Cell Cycle
ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20
Functional gains in energy and cell metabolism
after TSPO gene insertion
Guo-Jun Liu, Ryan J. Middleton, Winnie Wai-Ying Kam, David Y. Chin, Claire R.
Hatty, Ronald H. Y. Chan & Richard B. Banati
To cite this article: Guo-Jun Liu, Ryan J. Middleton, Winnie Wai-Ying Kam, David Y.
Chin, Claire R. Hatty, Ronald H. Y. Chan & Richard B. Banati (2017) Functional gains in
energy and cell metabolism after TSPO gene insertion, Cell Cycle, 16:5, 436-447, DOI:
10.1080/15384101.2017.1281477
To link to this article:  https://doi.org/10.1080/15384101.2017.1281477
© 2017 ANSTO View supplementary material 
Published online: 10 Feb 2017. Submit your article to this journal 
Article views: 2261 View related articles 
View Crossmark data Citing articles: 18 View citing articles 
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=kccy20
CELL CYCLE
2017, VOL. 16, NO. 5, 436–447
http://dx.doi.org/10.1080/15384101.2017.1281477
REPORT
Functional gains in energy and cell metabolism after TSPO gene insertion
Guo-Jun Liu a,b, Ryan J. Middleton a, Winnie Wai-Ying Kam a,c, David Y. Chind, Claire R. Hattya,b,
Ronald H. Y. Chana,b, and Richard B. Banati a,b
aAustralian Nuclear Science and Technology Organisation, Lucas Heights, NSW, Australia; bFaculty of Health Science and Brain and Mind Centre,
University of Sydney, NSW, Australia; cDepartment of Health Technology and Informatics, Hong Kong Polytechnic University, Hung Hom, Hong Kong,
China; dNCRIS Biologics Facility, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, QLD, Australia
ABSTRACT ARTICLE HISTORY
Recent loss-of-function studies in tissue-specific as well as global Tspo (Translocator Protein 18 kDa) Received 3 October 2016
knockout mice have not confirmed its long assumed indispensability for the translocation of cholesterol Revised 13 December 2016
across the mitochondrial inter-membrane space, a rate-limiting step in steroid biosynthesis. Instead, Accepted 5 January 2017
recent studies in global Tspo knockout mice indicate that TSPO may play a more fundamental role in KEYWORDS
cellular bioenergetics, which may include the indirect down-stream regulation of transport or metabolic cell metabolism; energy
functions. To examine whether overexpression of the TSPO protein alters the cellular bioenergetic profile, production; mitochondria;
Jurkat cells with low to absent endogenous expression were transfected with a TSPO construct to create a patch clamp; TSPO
stable cell line with de novo expression of exogenous TSPO protein. Expression of TSPO was confirmed by
RT-qPCR, radioligand binding with [3H]PK11195 and immunocytochemistry with a TSPO antibody. We
demonstrate that TSPO gene insertion causes increased transcription of genes involved in the
mitochondrial electron transport chain. Furthermore, TSPO insertion increased mitochondrial ATP
production as well as cell excitability, reflected in a decrease in patch clamp recorded rectified K channel
currents. These functional changes were accompanied by an increase in cell proliferation and motility,
which were inhibited by PK11195, a selective ligand for TSPO. We suggest that TSPO may serve a range of
functions that can be viewed as downstream regulatory effects of its primary, evolutionary conserved role
in cell metabolism and energy production.
Introduction
Recent observations in mice with global and conditional
The translocator protein 18 kDa (TSPO) or peripheral ben- deletions of Tspo with apparently normal phenotypes in
zodiazepine receptor (PBR) is a highly conserved,1-3 independent laboratories14-17 unexpectedly failed to confirm
multi-functional,4 mitochondrial, 5 transmembrane-domain an essential role of TSPO/PBR in cholesterol import into
protein.5-8 It is thought to interact with the voltage-dependent mitochondria and steroid synthesis. Additionally, the role of
anion channel (VDAC; 32 kDa),4,9-11 the adenine nucleotide TSPO in the regulation of mPTP also failed to be con-
transporter (ANT; 30 kDa).4,9-11 and the mitochondrial perme- 16firmed. Our observations over 24 months in 700 animals
ability transition pore (mPTP).4,9-11 The protein structure of of the GuwiyangWurra (‘Fire Mouse’) strain of Tspo knock-
the bacterial homolog RsTspO8 and Bacillus cereus (BcTspO)6 out mice revealed no differences in growth rate, fertility,
has been crystalized. Based on in vivo and in vitro observations, cholesterol transport and steroid biosynthesis, or blood lev-
the ‘translocation’ of cholesterol across the mitochondrial els of the endogenous TSPO ligand, protoporphyrin IX
inter-membrane space has been identified as a rate-determining (PPIX) compared with littermate wild-type animals. How-
step in steroid biosynthesis and as the most prominent, and ever, a decreased level of ATP production by mitochondria
essential for life, function of the TSPO,4,12 thus providing the in microglia extracted from the knockout animals indicates
rationale for its renaming as the Translocator Protein 18 kDa the potential existence of a latent phenotype that may come
(TSPO). The conceptual history of the ‘translocator protein’ to the fore under disease rather than normal physiological
and scientific publication trends (as reviewed by13) illustrate conditions.14 As a complement to the loss-of-function phe-
the emerging role of this protein as a diagnostic biomarker of notypic data in mitochondrial energy production from the
active disease in the nervous system, and a potential therapeutic GuwiyangWurra Tspo¡/¡ animals,13,14,18,19 we re-examined
target for a broad range of inflammatory, neurodegenerative, the role of the TSPO/PBR in energy production and cell
neoplastic, metabolic and behavioral diseases.13 metabolism in an in vitro model.
CONTACT Guo-Jun Liu gdl@ansto.gov.au; Richard B. Banati rib@ansto.gov.au Australian Nuclear Science and Technology Organisation, Locked Bag 2001,
Kirrawee DC, NSW 2232, Australia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy.
Supplemental data for this article can be accessed on the publisher’s website.
© 2017 ANSTO.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
CELL CYCLE 437
Figure 1. Confirmation of TSPO expression in Jurkat cells after stable transfection of TSPO expressing plasmids. (A) Relative abundance of exogenous TSPO mRNA expres-
sion in genetically modified Jurkat cell lines. Bar graph shows exogenous TSPO mRNA levels normalized to GAPDH and b-actin mRNAs. Expression of endogenous TSPO
mRNA in MDA-MB-231 cells was set to 1.0. Jurkat cells transfected with TSPO expressed a comparable level of TSPOmRNA (exogenous) with that of the MDA-MB-231 cells
(endogenous). No exogenous TSPO mRNA could be detected in wild-type and empty plasmid Jurkat cells. The data are presented as means § SD (n D 4). (B) The level of
TSPO protein expression in Jurkat cells was measured by radioligand binding using the TSPO specific ligand [3H]PK11195. The absence of specific binding in the wild-
type and empty plasmid Jurkat cells confirms the negiligible level of TSPO protein. TSPO-Jurkat cells exhibited specific [3H]PK11195 binding though its Bmax is about one-
third of that obtained from the positive control MDA-MB-231 cells. (C) Endogenous and exogenous expression of TSPO in Jurkat cells was determined by immunostaining
with a TSPO specific antibody (green). There was no positive immunostaining observed in both wild-type and empty plasmid transfected Jurkat cells. However, strong
staining with localized intracellular distribution along with mitochondria (red) was observed in TSPO-Jurkat cells. The blue color indicates the staining of nuclei. The yellow
fluorescence in the right image demonstrates the overlap between TSPO and mitochondria. Scale bars: 10 mm.
To this end, we stably transfected a T-cell line, Jurkat Results
cells, with the human TSPO gene. Jurkat cells have low or
Confirmation of stable TSPO transfection
absent expression of TSPO due to a high degree of pro-
moter methylation as revealed in our previous study.20 Con- Jurkat cell lines, derived from human leukemia cells, have pre-
firming the stable expression of the inserted exogenous viously been reported to have very low or absent TSPO expres-
human TSPO gene by PCR, RT-qPCR, membrane receptor sion.21 Wild-type Jurkat cells used in this study were 82%
binding with TSPO-specific ligand [3H]PK11195, and identical to Jurkat Clone E6–1 (ATCC: TIB-152), determined
immunohistochemistry with a TSPO antibody, we describe using short tandem repeat (STR) profiling (Table S1). The pro-
significant gain-of-function effects in the mitochondrial moter of TSPO in our Jurkat cell line is highly methylated in
electron transport chain, cell membrane excitability, as well the region surrounding the transcription start site,20 resulting
as marked changes in the highly energy-demanding func- in the expression of TSPO mRNA and TSPO protein at barely
tions of cell proliferation and motility. detectable levels in the wild-type Jurkat cells (Fig. 1).
438 G.-J. LIU ET AL.
Figure 2. Changes in mRNA abundance following TSPO transfection. (A) Microarray analysis was used to detect differences in gene expression between the 3 Jurkat cell
types. GSEA results comparing empty plasmid and TSPO-Jurkat cells were visualised using Enrichment Map Cytoscape Plug-in. Gene sets are represented by circular nodes
that are proportional to the number of genes present in the gene set. The thickness of the lines between nodes is proportional to the overlap between gene sets. Nodes
with highly similar gene sets are placed close to each other to form clusters. Red nodes are upregulated and blue nodes are downregulated. Expanded clusters in high
resolution are shown in Figs. S1–S6. (B) Heat map of the leading edge genes from the mitochondrial electron transport chain cluster. The top 60 genes are shown with
high expression represented in red. (C) Gene expression of mitochondrial respiratory chain complex IV (COX IV, subunit 2) in the TSPO-Jurkat cells was decreased by the
TSPO ligand PK11195 (100 nM) when compared with control (without PK11195 but containing 0.1% DMSO). The same concentration of PK11195 had no effect on the
wild-type and empty plasmid Jurkat cells.
Stable TSPO transfection of Jurkat cells (TSPO-Jurkat cells) control, the level of TSPO mRNA (Fig. 1A) was compared with
was initially confirmed by RT-qPCR using primers specifically the MDA-MB-231 cell line, a breast cancer cell line with high
designed for exogenous TSPO mRNA. Since the exogenous TSPO mRNA and protein expression.22,23
TSPO mRNA was codon-optimised, primers could be designed The level of TSPO protein in wild-type, empty plasmid-
to allow discrimination between exogenous and endogenous transfected and exogenous TSPO-transfected Jurkat cells, and
TSPO variants (Table S2). Insertion of TSPO into wild-type positive control MDA-MB-231 cells was measured by mem-
Jurkat cells and selection to create a stable cell line did not brane receptor binding using the TSPO ligand [3H]PK11195.24
change the Jurkat cell profile that was still 82% identical to No difference between total and non-specific binding of [3H]
Jurkat, Clone E6–1 (ATCC: TIB-152) (Table S3). As a positive PK11195 was found in the wild-type Jurkat cells, confirming
CELL CYCLE 439
the barely detectable level of endogenous TSPO protein in these
cells (Fig. 1B). This was also the case for Jurkat cells transfected
with the empty plasmid (Fig. 1B). In contrast, Jurkat cells trans-
fected with TSPO showed specific [3H]PK11195 binding
(Fig. 1B). Scatchard analysis revealed a maximal number of
binding sites (Bmax) of 4.8 § 0.3 pmol/mg protein with a disso-
ciation constant (KD) of 17.8 § 2.2 nM in the TSPO-Jurkat
cells, which is approximately a third of the expression levels
seen in the highly expressing tumor cell line MDA-MB-231
(Bmax of 15.4 § 0.8 pmol/mg protein and a Kd of 8.9 §
1.1 nM), similar to previous reports.23,25 Scatchard analysis of
wild-type and empty plasmid-containing Jurkat cells was not
possible due to the absence of specific binding.
Expression of TSPO in the wild-type, TSPO-transfected, and
empty plasmid-transfected Jurkat cells was examined by immu-
nostaining using a TSPO specific antibody.14,17 Undetectable to
faint non-localized background staining was observed in wild-
type (Fig. 1C) and empty plasmid Jurkat cells (Fig. 1C). In con-
trast, staining was clearly visible in the TSPO-Jurkat cells
(Fig. 1C). The intracellular distribution of TSPO expression
closely matched that of mitochondria, indicating that the exog-
enous TSPO co-localized with mitochondria in the transfected
Jurkat cells (Fig. 1C). The morphology of mitochondria in the
wild-type and empty plasmid Jurkat cells did not show a dis-
cernible difference compared with TSPO-Jurkat cells (Fig. 1C). Figure 3. TSPO increased mitochondrial ATP production. (A) Averaged time-course
Thus, TSPO overexpression in Jurkat cells did not appear to of ATP production from permeabilised Jurkat cells was measured with luciferin-
induce mitochondrial stress, which might cause non-speci c luciferase and a microplate reader. ATP production after injection of 2 mM ADPfi was markedly increased in all 3 cell types (wild-type, empty plasmid and TSPO-
differences from the control cells. Jurkat cells). ATP production in TSPO-Jurkat cells increased at a greater rate than
that in wild-type and empty plasmid Jurkat cells. (B) Accumulated ATP following
injection of ADP in TSPO-Jurkat cells, calculated by subtracting the baseline (before
Genes involved in the mitochondrial electron transport ADP injection), was significantly greater than that observed in wild-type and
chain are upregulated following TSPO transfection empty plasmid Jurkat cells.
Gene set enrichment analysis (GSEA) and Enrichment Map
were used to identify functional signatures within the expres-
sion data. As shown in Fig. 2A, B, TSPO transfection resulted (25 mM) was used to inhibit adenylate kinase which prevents
in an enrichment of clusters related to translation, transcrip- the inter-conversion of ATP to ADP and AMP. Iodoacetate
tion, protein folding, nucleotide excision repair, the electron (25 mM) was used to inhibit glyceraldyde-3-phosphate dehy-
transport chain and the proteasome. The role of TSPO in drogenase to block glycolysis-induced ATP production. Biolu-
energy production and cell metabolism, as indicated by the minescent luciferin-luciferase was then used to detect ATP by
upregulation of genes associated with these functions, was sub- generating photons correlating with the number of ATP mole-
sequently examined. cules present.
To further examine changes in the mitochondrial respira- ATP production increased markedly after the addition of
tory chain following TSPO gene insertion, RT-qPCR was used 2 mM ADP (Fig. 3A) in all 3 cell types (wild-type, empty
to detect mRNA of complex IV of the mitochondrial respira- plasmid and TSPO-Jurkat cells). However, the ATP produc-
tory chain (cytochrome c oxidase). The mRNA level of the tion in TSPO-Jurkat cells rose significantly higher (accumu-
COX IV subunit 2 in TSPO overexpressed Jurkat cells was lated ATP: 5.6 § 0.28 mg/mg protein, n D 6 wells from 3
higher, but not significantly, than those of the wild-type and batches of cultures) than in wild-type (3.3 § 0.14 mg/mg
empty plasmid Jurkat cells (Fig. 2C). The mRNA level of the protein, n D 3 wells, p < 0.001) and empty plasmid Jurkat
COX IV subunit 2 was significant decreased by the TSPO ligand cells (3.5 § 0.30 mg/mg protein, n D 6 wells, p < 0.001)
PK11195 at 100 nM (p < 0.05, n D 4), while the mRNA levels (Fig. 3A, B). No apparent ATP production was observed in
of the COX IV subunit 2 in wild-type and empty plasmid Jurkat either TSPO transfected, wild-type or empty plasmid Jurkat
cells were not significantly altered by PK11195 at the same con- cells after the addition of the buffer solution used for dis-
centration (Fig. 2C). solving ADP and luciferin-luciferase, or in cell-free lucif-
erin-luciferase solution after addition of ADP (Fig. 3A).
Moreover, there was no observable bioluminescence in the
Increased mitochondrial ATP production in Jurkat cells
Jurkat cells in the absence of luciferin-luciferase, nor any
with TSPO transfection
observable ATP contamination in the pure ADP prepara-
The role of TSPO in ATP production was examined using per- tion. The results suggest that TSPO transfection increased
meabilised Jurkat cells with intact mitochondria. Ap5A mitochondrial ATP production.
440 G.-J. LIU ET AL.
Increased cell motility in TSPO transfected Jurkat cells
The motility of TSPO-Jurkat cells was examined by measuring
spontaneous activity and induced cell migration through
Transwell (Boyden) chambers. TSPO-Jurkat cells (38 § 2.6
cells, n D 13 Transwells) had significantly higher spontaneous
movement/migration than wild-type (10 § 1.2 cells, n D 11
Transwells, p < 0.001) and empty plasmid Jurkat cells (13 §
2.5 cells, n D 11, p < 0.001) (Fig. 4B), as determined by the
number of cells passing through the Transwell chamber. There
was no significant difference in spontaneous movement/migra-
tion between wild-type and empty plasmid Jurkat cells
(Fig. 4B). The spontaneous activity of TSPO-Jurkat cells, but
not wild-type and empty plasmid Jurkat cells, was significantly
attenuated by the TSPO specific ligand PK11195 at 100 nM (23
§ 1.8 cells, n D 9 for TSPO C PK11195, p < 0.001; Fig. 4B).
The addition of 0.1% foetal bovine serum (FBS), which acts
as a chemo-attractant, significantly increased the number of
cells that migrated through the Transwell membrane for all 3
cell types – wild-type, empty plasmid and TSPO-Jurkat cells,
compared with cells in DMEM only (Fig. 4C). However, the
FBS-induced increase in the migration of TSPO-Jurkat cells
was significantly attenuated by the addition of 100 nM
PK11195, which had no effect on the wild-type and empty plas-
mid Jurkat cells (277 § 24 cells, n D 6 for FBS C TSPO-Jurkat
cells compared with 115 § 5 cells, n D 9 for PK11195 C FBS C
TSPO-Jurkat cells, p < 0.001; Fig. 4C). This suggests that the
effect of PK11195 on motility is TSPO-specific.
Decreased outward rectified KC currents in TSPO
transfected Jurkat cells
Changes in cell excitability as a result of altered membrane
Figure 4. TSPO increased cell proliferation and motility. (A) Time course of Jurkat potential were determined by patch clamp measurements of
cell proliferation normalized to day 0, i.e., 1 hour after plating each of the cell the electrophysiological cell membrane properties in a whole-
types. TSPO-Jurkat cells proliferated significantly faster than wild-type and empty cell mode configuration. Episodic traces of voltage-activation
plasmid Jurkat cells at all time points measured (i.e. days 1–3). There was no differ-
ence in proliferation rate between wild-type and empty plasmid Jurkat cells. (B) were applied from ¡100 mV to C80 mV, increasing by 20 mV
TSPO-Jurkat cells had significantly higher spontaneous movement/migration com- increments. The membrane potential was held at ¡60 mV.
pared with wild-type and empty plasmid Jurkat cells measured with the Boyden Outwardly rectified whole-cell currents were then recorded in
chamber (Transwell). The inhibitory effect of TSPO specific ligand PK11195 on
spontaneous movement/migration was only seen in the TSPO-Jurkat cells, indicat- all 3 cell types (i.e., wild-type, empty plasmid and TSPO-Jurkat
ing the effect is TSPO-specific. (C) Foetal bovine serum (FBS, 0.1%) in DMEM, a cells; Fig. 5A). The shape and amplitude of voltage-induced
chemo-attractant, non-specifically attracted all 3 types of Jurkat cells. However, outward currents in wild-type (220 § 41 pA at 0 mV, n D 4)
PK11195 at 100 nM significantly decreased FBS-induced cell migration in TSPO-
Jurkat cells only. and empty plasmid (197 § 20 pA at 0 mV, n D 10) Jurkat cells
are comparable, while the amplitude in TSPO-Jurkat cells was
markedly smaller (78 § 17 pA at 0 mV, n D 12, p < 0.001
when compared with wild-type or empty plasmid Jurkat cells).
It should be noted that outward currents in empty plasmid
Increased cell proliferation in TSPO transfected Jurkat cells
Jurkat cells peaked at ¡20 mV (261 § 51 pA, n D 10), while in
Observed over 3 days, all 3 cell types proliferated exponentially wild-type and TSPO-Jurkat cells it peaked at 0 mV (Fig. 5B).
(R2 D 0.986, y D 0.9342e0.282x for wild-type; R2 D 0.987, y D The amplitude of voltage-gated outward currents in TSPO-
0.955e0.253x for empty plasmid Jurkat cells and R2 D 0.993, y D Jurkat cells was significantly smaller (particularly at ¡20 mV to
0.9637e0.282x for TSPO-Jurkat cells). However, the TSPO-Jurkat 20 mV) than both wild-type and empty plasmid Jurkat cells
cells proliferated significantly faster than wild-type and empty (Fig. 5B). This suggests that TSPO transfection increased mem-
plasmid Jurkat cells across all measurements (i.e. 24, 48 and brane excitability by decreasing the voltage-activated outwardly
72 hours, p < 0.01) (Fig. 4A). There was no difference in prolif- rectified current.
eration rate between wild-type and empty plasmid Jurkat cells To examine whether the outward rectified currents were due
(Fig. 4A). to the efflux of KC, current traces from empty plasmid Jurkat
CELL CYCLE 441
Figure 5. TSPO decreased whole-cell outward recti Cfied K currents. (A) Episodic current traces of voltage-activated (from ¡100 mV to C80 mV with 20 mV incre-
ment for A, C) outward currents recorded in wild-type, empty plasmid, and TSPO-Jurkat cells. The membrane potential was held at ¡60 mV. The shape and ampli-
tude of voltage induced outward currents in wild-type and empty plasmid Jurkat cells were comparable, while the amplitude in TSPO-Jurkat cells was smaller. (B)
Current-voltage (I-V) curves of voltage-activated currents shown in (A). The amplitude of voltage-gated outward currents in TSPO-Jurkat cells was significantly
smaller at ¡20 mV to 20 mV than either wild-type or empty plasmid Jurkat cells. (C) Current traces of empty plasmid Jurkat cells in normal bath solution (contain-
ing NaCl) and normal pipette solution (containing KCl), in normal bath solution with addition of 20 mM TEA-Cl, in normal pipette solution with addition of 20 mM
TEA-Cl, and in a pipette solution with CsCl replacing KCl. TEA in the bath solution partly inhibited the voltage-gated outward current, but a greater effect was
seen when TEA was in the pipette solution. CsC replacement of KC in the pipette solution abolished the outward currents. (D) The I-V curves of corresponding
traces shown in (C).
cells with the addition of KC channel inhibitors, TEA-Cl and abnormalities in these mice in terms of reproduction and life
CsCl were studied. Adding TEA-Cl (20 mM to replace equal span up to 24 months. Our study using microglia isolated from
molar amounts of NaCl) to the bath solution partly inhibited global Tspo knockout mice had demonstrated that mitochon-
the voltage-gated outward current, but a greater effect was seen drial respiration and ATP production may be different from
when TEA-Cl (20 mM) was in the pipette solution (Fig. 5C, D). wild-type littermates.14 This finding has prompted us to further
CsCl replacement of KCl in the pipette solution abolished the investigate the functions of TSPO in mitochondrial energy/
outward currents (Fig. 5C, D). These results suggest that the ATP production and associated high energy intensive cell func-
outwardly rectified whole-cell currents were due to the efflux of tions. In contrast to the Tspo knockout method, a loss-of-func-
KC. tion approach, we opted for a gain-of-function approach by
inserting an additional TSPO gene into Jurkat cells, a human T-
lymphocyte leukemia cell line, which we had observed to have
Discussion
a barely detectable TSPO protein expression.
TSPO/PBR-dependent regulation has been reported for a num- The wild-type Jurkat cells used for transfection of the
ber of biological processes, including steroid biosynthesis,4,26 human TSPO gene in this study have shown TSPO
cell division,23,27 energy metabolism,9,13,14,28,29 production of expression at barely detetctable levels, confirmed by radioligand
reactive oxygen species,9,11,30 cell apoptosis,9,31-33 immune receptor membrane binding with [3H]PK11195 and immuno-
responses,34,35 and hematopoiesis.36 It is well established that cytochemistry with the most specific TSPO antibody (Abcam)
the expression of TSPO increases significantly after injury and that we have used in our laboratory. A high degree of TSPO
under neuropathological conditions, such as multiple sclerosis, promoter methylation in wild-type Jurkat cells contributes to
or neurodegenerative conditions such as Alzheimer’s dis- the low TSPO expression observed in these cells.28 Our barely
ease.13,37-46 Recently, the global and conditional Tspo knockout detectable TSPO expression is consistent with the finding of
mouse models created in different laboratories have not con- Bertomeu et al.21 who demonstrated negligible TSPO expres-
firmed the previously identified putative function of TSPO in sion in the clone E6.1 Jurkat cells and also overexpressed TSPO
cholesterol transport, biosynthesis, and the mitochondrial per- for studying the role of TLN-4601, a farnesylated dibenzodiaze-
meability transmission pore (mPTP).14-17,47 There were also no pinone. The affinity constant (Kd) for receptor membrane
442 G.-J. LIU ET AL.
binding with [3H]PK11195 in the TSPO overexpressed Jurkat overexpression of TSPO along with positive cell metabolism/
cells are in the low nano-molar range in both studies. However, energy production negatively correlates with the level of expres-
our study does not support the ndings by Costa et al,48fi who sion of AMPK/sirtuins. This also means that high expression
have shown TSPO expression in Jurkat cells. The differences levels of AMPK and sirtuins in reduced energy expenditure
are (i) they used Jurkat cells sourced from the Interlab Cell Line and halted cell cycles should correlate with lower TSPO expres-
Collection without providing the clone name; (ii) unusual bind- sion. To validate this conjecture, the levels of AMPK/sirtuins in
ing of [3H]PK11195 with Kd values in the micro-molar range; TSPO over-expressing Jurkat cells and in TSPO knockout ani-
(iii) all their studies on TSPO protein expression (Western blot, mals needs to be examined.
immunocytochemistry and electron microscopy) were based The increased cell proliferation and motility observed in
on the use of their TSPO antibody, which showed diffused TSPO-Jurkat cells is consistent with observations in other can-
staining throughout whole cells (even though they claimed cer cell lines.23,49,58-60 TSPO overexpression in C6 rat glioblas-
mitochondrial and nuclear membrane co-localization). This toma cells increases cell motility and proliferation.61
diffused staining is in stark contrast to our results in TSPO Overexpression of TSPO in the breast cancer cell line MCF7
overexpressed Jurkat cells which demonstrated mitochondrial also increases cell proliferation/migration and inhibits Mam-
co-localization of TSPO using a known highly specific TSPO mary Epithelial morphogenesis.62 However, these results
antibody. should be interpreted carefully as the cells already had high
The successful transfection of TSPO was confirmed by endogenous TSPO expression before they were manipulated,
PCR, RT-qPCR, membrane receptor binding with the TSPO- possibly resulting in different functional changes compared
specific ligand [3H]PK11195, and immunohistochemistry with cell lines with little to no endogenous TSPO.
with a TSPO antibody. We con Cfirmed that the TSPO protein Outward rectified K channel currents were recorded in
in transfected cells was localized to the mitochondria. Using whole Jurkat cells, and were found to be significantly
microarray gene expression profiling and gene set enrich- decreased in amplitude in TSPO-Jurkat cells. It is possible
ment analysis (GSEA), we found that a set of genes involved that increased ATP production could cause increased cell
in mitochondrial energy production via the mitochondrial excitability by decreasing whole-cell outward recti Cfied K
electron transport chain were upregulated in the TSPO- channel currents. It is also possible that the currents might
transfected Jurkat cells. This upregulation was associated be ATP sensitive KC channel (KATP) currents as they have
with increased mitochondrial ATP synthesis and an increase the same outward rectified shape,63,64 although this needs to
in cell excitability by decreased recti Cfied K channel currents. be confirmed using the KATP channel blocker glibencli-
These contributed to the increase in cell proliferation and made.63 Mitochondria also possess KATP channel cur-
motility. rents65,66 that may contribute to whole cell KATP currents,
The finding that the expression of TSPO correlates with which could directly influence other cellular functions (i.e.
ATP production and the upregulation of genes involved in ATP secretion, cell proliferation and motility).
mitochondrial respiration are consistent with our previous The TSPO/PBR antagonist PK11195 has been confirmed as
report using global Tspo knockout GuwiyangWurra (‘Fire a highly selective TSPO ligand using the global Tspo knockout
Mouse’) animals.14 In the microglia from Tspo knockout mice, GuwiyangWurra mouse.14 However, it has been reported that
we observed lower ATP production and basal oxygen con- PK11195 may exert functions independently from TSPO,47
sumption.14 Similar findings on the role of TSPO in ATP pro- particularly at higher concentrations. Using Langmuir mono-
duction have been previously reported.9,11,49-54 The observed layers, quartz crystal microbalance with dissipation monitoring
TSPO–dependent modulations in energy metabolism have and neutron reflectometry, we have previously investigated the
been suggested to occur through its interaction with VDAC on behavior of PK11195 in lipid bilayers with and without inte-
the outer mitochondrial membrane and ANT on the inner grated TSPO,67 indicating that there might be a synergistic
mitochondrial membrane. Krestinina et al.51 demonstrated that effect resulting from the oriented insertion of PK11195 into the
TSPO ligands can modulate energy production in mitochon- bi-lipid layer and its high affinity binding to TSPO. In the pres-
dria by controlling phosphorylation of the FoF1-ATPase sub- ent study, we show that PK11195 at concentrations less than
unit c. TSPO may modulate ATP synthase via a direct 10 mM has a significant antagonistic effect on the mRNA level
interaction between the ATP ‘synthasome’ (composed of ATP of COX IV subunit 2 and cell motility. It would be of great
synthase, phosphate carrier, and ANT) and the PBR complex interest to conduct further studies to determine whether other
composed of TSPO, VDAC, and ANT.55,56 Veenman et al.9,10 TSPO ligands, which do not bind to the PK11195 binding site
and Zeno et al.11 have suggested that reactive oxygen species or CRAC domain of TSPO protein, have similar effects. These
(ROS) generation is modulated by TSPO through regulation of ligands include endogenous ligands, such as cholesterol and
the Fo subunit of FoF1 ATP(synth)ase. protoporphyrin IX, and synthetic ligands, such as benzodiaze-
Both AMP-activated protein kinase (AMPK) and sirtuins pine Ro5–4864. The binding sites of Ro5–4864 and PK11195
(NADC-dependent histone/protein deacetylases) modulate on TSPO have distinct but overlapping binding sites that
energy balance in cells.57 They are activated by reduced cellular involve amino acid residues on the cytoplasmic loop and the C-
energy levels (e.g. increased AMP/ATP ratio).57 It would be of terminal.13
great interest to conduct future studies to examine the relation- In summary and outlook, the overexpression of TSPO in
ship between TSPO and AMPK/sirtuins, which has not yet Jurkat cells with barely detectable TSPO expression was suc-
been documented. Based on the reported functions, it can be cessfully achieved by stable transfection with a functional
speculated that under normal physiological conditions, human TSPO gene. This resulted in a gain-of-function
CELL CYCLE 443
compared with the non-transfected wild-type Jurkat cells, char- Total RNA was extracted from each of the Jurkat cell lines
acterized by increased cell metabolism, energy production, cell using the PureLink RNA mini kit (Invitrogen) following the
cycle, motility and cell membrane excitability. We speculate manufacturer’s protocol. The isolated RNA was further treated
that the broad range of reported regulatory effects associated with DNase using the PureLink DNase Set (Invitrogen), to
with the mitochondrial TSPO may be driven by its known evo- ensure the removal of contaminating DNA before RT-qPCR.
lutionary role in energy -the current study describes a range of The concentration of the RNA was determined using a Nano-
cellular effects due to the insertion of a TSPO gene, it is limited Drop 2000c Spectrophotometer (Thermo Fisher Scientific,
in regard to the precise effect of TSPO on the cell’s bioenerget- Waltham, MA, USA). The purity of the extracted RNA was
ics profile. Extensive studies, including the probing of the elec- assessed spectrophotometrically using the A260/A280 ratio,
tron transport chain under different substrate conditions, while RNA integrity was assessed using the 28s/18s ratio after
pharmacological dose range studies, and different compounds agarose gel electrophoresis.
across a range of cells types, are required to determine which First-strand cDNA was synthesized from 100 ng of total
functions of the TSPO are additional rather than merely resul- RNA by first incubating for 5 minutes at 65
C with 0.5 mM
tant from its potential primary role in bioenergetics. dNTP mix and 2.5 mM of oligo(dT)20 primer, this mix was
then placed on ice for at least 1 minute. After adding 1 x RT
buffer, 5 mM MgCl2, 10 mM DTT, 40 units RNaseOUT andMethods and materials 200 units of Superscript III (Invitrogen), the reaction (total vol-
Cell culture ume 20 ml) was incubated for 50 minutes at 50
C. The reverse
transcription was terminated by incubating the samples at
The wild-type Jurkat cells were donated by Professor Ian 85
C for 5 minutes. Samples without the addition of Super-
Campbell at the University of Sydney, Australia. Jurkat cells
C script III were included as control for genomic DNA contami-transfected with pcDNA3.1( ) empty vector, pcDNA3.1
C nation. The freshly prepared cDNA was further diluted by( )-TSPO (TSPO), and wild-type Jurkat cells were maintained DEPC-treated water so that approximately 1/20 of the original
in Dulbecco’s Modified Eagle’s Medium (DMEM), supple- stock was used for the subsequent RT-qPCR.
mented with 10% v/v fetal calf serum (FCS) (Invitrogen, Carls-
bad, CA, USA) and 2 mM glutamine in 5% CO2 at 37
C before
and during functional studies. The TSPO positive control cells Real-time PCR
MDA-MB-231 (a human breast cancer cell line) were cultured TSPO and COX IV subunit 2 mRNA expression in each of the
under the same conditions. Jurkat cell lines was assessed using a CFX 384 Real-Time PCR
Detection System (BioRad, Hercules, CA, USA). Primer-
Plasmid construction BLAST was used to design primers that detect the exogenous
and endogenous TSPO mRNA (Table S2). The primers for
Optimisation and synthesis of the human TSPO coding COX IV subunit 2 PCR were used according to a previous pub-
sequence, and subsequent cloning into the pMA vector, was lication68 (Table S2). Diluted cDNA (1 ml) was added to 4 ml of
performed by GeneArt (Life Technologies). The TSPO reaction mixture containing 2.5 ml of SsoFast supermix (Bio-
sequence in pMA was then subcloned into pcDNA3.1(C) (Invi- Rad) and forward and reverse primers at a final concentration
trogen). DNA sequencing was used to verify all of the con- of 0.5 mM each. PCR conditions were empirically optimised
structs (AGRF, Sydney, Australia). The plasmids were and the efficiency was further assessed at the selected annealing
linearized before stable transfection. temperature. The PCR conditions were 98
C for 30 seconds,
followed by 45 cycles at 98
C for 5 seconds and 63
C for 10 sec-
Cell transfection and selection onds. At the end of the 45
th cycle, the temperature was raised to
72
C for 10 minutes to ensure complete extension of the PCR
Jurkat cells were transfected using the Amaxa Nucleofection products. A melt curve analysis was subsequently performed to
system according to instructions provided by the manufac- confirm the specificity of the results. The PCR was repeated
turer (Lonza, Basel, Switzerland). Briefly, 1 £ 106 cells were without melt curve analysis for resolving the PCR product on a
resuspended in Nucleofector Solution V, mixed with 2 mg 1.5% agarose gel.
of plasmid DNA and transfected using program X-001. b-actin (ACTB)68 as well as glyceraldehyde 3-phosphate
To create stable cell lines, G418 was added to the culture dehydrogenase (GAPDH)69 were used as internal standards (i.e.
media 2 days after transfection. G418 was added at 1 mg/ housekeeping genes (Table S2)). Samples were run in tripli-
mL to the culture media of cells transfected with the TSPO cates. The relative expression of target genes was quantified
containing plasmid or the empty vector pcDNA3.1(C). using comparative Ct analysis incorporated into the CFX Man-
Once stable cell lines had been produced, cells were main- ager Software (version 1.5) (BioRad).
tained in media supplemented with a lower concentration
of G418 (0.5 mg/mL).
Microarray analysis
Total RNA was extracted from wild-type, empty plasmid
RNA isolation and reverse transcription
and TSPO-Jurkat cells using TRIzol and the PureLink RNA
Wild-type and stable transfected Jurkat cells were plated at mini kit (Invitrogen) with on-column DNase treatment fol-
1.6 £ 105 cells per well in 24-well plates and grown overnight. lowing the manufacturer’s protocol. The quality of the RNA
444 G.-J. LIU ET AL.
was assessed using an Agilent 2100 Bioanalyzer. Sample Cell permeabilisation
labeling, hybridization to Affymetrix Human Gene 1.0 ST
Jurkat cells used for measuring mitochondrial ATP produc-
Arrays, washing and scanning was performed by the Rama-
tion were permeabilised according to Milakovic and Johnson
ciotti Centre for Gene Function Analysis at the University
(2005).71 Briefly, Jurkat cells were pelleted and resuspended
of New South Wales. Two biological replicates for each
in media A (20 mM HEPES, 10 mM MgCl , 250 mM
sample were processed. The GenePattern platform (Broad 2
sucrose, pH 7.3). The cells (1£107 cells for each cell line)
Institute, MIT) was used to analyze the microarray data.
were then incubated in media A containing digitonin
Normalization was carried out using RMA via the Normali-
(Sigma-Aldrich, final concentration 100 mg/ml) and mixed
zeAffymetrixST module followed by the identification of
for 1 minute at room temperature. After pelleting the perme-
differentially expressed genes using the LimmaGP module
ablised Jurkat cells, the digitonin-containing media A was
(http://pwbc.garvan.unsw.edu.au/gp). Using the t-statistic
removed, and the permeabilised cells washed with 15 ml of
from LimmaGP, gene set enrichment analysis (GSEA) was per-
media A for 3 times to remove any remaining digitonin. The
formed in pre-ranked mode with 1000 permutations. The
permeablised Jurkat cells were finally resuspended in respira-
GSEA results were visualized using the Cytoscape plug-in
tory buffer (media A plus 0.72 mM KH PO , 1.28 mM
Enrichment Map. Only gene sets with a FDR value of less than 2 4
K HPO , pH 7–7.1, plus 1% BSA) in preparation for detect-
0.05 are shown. 2 4
ing mitochondrial ATP production.
Cell membrane preparation and radioligand binding
Detection of mitochondrial ATP production
Membranes of wild-type, empty plasmid and TSPO-Jurkat cells
for radioligand binding experiments were prepared as follows. ATP production was determined in Jurkat cells that were per-
Cells were lysed in 20 volumes of ice-cold 50 mM Tris-HCl pH meabilised but had intact mitochondria. Other possible ATP
7.4 using 50 strokes of a Potter-Elvehjem homogenizer. Cell production pathways, besides aerobic metabolism, were
lysate was centrifuged at 48 000 £ g for 20 minutes at 4
C and blocked with Ap5A (25 mM) to inhibit adenylate kinase, which
supernatant discarded. The membrane fraction collected was prevents the inter-conversion of ATP, ADP and AMP, and
homogenized in fresh buffer, centrifuged, and the final mem- with iodoacetate (25 mM) to inhibit glyceraldyde-3-phosphate
brane preparation resuspended in 50 mM Tris-HCl pH 7.4. dehydrogenase to block glycolysis-induced ATP production.
The protein concentration of membrane preparations was mea- ATP production was detected with the luciferin-luciferase bio-
sured using the BCA protein assay (Thermo Fisher Scientific). luminescence assay, which generates photons correlating to the
71
The saturation binding of [3H] PK11195 in MDA-MB-231 cells number of ATP molecules present.
and wild-type, empty plasmid, and TSPO-Jurkat cells was per- The permeabilised Jurkat cells were re-suspended in the
formed according to Banati et al.14 respiratory buffer containing Ap5A and iodoacetate and respi-
ratory substrates including 10 mM glutamate (for complex I of
the respiratory chain), 10 mM succinate (for complex II),
Determination of TSPO expression and subcellular 10 mM ascorbate and 25 mM TMPD (for complex IV). Ali-
distribution quots of 50 ml (5£106 cells/ml density) were placed into an
Immunocytochemistry was performed according to Liu et al.70 optical Nunk 96-well plate (Thermo Scientific) and 50 ml ATP
Briefly, cells from each of the Jurkat cell lines were allowed to assay mix (luciferin-luciferase, Sigma-Aldrich) was added.
settle-down onto glass coverslips and were then fixed with 3.7% After a 30 minute incubation (to allow luciferase to consume
paraformaldehyde for 10 minutes. After 3 washes with phos- the free ATP in the aliquots), the plate was placed in the BMG
phate buffered saline (PBS, 5 minutes per wash), the cell mem- plate reader. After an initial recording, 200 mM of ultra-pure
branes were permeabilised with 0.1% Triton X-100 for ADP (> 99.99%, Cell Technology, Mountain View, CA, USA)
1 minute. The cells were then incubated with 2% bovine serum was injected, and the recording continued for 2 more minutes.
albumin (BSA) for 30 minutes before being incubated with pri- At least 3 repeats for each cell line were performed. The
mary antibodies (rabbit anti-TSPO monoclonal antibody amount of protein from permeabilised cells was also quantified
(Abcam, Cambridge, UK) and mouse anti-human/mouse mito- using a NanoDrop (Thermo Scientific) with the Pierce BCA
chondrial electron transport chain complex IV antibody protein assay kit (Thermo Scientific). The photon emission was
(Abcam) overnight at 4
C. After 3 washes, the cells were incu- normalized against the amount of protein in each sample.
bated with the secondary antibodies Alexa Fluor (AF) 488-con-
jugated goat anti-rabbit antibody (Life Technologies) and
Cell proliferation
AF594-conjugated goat anti-mouse antibody (Life Technolo-
gies) for 1 hour. After 3 washes, the cells on coverslips were Jurkat cell proliferation was measured using a Scepter cell
mounted with ProLong Gold antifade reagent containing DAPI counter (Millipore, Billerica, MA, USA), which is based on
(Life Technologies) and viewed under a BX61WI Olympus Coulter Count-impedance measurement technology. Wild-
microscope. Images were acquired with a digital camera (Cool- type, empty plasmid and TSPO-Jurkat cells were all seeded at
SNAP, Photometrics, Tucson, AZ, USA) and the Image InVivo the same density of 3 £ 105 cells/ml before the experiment.
program (Photometrics). Deconvolution of images was per- Three days later, cells from each Jurkat cell line were counted
formed with the Auto Deblur program (Photometrics) and fur- with the Scepter cell counter and plated at 3 £ 105 cells/ml into
ther processed with ImageJ (NIH, Baltimore, MD, USA). 24-well culture plates (0.5 ml/well, 4 repeats for each cell line
CELL CYCLE 445
and for each time point). One hour after plating, the cells from unpaired Student’s t-tests were also used to evaluate statistical
each cell line were counted with the Scepter cell counter and significance. Results with p < 0.05 were considered significant.
recorded as day zero. The cells were then counted at 24, 48 and
72 hours after plating. The number of cells in each cell line was
normalized to the average number of cells at day 0. The cell Disclosure of Potential Conflicts of Interest
proliferation for each cell line was then plotted with an expo- We have no competing interests.
nential function and compared across groups.
Acknowledgments
Chemotaxis
The authors would like to thank their colleagues at the Australian Nuclear
Chemotaxis of Jurkat cells was investigated using the Transwell Science and Technology Organisation (ANSTO): C. Betlazar, M. Harrison-
migration assay (also called Boyden chamber) which consisted Brown, G. Dhand, E. Dodson, for technical laboratory support and assis-
of 24-well polyethylene terephthalate membranes with 5¡mm tance; RBB holds an ANSTO Distinguished Research Fellowship.
pores (Falcon, Franklin Lakes, NJ, USA). One hundred thou-
sand cells in 0.4 ml DMEM were added to the upper reservoir Authors’ contributions
and 0.7 mL DMEM was placed in the lower reservoir with
either a TSPO speci c ligand or 0.1% FBS as a general chemoat- G.-J.L. and R.B.B. devised and supervised the project. G.-J.L. performedfi
most of the experiments. R.J.M. created the TSPO-Jurkat cells, performed
tractant. To investigate the role of TSPO in FBS-induced che- cell culture and analyzed microarray data. D.Y.C. designed and created the
motaxis, 100 nM was added to both the upper and lower TSPO plasmid. W.W.-Y.K. performed RT-qPCR. C.R.H. and RC contrib-
reservoirs while 0.1% FBS was only placed in the lower reser- uted to radioligand membrane receptor binding studies. G.-J.L. wrote the
voir. After 4 hours of incubation at 37
C and 5% CO , the plate manuscript with assistance by R.B.B. and R.J.M.2
containing the Transwell chambers was shaken 5 times to free
the cells that had migrated but were still attached to the Trans- ORCID
well membrane in the lower reservoir. The migrated cells on
the bottom of each well of the plate were viewed and images Guo-Jun Liu http://orcid.org/0000-0002-4278-8372
were captured using an Olympus microscope (BX61WI, Olym- Ryan J. Middleton http://orcid.org/0000-0001-8024-7772
pus) mounted with 4x objective and a CCD camera (CoolSNAP Winnie Wai-Ying Kam http://orcid.org/0000-0002-5508-2432
Richard B. Banati http://orcid.org/0000-0003-3558-597X
HQ2). Migrated cells were counted from at least 3 Transwell
chambers (3 fields per chamber) with Image J.
References
Patch clamp electrophysiology [1] Eshleman AJ, Murray TF. Differential binding properties of the
peripheral-type benzodiazepine ligands [3H]PK 11195 and [3H]Ro
Whole-cell patch-clamp recordings from potassium channels in 5-4864 in trout and mouse brain membranes. J Neurochem 1989;
Jurkat cells were made using a Multiclamp 700B amplifier (Molec- 53:494-502; PMID:2746235; http://dx.doi.org/10.1111/j.1471-4159.
ular Devices, MDS Analytical Technologies, Toronto, Canada) at 1989.tb07361.x
room temperature (22–24
C) as described previously.63 Brie y, [2] Fan J, Lindemann P, Feuilloley MG, Papadopoulos V. Structural andfl
functional evolution of the translocator protein (18 kDa). Curr Mol
resistances of glass pipettes were 4–8 Mohm when a patch pipette Med 2012; 12:369-86; PMID:22364126
was filled with pipette solution containing 140 mM KCl, 1 mM [3] Snyder MJ, Van Antwerpen R. Evidence for a diazepam-binding
MgCl2, 10 mM EGTA, 10 mM HEPES and pH 7.3; the external inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdyster-
bath solution contained 135 mM NaCl, 5 mM KCl, 2 mM CaCl , oidogenesis by the insect prothoracic gland. Cell Tissue Res 1998;2
1 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4). To
294:161-8; PMID:9724466; http://dx.doi.org/10.1007/s004410051166
C C [4] Papadopoulos V, Baraldi M, Guilarte TR, Knudsen TB, Lacapere JJ,verify K channels, general 20 mM K blocker TEA was added to Lindemann P, Norenberg MD, Nutt D, Weizman A, Zhang MR,
the bath solution or in the normal pipette solution, or CsCl et al. Translocator protein (18kDa): new nomenclature for the
replaced KCl in the pipette solution. Episodic voltage-activated peripheral-type benzodiazepine receptor based on its structure and
current traces (from ¡100 mV to C100 mV with 20 mV incre- molecular function. Trends Pharmacol Sci 2006; 27:402-9;
ment) were recorded in wild-type, empty plasmid and TSPO- PMID:16822554; http://dx.doi.org/10.1016/j.tips.2006.06.005
¡ [5] Antkiewicz-Michaluk L, Guidotti A, Krueger KE. Molecular charac-Jurkat cells. The membrane potential was held at 60 mV. terization and mitochondrial density of a recognition site for periph-
Whole-cell currents were also recorded in gap-freemode at a hold- eral-type benzodiazepine ligands. Mol Pharmacol 1988; 34:272-8.
ing membrane potential of ¡60 mV. The currents were sampled PMID: 2843747
online using a Digidata 1400 interface and pClamp10 program [6] Guo Y, Kalathur RC, Liu Q, Kloss B, Bruni R, Ginter C, Kloppmann
(Molecular Devices). E, Rost B, Hendrickson WA. Structure and activity of tryptophan-
rich TSPO proteins. Science 2015; 347:551-5; PMID:25635100;
http://dx.doi.org/10.1126/science.aaa1534
Statistics [7] Jaremko L, Jaremko M, Giller K, Becker S, Zweckstetter M. Structure
of the mitochondrial translocator protein in complex with a diagnos-
Data presented are the averages of at least 4 repeat measure- tic ligand. Science 2014; 343:1363-6; PMID:24653034; http://dx.doi.
ments. Results are expressed as means § standard deviation. org/10.1126/science.1248725
[8] Li F, Liu J, Zheng Y, Garavito RM, Ferguson-Miller S. Crystal struc-
The statistical analysis of significance of results was carried out tures of translocator protein (TSPO) and mutant mimic of a human
with one-way analysis of variance (ANOVA) and Post-hoc polymorphism. Science 2015; 347:555-8; PMID:25635101; http://dx.
comparisons were made using Tukey’s HSD test. Two-tailed doi.org/10.1126/science.1260590
446 G.-J. LIU ET AL.
[9] Veenman L, Alten J, Linnemannstons K, Shandalov Y, Zeno S, expression, nuclear localization, and PBR-mediated cell proliferation
Lakomek M, Gavish M, Kugler W. Potential involvement of F0F1- and nuclear transport of cholesterol. Cancer Res 1999; 59:831-42;
ATP(synth)ase and reactive oxygen species in apoptosis induction by PMID:10029072
the antineoplastic agent erucylphosphohomocholine in glioblastoma [24] Le Fur G, Vaucher N, Perrier M, Flamier A, Benavides J, Renault C,
cell lines : a mechanism for induction of apoptosis via the 18 kDa DubroeucqM, Gueremy C, Uzan A. Differentiation between two ligands
mitochondrial translocator protein. Apoptosis 2010; 15:753-68; for peripheral benzodiazepine binding sites,[3 H] R05-4864 and [3 H]
PMID:20107899; http://dx.doi.org/10.1007/s10495-010-0460-5 PK 11195, by thermodynamic studies. Life Sci 1983; 33:449-57;
[10] Veenman L, Gavish M. The role of 18 kDa mitochondrial transloca- PMID:6308375; http://dx.doi.org/10.1016/0024-3205(83)90794-4
tor protein (TSPO) in programmed cell death, and effects of steroids [25] Hardwick M, Cavalli LR, Barlow KD, Haddad BR, Papadopoulos V.
on TSPO expression. Curr Mol Med 2012; 12:398-412; Peripheral-type benzodiazepine receptor (PBR) gene amplification in
PMID:22348610 MDA-MB-231 aggressive breast cancer cells. Cancer Genet Cytoge-
[11] Zeno S, Veenman L, Katz Y, Bode J, Gavish M, Zaaroor M. The net 2002; 139:48-51; PMID:12547158; http://dx.doi.org/10.1016/
18 kDa mitochondrial translocator protein (TSPO) prevents accu- S0165-4608(02)00604-0
mulation of protoporphyrin IX. Involvement of reactive oxygen spe- [26] Lacapere JJ, Papadopoulos V. Peripheral-type benzodiazepine recep-
cies (ROS). Curr Mol Med 2012; 12:494-501; PMID:22376065 tor: structure and function of a cholesterol-binding protein in steroid
[12] Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, and bile acid biosynthesis. Steroids 2003; 68:569-85;
Hardwick M, Li H, Vidic B, Brown AS, et al. Peripheral benzodiaze- PMID:12957662; http://dx.doi.org/10.1016/S0039-128X(03)00101-6
pine receptor in cholesterol transport and steroidogenesis. Steroids [27] Brown RC, Degenhardt B, Kotoula M, Papadopoulous V. Location-
1997; 62:21-8; PMID:9029710; http://dx.doi.org/10.1016/S0039-128X dependent role of the human glioma cell peripheral-type benzodiaze-
(96)00154-7 pine receptor in proliferation and steroid biosynthesis. Cancer Lett
[13] Liu GJ, Middleton RJ, Hatty CR, Kam WW, Chan R, Pham T, 2000; 156:125-32; PMID:10880761; http://dx.doi.org/10.1016/S0304-
Harrison-BrownM, Dodson E, Veale K, Banati RB. The 18 kDa trans- 3835(00)00451-1
locator protein, microglia and neuroinflammation. Brain Pathol 2014; [28] Alvarez-Maubecin V, Garcia-Hernandez F, Williams JT, Van Bock-
24:631-53; PMID:25345894; http://dx.doi.org/10.1111/bpa.12196 staele EJ. Functional coupling between neurons and glia. J Neurosci
[14] Banati RB, Middleton RB, Chan R, Hatty CR, Kam WWY, Quin C, 2000; 20:4091-8; PMID:10818144
Graeber MB, Parmar A, Zahra D, Callaghan P, et al. Positron emis- [29] Gut P, Baeza-Raja B, Andersson O, Hasenkamp L, Hsiao J, Hesselson
sion tomography and functional characterization of a complete PBR/ D, Akassoglou K, Verdin E, Hirschey MD, Stainier DYR. Whole-
TSPO-knockout. Nat Commun 2014; 5:5452; PMID:25406832; organism screening for gluconeogenesis identifies activators of fast-
http://dx.doi.org/10.1038/ncomms6452 ing metabolism. Nat Chem Biol 2013; 9:97-104; PMID:23201900;
[15] Morohaku K, Pelton SH, Daugherty DJ, Butler WR, Deng W, Selvaraj http://dx.doi.org/10.1038/nchembio.1136
V. Translocator protein/peripheral benzodiazepine receptor is not [30] Xiao J, Liang D, Zhang H, Liu Y, Li F, Chen YH. 40-Chlorodiazepam,
required for steroid hormone biosynthesis. Endocrinology 2014; a translocator protein (18 kDa) antagonist, improves cardiac func-
155:89-97; PMID:24174323; http://dx.doi.org/10.1210/en.2013-1556 tional recovery during postischemia reperfusion in rats. Exp Biol
[16] Sileikyte J, Blachly-Dyson E, Sewell R, Carpi A, Menabo R, Di Lisa F, Med (Maywood) 2010; 235:478-86; PMID:20407080; http://dx.doi.
Ricchelli F, Bernardi P, Forte M. Regulation of the mitochondrial org/10.1258/ebm.2009.009291
permeability transition pore by the outer membrane does not involve [31] Kugler W, Veenman L, Shandalov Y, Leschiner S, Spanier I, Lako-
the Peripheral Benzodiazepine Receptor (TSPO). J Biol Chem 2014; mek M, Gavish M. Ligands of the mitochondrial 18 kDa translocator
289:13769-81; PMID:24692541; http://dx.doi.org/10.1074/jbc. protein attenuate apoptosis of human glioblastoma cells exposed to
M114.549634 erucylphosphohomocholine. Cell Oncol 2008; 30:435-50;
[17] Tu LN, Morohaku K, Manna PR, Pelton SH, Butler WR, Stocco DM, PMID:18791274
Selvaraj V. Peripheral Benzodiazepine Receptor/Translocator protein [32] Veenman L, Papadopoulos V, Gavish M. Channel-like functions of
global knockout mice are viable with no effects on steroid hormone the 18-kDa translocator protein (TSPO): regulation of apoptosis and
biosynthesis. J Biol Chem 2014; 289:27444-54; PMID:24936060; steroidogenesis as part of the host-defense response. Curr Pharm
http://dx.doi.org/10.1074/jbc.M114.578286 Des 2007; 13:2385-405; PMID:17692008; http://dx.doi.org/10.2174/
[18] Gut P, Zweckstetter M, Banati RB. Lost in translocation: the functions of 138161207781368710
the 18-kD translocator protein. Trends Endocrinol Metab 2015; 26:349- [33] Zeno S, Zaaroor M, Leschiner S, Veenman L, Gavish M. CoCl(2)
56; PMID:26026242; http://dx.doi.org/10.1016/j.tem.2015.04.001 induces apoptosis via the 18 kDa translocator protein in U118MG
[19] Middleton RJ, Liu G-J, Banati RB. Guwiyang Wurra–‘Fire Mouse’: a human glioblastoma cells. Biochemistry 2009; 48:4652-61;
global gene knockout model for TSPO/PBR drug development, loss- PMID:19358520; http://dx.doi.org/10.1021/bi900064t
of-function and mechanisms of compensation studies. Biochem Soc [34] Gavish M, Bachman I, Shoukrun R, Katz Y, Veenman L, Weisinger
Trans 2015; 43:553-8; PMID:26551692; http://dx.doi.org/10.1042/ G, Weizman A. Enigma of the peripheral benzodiazepine receptor.
BST20150039 Pharmacol Rev 1999; 51:629-50; PMID:10581326
[20] Middleton RJ, Liu GJ, Banati RB. Epigenetic silencing of the human [35] Veenman L, Gavish M. The peripheral-type benzodiazepine receptor
18 kDa translocator protein (TSPO) in a T-cell leukemia cell line. and the cardiovascular system. Implications for drug development.
DNA Cell Biol 2017; 36:103-8; PMID:28004979; http://dx.doi.org/ Pharmacol Ther 2006; 110:503-24; PMID:16337685; http://dx.doi.
10.1089/dna.2016.3385 org/10.1016/j.pharmthera.2005.09.007
[21] Bertomeu T, Zvereff V, Ibrahim A, Zehntner S, Aliaga A, Rosa-Neto [36] Carayon P, Portier M, Dussossoy D, Bord A, Petitpretre G, Canat X,
P, Bedell B, Falardeau P, Gourdeau H. TLN-4601 peripheral benzodi- Le Fur G, Casellas P. Involvement of peripheral benzodiazepine
azepine receptor (PBR/TSPO) binding properties do not mediate receptors in the protection of hematopoietic cells against oxygen rad-
apoptosis but confer tumor-specific accumulation. Biochem pharma- ical damage. Blood 1996; 87:3170-8; PMID:8605331
col 2010; 80:1572-9; PMID:20655882; http://dx.doi.org/10.1016/j. [37] Banati RB. Visualising microglial activation in vivo. Glia 2002;
bcp.2010.07.018 40:206-17; PMID:12379908; http://dx.doi.org/10.1002/glia.10144
[22] Bribes E, Carriere D, Goubet C, Galiegue S, Casellas P, Simony- [38] Banati RB. Neuropathological imaging: in vivo detection of glial acti-
Lafontaine J. Immunohistochemical assessment of the peripheral vation as a measure of disease and adaptive change in the brain. Br
benzodiazepine receptor in human tissues. J Histochem Cytochem Med Bull 2003; 65:121-31; PMID:12697620; http://dx.doi.org/
2004; 52:19-28; PMID:14688214; http://dx.doi.org/10.1177/ 10.1093/bmb/65.1.121
002215540405200103 [39] Banati RB, Cagnin A, Brooks DJ, Gunn RN, Myers R, Jones T, Birch R,
[23] Hardwick M, Fertikh D, Culty M, Li H, Vidic B, Papadopoulos V. Anand P. Long-term trans-synaptic glial responses in the human thala-
Peripheral-type benzodiazepine receptor (PBR) in human breast can- mus after peripheral nerve injury. Neuroreport 2001; 12:3439-42;
cer: correlation of breast cancer cell aggressive phenotype with PBR PMID:11733686; http://dx.doi.org/10.1097/00001756-200111160-00012
CELL CYCLE 447
[40] Banati RB, Goerres GW, Tjoa C, Aggleton JP, Grasby P. The func- ATP synthase and carriers for Pi and ADP/ATP. J Biol Chem 2003;
tional anatomy of visual-tactile integration in man: a study using 278:12305-9; PMID:12560333; http://dx.doi.org/10.1074/jbc.
positron emission tomography. Neuropsychologia 2000; 38:115-24; C200703200
PMID:10660224; http://dx.doi.org/10.1016/S0028-3932(99)00074-3 [56] McEnery MW, Snowman AM, Trifiletti RR, Snyder SH. Isolation of
[41] Cagnin A, Brooks DJ, Kennedy AM, Gunn RN, Myers R, Turkheimer the mitochondrial benzodiazepine receptor: association with the volt-
FE, Jones T, Banati RB. In-vivo measurement of activated microglia age-dependent anion channel and the adenine nucleotide carrier.
in dementia. Lancet 2001; 358:461-7; PMID:11513911; http://dx.doi. Proc Natl Acad Sci U S A 1992; 89:3170-4; PMID:1373486; http://dx.
org/10.1016/S0140-6736(01)05625-2 doi.org/10.1073/pnas.89.8.3170
[42] Cagnin A, Myers R, Gunn RN, Lawrence AD, Stevens T, Kreutzberg [57] Ruderman NB, Xu XJ, Nelson L, Cacicedo JM, Saha AK, Lan F, Ido
GW, Jones T, Banati RB. In vivo visualization of activated glia by [11C] Y. AMPK and SIRT1: a long-standing partnership? Am J Physiol
(R)-PK11195-PET following herpes encephalitis reveals projected neu- Endocrinol Metab 2010; 298:E751-60; PMID:20103737; http://dx.
ronal damage beyond the primary focal lesion. Brain 2001; 124:2014- doi.org/10.1152/ajpendo.00745.2009
27; PMID:11571219; http://dx.doi.org/10.1093/brain/124.10.2014 [58] Casellas P, Galiegue S, Basile AS. Peripheral benzodiazepine recep-
[43] Kuhlmann AC, Guilarte TR. Cellular and subcellular localization of tors and mitochondrial function. Neurochem Int 2002; 40:475-86;
peripheral benzodiazepine receptors after trimethyltin neurotoxicity. PMID:11850104; http://dx.doi.org/10.1016/S0197-0186(01)00118-8
J Neurochem 2000; 74:1694-704; PMID:10737628; http://dx.doi.org/ [59] Corsi L, Geminiani E, Avallone R, Baraldi M. Nuclear location-
10.1046/j.1471-4159.2000.0741694.x dependent role of peripheral benzodiazepine receptor (PBR) in
[44] Pedersen MD, Minuzzi L, Wirenfeldt M, Meldgaard M, Slidsborg C, hepatic tumoral cell lines proliferation. Life Sci 2005; 76:2523-33;
Cumming P, Finsen B. Up-regulation of PK11195 binding in areas of PMID:15769477; http://dx.doi.org/10.1016/j.lfs.2004.08.040
axonal degeneration coincides with early microglial activation in [60] Mendonca-Torres MC, Roberts SS. The translocator protein (TSPO)
mouse brain. Eur J Neurosci 2006; 24:991-1000; PMID:16930426; ligand PK11195 induces apoptosis and cell cycle arrest and sensitizes
http://dx.doi.org/10.1111/j.1460-9568.2006.04975.x to chemotherapy treatment in pre- and post-relapse neuroblastoma
[45] Venneti S, Lopresti BJ, Wiley CA. The peripheral benzodiazepine cell lines. Cancer Biol Ther 2013; 14:319-26; PMID:23358477; http://
receptor (Translocator protein 18kDa) in microglia: from pathology dx.doi.org/10.4161/cbt.23613
to imaging. Prog Neurobiol 2006; 80:308-22; PMID:17156911; http:// [61] RechichiM, Salvetti A, Chelli B, Costa B, Da Pozzo E, Spinetti F, LenaA,
dx.doi.org/10.1016/j.pneurobio.2006.10.002 Evangelista M, Rainaldi G, Martini C, et al. TSPO over-expression
[46] Wilms H, Claasen J, Rohl C, Sievers J, Deuschl G, Lucius R. Involve- increases motility, transmigration and proliferation properties of C6 rat
ment of benzodiazepine receptors in neuroinflammatory and neuro- glioma cells. Biochim Biophys Acta 2008; 1782:118-25;
degenerative diseases: evidence from activated microglial cells in PMID:18190798; http://dx.doi.org/10.1016/j.bbadis.2007.12.001
vitro. Neurobiol Dis 2003; 14:417-24; PMID:14678758; http://dx.doi. [62] Wu X, Gallo KA. The 18-kDa translocator protein (TSPO) disrupts
org/10.1016/j.nbd.2003.07.002 mammary epithelial morphogenesis and promotes breast cancer cell
[47] Tu LN, Zhao AH, Stocco DM, Selvaraj V. PK11195 effect on ste- migration. PLoS One 2013; 8:e71258; PMID:23967175; http://dx.doi.
roidogenesis is not mediated through the translocator protein org/10.1371/journal.pone.0071258
(TSPO). Endocrinology 2015; 156:1033-39; PMID:25535830; http:// [63] Liu GJ, Simpson AM, Swan MA, Tao C, Tuch BE, Crawford RM,
dx.doi.org/10.1210/en.2014-1707 Jovanovic A, Martin DK. ATP-sensitive potassium channels induced
[48] Costa B, Salvetti A, Rossi L, Spinetti F, Lena A, Chelli B, Rechichi M, in liver cells after transfection with insulin cDNA and the GLUT 2
Da Pozzo E, Gremigni V, Martini C. Peripheral Benzodiazepine transporter regulate glucose-stimulated insulin secretion. Faseb J
Receptor: Characterization in Human T-Lymphoma Jurkat Cells. 2003; 17:1682-4; PMID:12958175
Mol Pharmacol 2006; 69:37-44; PMID:16189298 [64] Tamarina NA, Kuznetsov A, Fridlyand LE, Philipson LH. Delayed-
[49] Choi J, Ifuku M, Noda M, Guilarte TR. Translocator protein (18 rectifier (KV2.1) regulation of pancreatic beta-cell calcium responses
kDa)/peripheral benzodiazepine receptor specific ligands induce to glucose: inhibitor specificity and modeling. Am J Physiol Endocri-
microglia functions consistent with an activated state. Glia 2011; nol Metab 2005; 289:E578-85; PMID:16014354; http://dx.doi.org/
59:219-30; PMID:21125642; http://dx.doi.org/10.1002/glia.21091 10.1152/ajpendo.00054.2005
[50] Eckert GP, Schiborr C, Hagl S, Abdel-Kader R, Muller WE, Rimbach [65] Liu Y, Sato T, O’Rourke B, Marban E. Mitochondrial ATP-depen-
G, Frank J. Curcumin prevents mitochondrial dysfunction in the dent potassium channels: novel effectors of cardioprotection? Circu-
brain of the senescence-accelerated mouse-prone 8. Neurochem Int lation 1998; 97:2463-9; PMID:9641699; http://dx.doi.org/10.1161/01.
2013; 62:595-602; PMID:23422877; http://dx.doi.org/10.1016/j. CIR.97.24.2463
neuint.2013.02.014 [66] O’Rourke B. Evidence for mitochondrial KC channels and their role
[51] Krestinina OV, Grachev DE, Odinokova IV, Reiser G, Evtodienko in cardioprotection. Circ Res 2004; 94:420-32; PMID:15001541;
YV, Azarashvili TS. Effect of peripheral benzodiazepine receptor http://dx.doi.org/10.1161/01.RES.0000117583.66950.43
(PBR/TSPO) ligands on opening of Ca2C-induced pore and phos- [67] Hatty CR, Le Brun AP, Lake V, Clifton LA, Liu GJ, James M, Banati
phorylation of 3.5-kDa polypeptide in rat brain mitochondria. Bio- RB. Investigating the interactions of the 18kDa translocator protein
chemistry (Mosc) 2009; 74:421-9; PMID:19463096; http://dx.doi.org/ and its ligand PK11195 in planar lipid bilayers. Biochim Biophys
10.1134/S0006297909040105 Acta 2014; 1838:1019-30; PMID:24374318; http://dx.doi.org/
[52] Rosenberg N, Rosenberg O, Weizman A, Veenman L, Gavish M. In 10.1016/j.bbamem.2013.12.013
vitro catabolic effect of protoporphyrin IX in human osteoblast-like [68] Cheng KT, Hou WC, Huang YC, Wang LF. Baicalin induces differ-
cells: possible role of the 18 kDa mitochondrial translocator protein. ential expression of cytochrome C Oxidase in human lung H441
J Bioenerg Biomembr 2013; 45:333-41; PMID:23475134; http://dx. Cell. J Agric Food Chem 2003; 51:7276-9; PMID:14640570; http://dx.
doi.org/10.1007/s10863-013-9501-4 doi.org/10.1021/jf0301549
[53] Seneviratne MS, Faccenda D, De Biase V, Campanella M. PK11195 [69] Dveksler GS, Basile AA, Dieffenbach CW. Analysis of gene expres-
inhibits mitophagy targeting the F1Fo-ATPsynthase in Bcl-2 knock- sion: use of oligonucleotide primers for glyceraldehyde-3-phosphate
down cells. Curr Mol Med 2012; 12:476-82; PMID:22348615 dehydrogenase. PCR Methods Appl 1992; 1:283-5; PMID:1477664;
[54] Veenman L, Gavish M, Kugler W. Apoptosis induction by erucyl- http://dx.doi.org/10.1101/gr.1.4.283
phosphohomocholine via the 18 kDa mitochondrial translocator [70] Liu GJ, Nagarajah R, Banati RB, Bennett MR. Glutamate induces
protein: implications for cancer treatment. Anticancer Agents Med directed chemotaxis of microglia. Eur J Neurosci 2009; 29:1108-18;
Chem 2014; 14:559-77; PMID:24628235; http://dx.doi.org/10.2174/ PMID:19302147; http://dx.doi.org/10.1111/j.1460-9568.2009.06659.x
1871520614666140309230338 [71] Milakovic T, Johnson GVW. Mitochondrial respiration and ATP
[55] Ko YH, Delannoy M, Hullihen J, Chiu W, Pedersen PL. Mitochondrial production are significantly impaired in striatal cells expressing
ATP synthasome. Cristae-enriched membranes and a multiwell deter- mutant Huntingtin. J Biol Chem 2005; 280:30773-82;
gent screening assay yield dispersed single complexes containing the PMID:15983033; http://dx.doi.org/10.1074/jbc.M504749200