Radiolabeled Cu-ATSM as a novel indicator of overreduced intracellular state due to mitochondrial dysfunction: studies with mitochondrial DNA-less ρ0 cells and cybrids carrying MELAS mitochondrial DNA mutation
Introduction
Mitochondria play critical roles in cell survival, and abnormalities caused by mutations in mitochondrial DNA (mtDNA) lead to various disorders, such as mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Parkinson's disease and Alzheimer's disease [1], [2], [3], [4], [5]. In these disorders, impairment of the respiratory chain generates excess electrons, which cause an overreduced intracellular state, thereby generating reactive oxygen species, increasing oxidative stress and damaging surrounding cells [6], [7], [8]. Therefore, it is important to detect overreduced intracellular states for the diagnosis of disorders caused by mitochondrial dysfunction and the evaluation of pathophysiological conditions.
Among these disorders, the disease mechanisms of MELAS syndrome have been studied in great detail. It has been reported that an A-to-G point mutation at nucleotide position 3243 (A3243G) in mtDNA is the main cause of MELAS syndrome [2], [5]. The A-to-G point mutation in MELAS syndrome induces mitochondrial and respiratory dysfunction at a cellular level, which leads to clinical symptoms, such as stroke-like episodes, myopathy, lactic acidosis, diabetes mellitus and cardiomyopathy, in MELAS patients [9].
In order to study mitochondrial dysfunction, human mtDNA-less ρ0 cells have been established [10] (Fig. 1). ρ0 cells are depleted of mtDNA by long-term exposure to ethidium bromide, while maintaining the same nuclear genetic background as the original cells. ρ0 cells are therefore considered to be useful models for studying chronic mitochondrial impairment and deficient respiration. In addition, to analyze the pathological conditions of MELAS syndrome, cybrids were constructed by transfer of cytoplasts possessing mutated mitochondria from MELAS patients to ρ0 cells, and their mitochondrial respiratory functions have been evaluated [11] (Fig. 1). Cybrids carrying mutated mitochondria show mitochondrial impairment when compared to those carrying normal mitochondria, and these cells are also considered to represent a useful in vitro culture model for studying the characteristics of mitochondrial dysfunction, as seen in MELAS syndrome. It is so far reported that mtDNA deletion or mutation of mtDNA as seen in ρ0 cells and cybrids carrying MELAS mutated mitochondria causes marked defects in mitochondrial protein synthesis related to respiratory function, which lead to defect of the respiratory activity (e.g., mitochondrial electron transport chain and O2 consumption) [10], [11], [12]; this is because mitochondrial proteins involved in electron transport chain, such as NADH dehydrogenase subunits, cytochrome c oxidase subunits and cytochrome b, are not synthesized in ρ0 cells and considerably decreased in cybrids carrying MELAS mutated mitochondria.
Radiolabeled Cu-diacetyl-bis (N4-methylthiosemicarbazone) (⁎Cu-ATSM), including 60/62/64Cu-ATSM, is a noninvasive imaging agent for detection of hypoxic tumors on positron emission tomography (PET) [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] (Fig. 2A). Clinical studies have evaluated the feasibility and practical usefulness of ⁎Cu-ATSM for delineating hypoxic tumors and the related pathological conditions, such as the tumor's therapeutic resistance and metastatic potential [23], [24], [25], [26], [27]. The mechanism of ⁎Cu-ATSM accumulation in hypoxic tumor cells has been studied previously [13], [28] (Fig. 2B). ⁎Cu-ATSM has a low molecular weight, high membrane permeability and low redox potential, which allows penetration into cells and stability under normal oxygen-conditioned tissues; on the other hand, in tumor cells in overreduced states caused by hypoxia, ⁎Cu(II) in ⁎Cu-ATSM is reduced to ⁎Cu(I), is instantly released from the ATSM ligand and is trapped in the cells. It is also reported that the reduction process of ⁎Cu-ATSM is conducted in a manner dependent on biological reductants NADH and NADPH, and these reactions were mainly catalyzed by cytoplasmic bioreductive enzymes, NADH-dependent cytochrome b5 reductase and NADPH-dependent cytochrome P450 reductase [28]. This suggests that ⁎Cu-ATSM can act as an indicator of overreduced intracellular states generated by increase of NAD(P)H levels in hypoxic tumors.
In addition, we recently reported that 62Cu-ATSM-PET successfully visualizes stroke-like episodes in the brains of patients with MELAS syndrome [29]. This study demonstrated that lesions of stroke-like episodes in the brain of MELAS patients show high accumulation of 62Cu-ATSM while maintaining cerebral blood flow, which indicates that 62Cu-ATSM accumulates in lesions of stroke-like episodes even in the presence of oxygen. ⁎Cu-ATSM accumulation was therefore thought to be related to an overreduced state in such lesions, despite oxygenic conditions; however, this remains uncertain. In this study, to evaluate ⁎Cu-ATSM as an indicator of overreduced intracellular states in mitochondrial dysfunction, we examined the relationship between ⁎Cu-ATSM accumulation and reduced intracellular states using human mtDNA-less ρ0 cells and cybrids carrying mutated mitochondria from a MELAS patient in vitro. We then discussed the feasibility of ⁎Cu-ATSM for detection of overreduced states in mitochondrial disorders.
Section snippets
Cell lines and media
A human osteosarcoma cell line (143B) (ATCC CRL8303) and the mtDNA-depleted derivative cell line (ρ0206) were used in this study (Fig. 1). The ρ0206 cells used in this study were established by Drs. King and Attardi [10]. The ρ0206 cells have completely depleted mtDNA, but possess the same nuclear genetic background as the parental 143B cells [10]. Also, they are reported to lack synthesis of mitochondrial proteins and show little O2 consumption [10], [11], [12]. The cybrids 2SA and 2SD were
64Cu-ATSM uptake and levels of NAD(P)H in 143B and ρ0206 cells
The 64Cu-ATSM uptake by the 143B cells and ρ0206 cells under each treatment condition is shown in Fig. 3. Under normoxic conditions (20% O2), mtDNA-depleted ρ0206 cells showed 2.7-fold higher 64Cu-ATSM uptake than parental 143B cells (P<.001), whereas there was no significant difference in 64Cu-ATSM uptake between ρ0206 cells under normoxia and those under hypoxia (1% O2). The 143B cells treated under 1% O2 hypoxia and treated in growth medium containing rotenone showed 2.1-fold and 3.2-fold
Discussion
We characterized 64Cu-ATSM uptake in cells that are in overreduced states, such as mtDNA-less ρ0 cells and cybrids carrying mutated mitochondria from a MELAS patient. Based on studies in 143B and ρ0206 cells, it was revealed that cells with high levels of NADH and NADPH, such as 143B cells under hypoxia, 143B cells with inhibited mitochondrial respiration by rotenone and mtDNA-depleted ρ0206 cells, showed increased 64Cu-ATSM uptake when compared with control 143B cells under normoxic
Conclusion
Consequently, in this paper, we found that ⁎Cu-ATSM uptake reflects overreduced intracellular states, even under normoxic conditions, and ⁎Cu-ATSM is thus considered to be a promising marker for the pathophysiologic evaluation of human disorders with mitochondrial dysfunction, such as mitochondrial encephalomyopathies, and neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease. Further studies on the clinical application of ⁎Cu-ATSM-PET for these disorders are
Acknowledgments
We would like to thank the staff of the Biomedical Imaging Research Centre of the University of Fukui, Japan, for their helpful discussion. This work was funded in part by Grants-in-Aid for Scientific Research (B) (H.O.); Scientific Research on Innovative Areas (M.Y.); Young Scientists (B) (to Y.Y., M.I.); the 21st Century COE Program (Medical Science) from the Japan Society for the Promotion of Science, Japan (JSPS); and Japan Advanced Molecular Imaging Program from the Ministry of Education,
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