LC3B, suggesting that mitochondria-targeting KillerRed may be a potential tool for studying Parkin-mediated mitophagy [11], [12]. Additionally, Bulina et al. reported that B16 melanoma cells expressing mitochondria-targeting KillerRed were killed by light irradiation, while these cells survived identical irradiation and preserved their normal shape for at least 1.5 h afterward when preincubated with the pan-caspase inhibitor zVAD, suggesting that mtROS might induce apoptosis in B16 melanoma cells [5], [6].

Although previous studies have shown that light irradiation of mammalian cells expressing mitochondria-targeting KillerRed can induce cytotoxicity, it is still unclear exactly how mitochondrial damage is caused by KillerRed-mediated production of ROS in the mitochondria (mtROS) and how mtROS generated by KillerRed affect the fate of cells, that is, whether activation of the cell death mechanisms result in apoptosis and/or necrosis. In the present study, we generated two human cell lines stably expressing mitochondria-targeting KillerRed, and investigated the effects of mtROS generated by KillerRed on the mitochondria and on the fate of these cells. Additionally, several authors have reported that excessive mitochondrial ROS production contributes to the pathogenesis of a wide range of diseases including diabetes, Alzheimer’s disease, and Parkinson’s disease, as well as being involved in the aging process [13]. To investigate role of mtROS in such pathology, we also generated transgenic Caenorhabditis elegans expressing mtKillerRed.

Section snippets

Human cell cultures

HEK293T and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C under 5% CO2.

C. elegans

The C. elegans strains used in this study were maintained at 20 °C on nutrient growth medium (NGM) agar plates seeded with E. coli HB101, according to standard techniques [14]. Worms were grown under dark conditions except when they were irradiated with light. Wild-type Bristol N2, SJ4100 (zcIs13[

mtKillerRed produces ROS after irradiation

We generated human HEK293T and HeLa cell lines that stably expressed mitochondria-targeting signal (MTS)-fused KillerRed protein (mtKillerRed) (293-mtK and HeLa-mtK cells) as described in Section 2 (Fig. S1). The localization of mtKillerRed in the mitochondria was confirmed by colocalization with MitoTracker Green-FM (a mitochondrial membrane-targeting dye). As a negative control, we also generated human cell lines stably expressing MTS-fused DsRed (mtDsRed), which does not produce enough ROS

Mitochondria are morphologically and functionally disrupted by mitochondrial ROS generation in human cells

The present study revealed that the photodynamic action of mtKillerRed, which generated mtROS, led to mitochondrial fragmentation and swelling in human cell lines. During 20 min of incubation after irradiation with light, confocal microscopy revealed that the majority of the mitochondria in HeLa-mtK and 293-mtK cells underwent fragmentation and then gradually became swollen, suggesting that mitochondrial fragmentation probably preceded swelling. In addition, electron microscopy clearly showed an

Abbreviations

ROSreactive oxygen species
mtROSmitochondrial ROS
mtKillerRedmitochondrial KillerRed
PDTphotodynamic therapy
ΔΨmmitochondrial membrane potential
PIpropidium iodide
NACN-acetyl-cysteine
MPTmitochondrial permeability transition
CsAcyclosporine A
MTSmitochondrial targeting signal
CCCPcarbonyl cyanide p-chlorophenylhydrazone
NGMnutrient growth medium
UPRmtmitochondrial unfolded protein response

Acknowledgements

We thank Drs. Y. Eguchi, K. Shinzawa, Y. Matsuoka, and other members of the Tsujimoto lab for their technical advice and assistance, reagents, and helpful discussion. We acknowledge Drs. Toshihiko Oka and Noriyuki Matsuda for providing the TOM20-GFP plasmid and GFP-Parkin plasmid, respectively. We are grateful to Dr. Hiroki Moribe for providing C. elegans strains, sharing the microinjection system, and to Dr. Takashi Hirose, Ms. Mayumi Shibuya for technical assistance, respectively. This work

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