In vitro inhibition of topoisomerase IIα by reduced glutathione.

Abstract

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 µM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity.

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Regular paper
In vitro inhibition of topoisomerase IIα by reduced glutathione
Zahid M. Delwar1, Marina Fernanda Vita1,*, Åke Siden2, Mabel Cruz1
and Juan Sebastian Yakisich1*
1Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden; 2Department of Neurology, Karolinska University Hospital,
Stockholm, Sweden
In most cells, the major intracellular redox buffer is glu-
tathione (GSH) and its disulfide-oxidized (GSSG) form.
The GSH/GSSG system maintains the intracellular re-
dox balance and the essential thiol status of proteins
by thiol disulfide exchange. Topoisomerases are thiol
proteins and are a target of thiol-reactive substances.
In this study, the inhibitory effect of physiological con-
centration of GSH and GSSG on topoisomerase IIα activ-
ity in vitro was investigated. GSH (0–10 mM) inhibited
topoisomerase IIα in a concentration-dependent manner
while GSSG (1–100 μM) had no significant effect. These
findings suggest that the GSH/GSSG system could have
a potential in vivo role in regulating topoisomerase IIα
activity.
Keywords: DNA topoisomerases, glutathione, redox regulation
Received: 21 March, 2011; 26 April, 2011; accepted: 04 June, 2011;
available on-line: 06 June, 2011
InTRoducTIon
DNA topoisomerases (TOPO) are key enzymes im-
plicated in nearly all events related to DNA metabolism
and are the primary cellular target for many effective an-
tineoplastic agents (Bates & Maxwell, 2007; Schoeffler
& Berger, 2008). The TOPOs, especially the TOPO IIα
isoform which is highly expressed in proliferating tissues
(Isaacs et al., 1998), are thiol proteins (Sng et al., 1999).
The human enzyme TOPO IIα and IIβ isoforms have
13 and 17 cysteine residues, respectively (Tsai-Pfugfelder
et al., 1988; Jenkins et al., 1992). SH-reactive agents (e.g.,
N-ethylmaleimide (NEM), menadione and disulfiram)
inhibit TOPOs (Tirumalai et al., 1996; Frydman et al.,
1997; Neder et al., 1998; Yakisich et al., 2001). This in-
dicates that sulfhydryl groups on cysteines are important
for the enzymatic activity and, thus, potential targets for
TOPO-inhibiting drugs. Since the cellular concentration
of protein SH groups is 10 mM to 30 mM (Kaplowitz
et al., 1985), it is likely that, due to the high number of
cysteine residues on TOPO IIα, the activity of this en-
zyme could be modulated in vivo by cellular thiol-reactive
substances. To assess this hypothesis, it is first essential
to determine the effect of natural occurring thiol-reactive
substances on the activity of purified TOPO enzymes.
The thiol-reduced form of glutathione (GSH), which is
also the biologically active form, is the dominant non-
protein thiol in mammalian cells and occurs in virtually
all animal cells, often at a relatively high (0.1–12 mM)
concentration (Meister, 1995; Pastore et al., 2003; Lu,
2009). It has been reported that expression of Bcl-2 in
HeLa cells produces redistribution of glutathione to the
nucleus to give a concentration of 16 mM (74 % of to-
tal cellular GSH within the nucleus compared to 32 %
when Bcl-2 expression is off) (Voehringer et al., 1998).
GSSG is present at much lower concentrations (20–40
µM) (Akerboom et al., 1982; Gilbert, 1995). Available
data also indicate that the GSH/GSSG redox potential
is likely to be more reduced in nuclei than in the cyto-
plasm (Go & Jones, 2008). The GSH/GSSG system has
important functions as an antioxidant, in detoxification
of xenobiotics, maintenance of intracellular redox bal-
ance, storage and transport of cysteine (Meister, 1995;
Lu, 1999; Dingren, 2000), and is essential for cell pro-
liferation (Poot et al., 1995; Lu, 1999; Dingren, 2000).
Many enzymes and other endogenous compounds have
been found to be modulated (activated or inhibited) by
GSH and GSSG (Wang & Bellatori, 1998). To the best
of our knowledge, except for plant mitochondrial TOPO
I (Konstantinov et al., 2001) and the use of high GSSG
concentration (10 mM) for trapping Type1A TOPO/
DNA complex (Li et al., 2001), the redox regulation of
TOPOs has not yet been evaluated.
MaTeRIal and MeTHodS
Dimethylsulfoxide (DMSO), reduced and oxidized
glutathione (GSH and GSSG, respectively), etoposide,
camptothecin were purchased from Sigma (Sweden),
TOPO I, TOPO IIα and pBR322 plasmid DNA were
purchased from Inspiralis (UK). All other reagents were
of analytical grade or the highest grade available. Etopo-
side and camptothecin were prepared as stock solutions
(25 mM) in DMSO. GSH and GSSG were prepared as
stock solutions (200 and 300 mM, respectively) in sterile
distilled water. Dithiothreitol (DTT) was purchased as 1
M stock solution. Except DTT (stored at 4 °C) all stock
solutions were stored at –20 °C and diluted accordingly
before use. Topoisomerase activity was measured as pre-
viously described (Yakisich et al., 2001). Briefly, TOPO I
and TOPO IIα activity was measured by the relaxation
activity of superhelical plasmid pBR322 (400 ng/reac-
tion) using appropriate DTT-free solutions and protocols
adapted to the supplier recommendations. For TOPO I,
the reactions were started by the addition of the enzyme
(1 U) and allowed to proceed at 37 °C for 30 min. For
TOPO IIα, the reactions were started by the addition
of the enzyme (5 U) and allowed to proceed at 30 °C
*e-mail: Sebastian.Yakisich@ki.se
*Present address: Centre de Recherche en Cancérologie de Mar-
seille (CRCM), France
abbreviations: GSH, reduced glutathione; GSSG, oxidized glutath-
ione; TOPO, topoisomerase; DMSO, dimethylsulfoxide; DTT, dithio-
threitol
Vol. 58, No. 2/2011
265–267
on-line at: www.actabp.pl

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266 2011
Z. M. Delwar and others
for 15 min. The reactions of
both assays were stopped by
the addition of 5 µl of loading
buffer. Aliquots (15 µl) were
run in 1 % agarose minigels
at 2 V/cm for 16–20 h and
stained with ethidium bromide
for visualization with ultravio-
let light. Quantitative determi-
nation of the bands was done
using ImageJ (http://rsbweb.
nih.gov/ij/).
ReSulTS and dIScuSSIon
In the present work, we
showed that physiologically
relevant concentrations
GSH inhibited human TOPO
IIα activity in vitro. Because
TOPO I was only inhibited
by high GSH concentration
(we used camptothecin as
positive control) of no physi-
ological relevance (not shown)
we did not further investigate
this enzyme. TOPO IIα ac-
tivity decreased with increas-
ing GSH concentration (≥ 6
mM), reaching a maximum
of inhibition at 10 mM GSH
(Fig. 1A). GSH concentra-
of
tions below 4 mM showed no inhibitory effect (Fig. 1
and data not shown). The lack of inhibitory effect at
low GSH concentrations is in agreement with a recent
article showing that 0.5 mM GSH had no effect on
the decatenation activity of topoisomerase II (Wu et al.,
2011). Figure 1B shows that TOPO IIα activity was not
inhibited by GSSG (1–100 μM). As described before,
the cellular concentration of GSH varies from 0.2 to 12
mM (and GSSG from 10–40 μM). This means that GSH
might affect in vivo the activity of TOPO IIα. The in-
hibitory effect of GSH was not affected by addition of
1 or 5 mM DTT (Fig. 2). However, it is possible that
the DTT/GSH ratio (5 mM/8 mM = 0.625) used in our
study may not be enough to prevent the inhibitory effect
of GSH on TOPO IIα. For instance, the inhibitory ef-
fect of disulfiram on TOPO IIα was partially prevented
by the addition of DTT to a high DTT/disulfiram (≥ 40)
molar ratio (Yakisich et al., 2001). A similar DTT/GSH
molar ratio for the lowest GSH concentration (8 mM)
that, in our study, showed a significant inhibitory effect
on TOPO IIα (Fig. 1) was not possible to achieve due
to the limited solubility of DTT (0.1 M at 20 °C). Thus,
a thiol-disulphide exchange may still be important for
the inhibitory effect of GSH as described for other SH-
reactive agents such as disulfiram (Yakisich et al., 2001).
TOPO II enzymes contain 13–17 cysteine residues
(Jenkins et al., 1992; Tsai-Pfugfelder et al., 1988; Wyck-
off et al., 1989). Interestingly, TOPO I has only eight
cysteine residues (D’Arpa et al., 1988). This suggests that
the number of cysteine residues might be important for
the modulatory effect of thiol-reactive substances that act
directly on the cysteine residues of the enzymes and sup-
ports the (selectively) high effect of GSH on TOPO IIα
activity (compared to TOPO I). The knowledge of the
endogenous regulation of TOPOs by naturally occurring
thiol substances, such as the GSH/GSSG system, might
Figure. 1. effect of GSH and GSSG on relaxation activity of ToPo IIα
Supercoiled (S) pBR322 plasmid DNA was incubated with different concentrations of GSH (A) or
GSSG (B). Equivalent concentrations of vehicle alone (H2O and DMSO) were used in control reac-
tions containing only plasmid and TOPO IIα (second lanes). Etoposide (Etop) was used as posi-
tive control (third lanes). Representative gels are shown. Quantitative data for GSH and GSSG
are shown in panels C and D, respectively. Data are the mean ± S.D. of three independent ex-
periments.
Figure 2. effects of dTT on inhibitory activity of GSH
Supercoiled (S) pBR322 plasmid DNA was incubated with indi-
cated concentrations of GSH alone or GSH+DTT and the relaxa-
tion activity of TOPO IIα was measured as described in Materials
and Methods. Equivalent concentrations of vehicle alone (H2O
and DMSO) were used in control reaction containing plasmid and
TOPO IIα (second lane). Representative gel is shown (A). Etoposide
(Etop) was used as positive control (third lane). Quantitative data
are shown in panel B. Data are the mean ± S.D. of three independ-
ent experiments.

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Vol. 58 267
Inhibition of topoisomerase IIα by gluthathione
be of importance for designing new therapeutic strate-
gies for cancer treatment, protection of normal cells and
for chemoprevention.
Acknowledgements
This work was supported by grants from the Swed-
ish Research Council, the Karolinska Institute and the
Stockholms Läns Landsting.
ReFeRenceS
Akerboom TP, Bilzer M, Sies H (1982) The relationship of biliary glu-
tathione disulfide efflux and intracellular glutathione disulfide con-
tent in perfused rat liver. J Biol Chem 257: 4248–4252.
Bates AD, Maxwell A (2007) Energy coupling in type II topoisomer-
ases: why do they hydrolyze ATP? Biochemistry 46: 7929–7941.
D’Arpa P, Machlin PS, Ratrie Hr, Rothfield NF, Cleveland DW, Earn-
shaw WC (1988) cDNA cloning of human DNA topoisomerase I:
catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl
Acad Sci USA 85: 2543–2547.
Dingren R (2000) Metabolism and functions of glutathione in brain.
Progress Neurobiol 62: 649–671.
Frydman B, Marton LJ, Sun JS, Neder K, Witiak DT, Liu AA, Wang
H–M, Mao YM, Wu H-Y, Sanders MM, Liu LF (1997) Induction
of DNA topoisomerase II-mediated DNA cleavage by B-lapachone
and related naphtoquinones. Cancer Res 57: 620–627.
Gilbert H (1995) Thiol/disulfide exchange equilibria and disulfide bond
stability. Methods Enzymol 251: 8–28.
Go YM, Jones DP (2008) Redox compartmentalization in eukaryotic
cells. Biochim Biophys Acta 1780: 1273–1290.
Isaacs RJ, Davies SL, Sandri MI, Redwood C, Wells NJ, Hickson ID
(1998) Physiological regulation of eukaryotic topoisomerase II. Bio-
chim Biophys Acta 1400: 121–137.
Jenkins JR, Ayton P, Jones T, Davies SL, Simmons DL, Harris AL,
Sheer D, Hickson ID (1992) Isolation of cDNA clones encoding
the beta isozyme of human DNA topoisomerase II and localization
of the gene to chromosome 3p24. Nucleic Acids Res 20: 5587–5592.
Kaplowitz N, Aw TY, Ookhtens M (1985) The regulation of hepatic
glutathione. Ann Rev Pharmacol Toxicol 25: 715–744.
Konstantinov YM, Tarasenko VI, Rogozin IB (2001) Redox modula-
tion of the activity of DNA topoisomerases I from carrot (Daucus
carota) mitochondria. Dokl Biochem Biophys 377: 82–84. Translated
from Dokl. Akad. Nauk. 377, 263–265 (2001).
Li Z, Mondragón A, DiGate RJ (2001) The mechanism of type IA
topoisomerase-mediated DNA topological transformations. Mol Cell
7: 301–307.
Lu SC (1999) Regulation of hepatic glutathione synthesis: current con-
cepts and controversies. FASEB J 13: 1169–1183.
Lu SC (2009) Regulation of glutathione synthesis. Mol Aspects Med 30:
42–59.
Meister A (1995) Glutathione methabolism. Methods Enzymol 251: 3–7.
Neder K, Marton LJ, Liu LF, Frydman B (1998) Reaction β-lapachone
and related naphthoquinones with 2-mercaptoethanol: a biomimetic
model of topoisomerase II poisoning by quinones. Cell Mol Biol 44:
465–474.
Pastore A, Federici G, Bertini E, Piemonte F (2003) Analysis of glu-
tathione: implication in redox and detoxification. Clin Chim Acta
333: 19–39.
Poot M, Teubert H, Rabinovitch PS, Kavanagh TJJ (1995) De novo syn-
thesis of glutathione is required for both entry into and progression
of the cell cycle. Cell Physiol 163: 555–560.
Schoeffler AJ, Berger JM ( 2008) DNA topoisomerases: harnessing and
constraining energy to govern chromosome topology. Q Rev Biophys
41: 41–101.
Sng J-H, Heaton VJ, Bell M, Maini P, Austin CA, Fisher LM (1999)
Molecular cloning and charachterizaton of the human topoisomer-
ase IIα and IIβ genes: evidence for isoform evolution through gene
duplication. Biochim Biophys Acta 1444: 395–406.
Tirumalai RS, Pargellis CA, Landy A (1996) Identification and charac-
terization of the N-ethylmaleimide-sensitive site in l-integrase. J Biol
Chem 271: 29599–29604.
Tsai-Pfugfelder M, Liu LF, Liu AA, Tewey KM, Whang-Peng J, Knut-
sen T, Huebner K, Croce CM, Wang JC (1988) Cloning and se-
quencing of cDNA encoding human DNA topoisomerase II and
localization of the gene to chromosome region 17g 21–22. Proc Natl
Acad Sci USA 85: 7177–7181.
Wang W, Bellatori N (1998) Endogenous glutathione conjugates: oc-
currence and biological functions. Pharmacol Rev 50: 335–355.
Voehringer DW, McConkey DJ, McDonnell TJ, Brisbay S, Meyn RE
(1998) Bcl-2 expression causes redistribution of glutathione to the
nucleus. Proc Natl Acad Sci USA 95: 2956–2960.
Wu X, Yalowich JC, Hasinoff BB (2011) Cadmium is a catalytic inhibi-
tor of DNA topoisomerase II. J Inorg Biochem 105: 833–838.
Wyckoff E, Natalie D, Nolan JM, Lee M, Hsieh T (1989) Structure of
the Drosophila DNA topoisomerase II gene. Nucleotide sequence
and homology among topoisomerases II. J Mol Biol 205: 1–13.
Yakisich JS, Sidén Å, Eneroth P, Cruz M (2001) Disulfiram is a potent
inhibitor of DNA topoisomerases. Biochem Biophys Res Commun 289:
586–590.