Cell-penetrating autoantibody induces caspase-mediated apoptosis through catalytic hydrolysis of DNA

Article abstract of DOI:10.1016/j.bmc.2006.12.037

In the present study, we investigated the substrate specificity of catalytic activity of a cytotoxic anti-DNA monoclonal autoantibody, G1-5, which was obtained from an MRL-lpr/lpr mouse by hybridoma technology. The antibody catalyzed hydrolysis of single-

Full text of DOI:10.1016/j.bmc.2006.12.037

Bioorganic & Medicinal Chemistry 15 (2007) 2016–2023
Cell-penetrating autoantibody induces caspase-mediated apoptosis
through catalytic hydrolysis of DNA
a
a
b
b
Eun-Jung Lee, Eun-Jung Jang, Eunhae Lee, Jaehoon Yu,
c
a,
*
Hee Yong Chung and Young-Ju Jang
a
Laboratory of Immunology, Institute for Medical Science and BK 21 Program, Ajou University, School of Medicine,
Suwon 443-721, Republic of Korea
b
Department of Chemistry and Education, Seoul National University, Seoul 151-742, Republic of Korea
Department of Microbiology, Hanyang University College of Medicine, Seoul 133-791, Republic of Korea
c
Received 9 November 2006; revised 22 December 2006; accepted 23 December 2006
Available online 30 December 2006
Abstract—In the present study, we investigated the substrate specificity of catalytic activity of a cytotoxic anti-DNA monoclonal
autoantibody, G1-5, which was obtained from an MRL-lpr/lpr mouse by hybridoma technology. The antibody catalyzed hydrolysis
of single- and double-stranded DNA with a higher substrate specificity for thymine than adenine by either b-glycosidic or phospho-
diester bond cleavage. The hydrolysis rate (kcat) showed maximum at acidic pH conditions, suggesting that the catalytic site of the
antibody contains essential carboxylic group(s). Treatment of cells with the antibody promoted cell death and induced the activation
of caspases. The cell death induced by the antibody was inhibited by the pan-caspase inhibitor. Furthermore, the antibody binds to
cell membrane and penetrates into the cells. Our results suggest that the cell death is initiated by antibodies penetrating to cells and
nucleus, hydrolyzing considerable amount of DNA, and mediating the caspase-dependent apoptotic pathway.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
properties of chemical bonds in DNA substrates that
hydrolyzed by anti-DNA autoantibodies isolated from
SLE-prone humans or animals have not been reported
except one case. Our group previously produced several
anti-double-stranded (dsDNA) monoclonal autoanti-
Circulating autoantibodies that interact with DNA are a
typical feature of systemic lupus erythematosus (SLE) in
humans and MRL-lpr/lpr mice. Some anti-DNA auto-
antibodies have been known to have nuclease or prote-
1
1
0
bodies from an MRL-lpr/lpr mouse and reported that
one of the autoantibodies, G1-2, has catalytic activities,
with a preference for the hydrolysis of b-glucosidic
2
,3
ase activity as well as cytotoxicity in vitro.
8
Antibodies (Abs) with nuclease or protease activities,
known as catalytic Abs, are found in autoimmune-prone
bonds.
4
,5
mice more frequently than in normal mice, suggesting
that catalytic activity participates in the unique function
and pathogenesis of autoantibodies. Catalytic Abs have
been generated by in vitro immunization in autoim-
mune-prone and normal mice using transition state ana-
Besides the catalytic activity, there are other interesting
features of the anti-DNA autoantibodies that may par-
ticipate in their pathogenic effects. Autoantibodies such
as anti-DNA and anti-RNP can penetrate into living
cells and interact with their intracellular and nuclear tar-
11–14
6
logs as haptens. Antibodies against these haptens could
catalyze hydrolysis of the intended nuclease or protease
gets.
Some of these autoantibodies possess multiple
positively charged amino acids in the complementarity
determining region 3 of H chain (CDR3H) in a sequence
7
activities. Natural autoantibodies also showed the same
8
,9
15
enzymatic activities.
However, the fine structural
that resembles a nuclear localization signal. Several
studies have demonstrated that the cell penetrating
1
6
Abs can induce tumor cell death. Most of these stud-
ies, however, have been performed using polyclonal
anti-DNA Abs that have heterogeneous binding proper-
ties and functions. Recently, however, monoclonal anti-
bodies (mAbs) have been utilized for studying the
Keywords: Anti-DNA autoantibody; Catalytic activity; Glycosidic
bond; Phosphodiester bond; Cytotoxicity.
*
Corresponding author. Tel.: +82 31 219 4516; fax: +82 31 219
401; e-mail: jangyj@ajou.ac.kr
4
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.037

E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
2017
involvement of signal pathways in penetrating anti-
1
2.2. Substrate specificity of enzymatic activity
7
DNA antibody-induced apoptosis.
Studies using
mAbs are necessary to more clearly determine not only
the mechanisms of cell death but also details of DNA
hydrolysis induced by anti-DNA autoantibodies. Such
studies are important because cytotoxic anti-DNA
antibodies are currently of great interest in the fields
of autoimmunity and cancer therapy.
To further analyze the DNA hydrolyzing activity of
mAb G1-5, we initially measured the cleavage of the
digoxigenin-labeled single-stranded oligonucleotides
((dA)40 or (dT) ) and double-stranded oligonucleo-
40
tides((dA) :(dT) ). The Ab hydrolyzed both ssDNA
4
0
40
and dsDNA substrates with sequence preference for thy-
mine over adenine (Fig. 2a). We also investigated the
cleavage of the same oligonucleotides by S1 nuclease
and DNase I. S1 nuclease cleaved both (dA)40 and
(dT)40 without a clear sequence specificity (Fig. 2b).
Whereas DNase I showed remarkable cleaving activity
against (dA) :(dT) , it cleaved either (dA) or (dT)40
In the present study, we not only measured the catalytic
properties of mAb G1-5 but also studied cytotoxic
mechanisms, because G1-5 showed the highest cytotoxic
activity for several cancer cell-lines among various
mAbs we produced. By analyzing catalytic activities of
DNA hydrolysis and mechanisms of cell death induced
by anti-DNA autoantibodies using G1-5, we propose
that the autoantibodies penetrate into the cells and
hydrolyze DNA and induce caspase-mediated
apoptosis.
4
0
40
40
at lower levels (Fig. 2c). By showing a difference of the
substrate specificity between mAb G1-5 and DNases,
we could eliminate a possibility of contaminations of
the purified antibody with natural enzymes. To examine
the activity in greater detail, we also measured the glyco-
sidase and phosphatase activities of the Ab using vari-
ous DNA substrates (Fig. 3a) and used the results to
calculate their kinetic constants (Table 1). Although
2
. Results and discussion
the kcat and K values were not as efficient as natural en-
m
2.1. DNA hydrolyzing activity of anti-DNA monoclonal
autoantibody G1-5
zymes, mAb G1-5 showed clearly glycosidase and phos-
phatase activities toward a variety of substrates. Both
the glycosidase and phosphatase showed saturation
kinetics. As demonstrated by the highest kcat/Km value,
the Ab had the highest catalytic specificity for pNP-b-
Gal for glycosidase activity, which has a same b-anomer
as the C–N glycosidic bond in DNA. The maximum
activity was observed at pH 4–4.8, suggesting the pres-
ence of an essential carboxylic group(s) in the catalytic
site of the Ab (Fig. 3b). Figure 3c shows a typical
Lineweaver–Burk plot used to calculate the kinetic
constants.
We examined the ability of mAb G1-5 to hydrolyze
dsDNA using plasmid pUC18 and double-stranded
M13mp18 as substrates. As shown in Figure 1a, G1-
5
IgG (100 lg/ml) transformed the supercoiled
pUC18 into an open circular form and cleaved the
M13mp18 in 3 h. Fab (100 lg/ml) produced by papain
digestion of G1-5 IgG also hydrolyzed both pUC18
and M13mp18 DNA, although the activity was lower
than that of the intact IgG. When pUC18 DNA was
treated with various concentrations of mAb G1-5, the
cleaving activity of the Ab was observed at an even
lower concentration (30 lg/ml), and some open circu-
lar DNA was converted to the linear form by
The mAb G1-5 has glycosidase and phosphatase activi-
ties toward both dsDNA and ssDNA with a higher spec-
ificity for thymine than adenine. The ability to cleave
glycosidic and phosphodiester bonds of DNA might
be an intrinsic property of anti-DNA Abs, because
these chemical bonds are the most frequent in DNA.
The exertion of the glycosidase activities toward
specific DNA structure such as C–N glycosidic bond
by mAb G1-5 might partly contribute to DNA
300 lg/ml G1-5 (Fig. 1b). Also, hydrolysis of pUC18
DNA was time-dependent (Fig. 1c). In addition, the
supercoiled form of pUC18 was reduced starting after
1
6
h of incubation and disappeared completely after
h, and the linear form appeared after 6 h or more
of incubation.
Figure 1. Cleavage of supercoiled pUC18 DNA or M13mp18 dsDNA by mAbG1-5. (a) pUC18 or M13mp18 dsDNA (1 lg) was incubated without
lanes 1 and 4) or with 100 lg/ml of G1-5 IgG (lanes 2 and 5) or G1-5 Fab (lanes 3 and 6) for 3 h. (b) pUC18 DNA (1 lg) was incubated with 0 (lane
), 30 (lane 2), 50 (lane 3), 100 (lane 4) or 300 (lane 5) lg/ml G1-5 for 3 h. (c) pUC18 DNA (1 lg) was incubated for 1, 3, 6, or 16 h (lanes 1–4,
respectively) with 100 lg/ml G1-5. SC, OC, and L represent supercoiled, open circular, and linear forms of plasmid DNA, respectively.
(
1

2018
E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
0
0
Figure 2. ALONA of mAb G1-5 and natural DNases. Double-labeled (5 -digoxigenin and 3 -biotin) single-stranded 40-mer (dA)40 or (dT)40 and
double-stranded 40-mer (dA)40:(dT)40 immobilized on a streptavidin-coated microtiter plate was used as DNA substrates. The amount of uncleaved
DNA after treatment of mAb G1-5 (a), S1 nuclease (b), or DNase I (c) was determined as explained in Section 4. The results are presented as the
percentage of cleavage in the absence of added G1-5 and are means ± SD of triplicate measurements.
Figure 3. (a) Structure of DNA substrates used for studying the catalytic activities of G1-5. (b) The pH dependence of the catalytic activity (kcat) of
m
G1-5 for pNP-b-Gal. (c) A typical Lineweaver–Burk plot to determine the Vmax and K values. Substrate is pNP-b-Gal.
cleavage-mediated cell death, leading to tissue damage
in autoimmune disease. We also showed that an essen-
tial carboxylic group(s) might be present in the catalytic
site of the Ab by the observation that the catalytic activ-
ity was optimal under acidic conditions. The carboxylic
acid moieties of the Glu residues in CDR3H, CDR2 of
light chain (CDR2L), and CDR3L of mAb G1-5
plored clearly whether the acidic amino acids contribute
to the catalytic activity, contrary to the fact that the pos-
itively charged amino acid residues play a role in DNA
binding by studies such as site-directed mutagenesis
and computer-modeling.
1
,18
Catalytic properties of mAb G1-5 are quite different
8
(Fig. 4) may contribute to the hydrolyzing ability by
functioning as general acid–bases. It is worth being ex-
from those of mAb G1-2 , which has very high sequence
identity with mAb G1-5 (Fig. 4). First, the kcat values of

E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
Table 1. Catalytic constants of Ab G1-5 for various substrates
2019
ꢀ1
ꢀ1
ꢀ1
Substrate
V
max (M/min)
k
cat (min
)
K
m
(lM)
kcat/K
m
(M min )
Reference
p-Nitrophenyl-a-D-glucopyranoside (pNP-a-Glc)
p-Nitrophenyl-b-D-glucopyranoside (pNP-b-Glc)
pNP-a-galactopyranoside (Gal)
pNP-b-Gal
pNP-a-mannopyranoside (Man)
pNP-b-Man
p-Nitrophenyl phosphate (pNPP)
—
—
—
—
ND
ꢀ
ꢀ
ꢀ
ꢀ
11
10
9
ꢀ4
ꢀ4
ꢀ3
ꢀ4
2
9.3 · 10
2.7 · 10
1.2 · 10
4.0 · 10
—
1.5 · 10
4.2 · 10
1.8 · 10
4.8 · 10
—
2.1 · 10
2.0 · 10
2.3 · 10
5.9 · 10
—
0.71
2.1
7.8
0.81
—
pH 5.0
pH 5.0
pH 5.0
pH 5.0
ND
2
2
2
10
ꢀ
ꢀ
9
9
ꢀ3
ꢀ4
2
1.1 · 10
1.3 · 10
—
1.8 · 10
2.0 · 10
—
3.8 · 10
1.4 · 10
—
4.7
0.14
—
pH 5.0
pH 7.5
ND
3
Bis-pNPP
ND, not detected. The assay was carried out at 37 ꢁC with 0.094 mg/mL G1-5 in 10 mM MES (pH 5) with 150 mM NaCl.
Figure 4. Alignment of amino acid sequences of the VH (a) and VL regions (b) of G1-5 and G1-2. Different amino acids and CDR regions are shown
in red and underlined in blue, respectively. The GenBank Accession Nos. are AF289182 (G1-5VH), AF289183 (G1-5 VL), AF289180 (G1-2 VH), and
AF289181 (G1-2 VL).
mAb G1-5 are about an order of magnitude lower than
those of mAb G1-2. Second, mAb G1-5 accepts both a-
and b-anomeric carbohydrates as substrates, whereas
mAb G1-2 is specific for b-anomeric substrates. Third,
the best substrates for mAbs G1-5 and G1-2 are b-galac-
toside and b-glucoside, respectively. The phosphatase
activity of mAb G1-5 is also an order of magnitude low-
er than that of mAb G1-2. Interestingly, there are only a
few different amino acids between the two Abs (one in
CDR3-VL, one in FR1-VH, and one in FR4-VH, pre-
sented as red colored letters in Fig. 4), although they
have quite different catalytic activities. This may be
due to a relatively large conformational change in the
HeLa) (Fig. 5). Cell death rates of the cell lines were not
so high even at the high concentration of the antibody.
However, HL60 cells were the most sensitive to the
Ab-induced cytotoxicity. From 200 lg/ml of the Ab,
approximately 30–35% of the HL60 cells were dead.
Normal mouse IgG was not cytotoxic to these cell lines
even at 1000 lg/ml (data not shown). To determine
whether caspases are involved in the cell death induced
by the Ab, we examined caspase-3 activation and cleav-
age of PARP, an endogenous substrate of caspase-3.
When HL60 cells were treated with the Ab, both cas-
pase-3 activation and PARP cleavage were detected
within 48 h (Fig. 6a). Activations of caspase-3 and -7
were also analyzed by fluorescence intensities (Fig. 6b).
Both mAb G1-5 and sTRAIL, a well-known inducer
of caspase activation, activated caspase-3/7, whereas
cells cultured in the absence of either reagent did not
activate them. The levels of caspase-3/7 activation and
PARP cleavage induced by the mAb, however, were
lower than those induced by sTRAIL. One of the possi-
ble reasons might be the lower level of cell death induced
by the mAb G1-5. Next, we tested the effect of zVAD-
fmk, a pan-caspase inhibitor, on mAb G1-5-induced cell
death. As shown in Figure 6c, zVAD-fmk dose-depen-
dently inhibited both sTRAIL- and mAb G1-5-induced
1
9
Abs as a result of mutations in the FR. Similar find-
ings were also previously reported for glycosidase anti-
6
bodies differing by only four amino acids in the FR.
It is suggested that anti-DNA autoantibodies found in
the sera of SLE patients and MRL-lpr/lpr mice have dis-
tinct abilities to cleave DNA even at the level of fine dif-
ference of chemical bonds and forms.
2
.3. Analysis of cell death induced by mAb G1-5
We analyzed cytotoxicity of the Ab in the range of 30–
000 lg/ml in several cell lines (HL60, L929, Jurkat, and
1

2020
E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
The mAb G1-5 induced cell death by mechanisms
involving activation of caspases. These findings are
essentially in agreement with recent reports showing
the importance of caspases in anti-DNA Ab-induced
1
7
apoptosis. Similarly, cytotoxic Abs against recoverin
also induced the death of retinal cells by activating a cas-
2
0
pase 3-dependent apoptosis pathway. However, we
could not exclude involvement of other mechanisms
such as caspase-independent apoptosis for following
reasons; compared to the experiments performed with
sTRAIL, the mAb G1-5 did not fully activate caspases,
and the cell death induced by the mAb was not com-
pletely inhibited by a pan-caspase inhibitor.
From above results, two mechanisms are suggested in
G1-5-induced cell death. First, upon penetrating the
cells, mAb G1-5 enters the nucleus, where it cleaves
DNA due to enzymatic activities, such as glycosidase
or phosphatase, resulting in cell death. Second, the
mAb induces cell death by triggering a signal transduc-
tion pathway (at least partly caspase-dependent) upon
binding to the cell membrane. It is possible that the
two mechanisms may work together to cause cell death
as suggested in recent report. Further analysis of the
DNA-hydrolyzing and cytotoxic activities of anti-
DNA antibodies will lead to a better understanding of
the mechanism of tissue damage and pathogenesis of
autoimmune diseases such as SLE.
Figure 5. Cytotoxicity of mAb G1-5 against various cell lines.
Percentage survival was determined by CCK-8 assay after incubation
of each cell line with indicated concentrations of G1-5 for 24 h. The
extent of cell survival after a 24-h culture without Ab, but with the
same volume of Ab dilution buffer, phosphate-buffered saline (PBS)
was regarded as 100%. Results shown are means ± SD of at least
triplicate measurements.
1
7
death of HL60 cells, although the level of the inhibition
by zVAD-fmk was relatively lower in case of treatment
of the Ab. These results suggest that the mechanism of
cell death induced by mAb G1-5 includes the activation
of caspases in some extent.
Figure 6. (a) Caspase-3 activation and PARP cleavage in HL60 cells treated with mAb G1-5 or sTRAIL. Cells were cultured in the absence (lane 1)
or presence of 100 lg/ml G1-5 (lane 2) or 1 lg/ml sTRAIL (lane 3) for 24 h. Cells were lysed in Laemmli sample buffer. Samples were normalized for
total protein content and were analyzed by Western blotting. (b) After a 24-h treatment with 100 lg/ml G1-5 or 1 lg/ml sTRAIL, caspase 3/7 activity
was measured in the cell culture supernatant using ApoONE homogeneous caspase-3/7 assay reagents and a fluorometric plate reader. The activity is
presented as the relative fluorescence units (RFLU) compared to cells incubated with the same volume of PBS (buffer used for dilution of G1-5 or
4
sTRAIL). (c) Effects of zVAD-fmk on the cytotoxicity of G1-5 or sTRAIL in HL60 cells. Cells (4 · 10 ) were treated with 100 lg/ml G1-5 or 1 lg/ml
sTRAIL for 24 h in the presence of the indicated concentrations of zVAD-fmk. Cell viability was measured by CCK-8 assay. Results shown are
means ± SD of triplicate measurements.

E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
2021
Figure 7. (a) Analysis of cell surface binding of G1-5 by FACS. After incubation of HL60 cells with various concentrations of G1-5 at 4 ꢁC for 3 h,
the cells were harvested, washed extensively, and fixed. Bound G1-5 was detected with fluorescein-EX-conjugated anti-mouse IgG using flow
cytometer. HL60 cells incubated with fluorescein-EX-anti mouse IgG alone were used as a control (filled bars). (b) Analysis of cell surface binding of
G1-5 by fluorescence microscopy. Image of L929 cells treated with 50 lg/ml of rhodamine Red-X-labeled G1-5 at 4 ꢁC for 2 h (200· magnification).
(c) Analysis of internalization of G1-5 into cells by FACS. HL60 cells were cultured at 37 ꢁC for 2 h in the presence of various concentrations of G1-5
labeled with fluorescein-EX. Cells incubated with PBS alone were used as control (filled bars). (d) Analysis of internalization of G1-5 into cells by
fluorescence microscopy. Images of L929 cells treated with 50 lg/ml of rhodamine Red-X-labeled G1-5 at 37 ꢁC for 10 min (left) or 2 h (right) (400·
magnification).
2.4. Cell surface binding and intracellular localization of
mAb G1-5
anti-DNA Abs from SLE patients contain multiple pos-
itively charged amino acids in the CDRs in a sequence
1
5
that resembles a nuclear localization signal. In fact,
mAb G1-5 has several Arg or Lys residues in the
CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, and
CDR3-VL regions, including two adjacent Arg residues
at the end of FR4 and CDR3 of VH (Fig. 4). It might be
suggested that mAb G1-5 is possibly localized to the
nuclear via the positively charged amino acids in the
antigen binding sites, although non-penetrating Abs
have also been known to contain the basic amino acids
We next determined whether the mAb could bind to the
cell surface and enter the cells. First, to examine the
binding of the mAb to the cell surface, we incubated
HL60 cells with mAb G1-5 at 4 ꢁC. The bound G1-5
was detected with fluorescein-EX-conjugated anti-
mouse IgG and performed flow cytometric analysis.
As shown in Figure 7a, mAb G1-5 bound to cell surface
in a dose-dependent manner from 0.5 up to 50 lg/ml. To
examine whether the mAb enters the cells, we cultured
HL60 cells in the presence of fluorescein-EX-labeled
G1-5 at 37 ꢁC. We found that the level of mAb G1-5
internalization increased with the concentration of Ab
in the range of 0.5–50 lg/ml (Fig. 7c). Cell surface bind-
ing and intracellular localization of the Ab were con-
firmed using adherent cell line L929 by fluorescence
microscopy. As shown in Figure 7b, when L929 cells
were treated with 50 lg/ml Rhodamine red-X-labeled
G1-5 for 2 h at 4 ꢁC, binding of the Ab to the cell surface
could be detected. As shown in Figure 7d, when the
L929 cells were incubated with the Rhodamine red-X-la-
beled G1-5 at 37 ꢁC, the Ab could be found inside the
cells within 10 min, although the level of inside Abs
was low. After 2 h, there were higher amounts of the
internalized Abs, and some of the labeled Abs were pres-
ent in the nucleus.
2
1
in the CDRs. Penetration of autoantibodies into living
cells and nuclear localization supports the possible inter-
action between the autoantibodies and nuclear DNA as
well as the role of the autoantibody in the pathogenesis
3
and tissue damage of autoimmune diseases.
3. Conclusion
Catalytic and cytotoxic properties of anti-DNA mono-
clonal autoantibody, G1-5, produced from an MRL-
lpr/lpr mouse were studied. The antibody preferentially
cleaves thymine-rich single- and double-stranded DNA
either by b-galactosidic or phosphodiester bond hydro-
lysis. The hydrolysis rate (kcat) was maximized at acidic
pH, suggesting that the general acid(s) is (are) involved
in the catalytic core. Treatment of cells with the autoan-
tibody promoted cell death, which is induced by the acti-
vation of caspases. Inhibition of apoptosis by a caspase
inhibitor proposes a mechanism that the antibodies
The mAb G1-5 entered the cells and was located in the
nucleus. It has been shown that the nuclear localizing

2022
E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
enter nucleus of cells, catalyzing the hydrolysis of DNA,
and then activating caspase-dependent apoptosis, result-
ing in cytotoxic effect.
oligonucleotides, heating at 65 ꢁC for 5 min, and cooling
slowly to room temperature. The amount of uncleaved
digoxigenin-labeled DNA was determined by adding
alkaline phosphatase-labeled anti-digoxigenin Fab frag-
ments (Roche), followed by p-nitrophenyl phosphate
(pNPP) substrate (Sigma). The absorbance of the solu-
tion in each well was then determined at 405 nm with
an ELISA plate reader. S1 nuclease and DNase I were
purchased from Amersham Pharmacia Biotech and
USB, respectively. Reaction of S1 nuclease (0.313–
10 U/well) with the oligonucleotides was performed at
pH 4.6 in 30 mM sodium acetate, 280 mM NaCl, and
4. Experimental
4
.1. Cell culture
Cancer cell lines HL60, Jurkat, L929, and HeLa were
obtained from the American Type Culture Collection.
Cell lines were maintained in either Roswell Park
Memorial Institute-1640 medium (HL60 and Jurkat)
or Dulbecco’s modified Eagle’s medium (L929 and
HeLa) supplemented with 10% fetal bovine serum (GIB-
CO BRL) and 1% penicillin/streptomycin (GIBCO
BRL) in a humidified atmosphere at 37 ꢁC and 5% CO2.
1 mM ZnSO for 7 min at 37 ꢁC. DNase I (0.008–5 lg/
4
ml) was incubated at pH 7.5 in 25 mM Tris–HCl,
10 mM MgCl for 30 min at 37 ꢁC.
2
4.5. Catalytic assays for mAb G1-5
4
.2. Production of monoclonal anti-DNA mAb G1-5 IgG
and G1-5 Fab fragments
Phosphatase activity was determined using an enzymatic
assay employing p-nitrophenyl phosphate (pNPP) or
bis-pNPP. Glycosidase activity was determined using
enzymatic assays with various substrates (Sigma) which
are illustrated in Figure 3a. Kinetic assays were per-
formed in 10 mM morpholinoethane sulfonic acid
(MES) (pH 3–7) or 10 mM EPPS (pH 7–8) in the pres-
ence of 0.6 mM of the Ab. Both buffers contained
100 mM NaCl, and the assays were performed at
37 ꢁC. Product conversion was determined by measuring
the amount of p-nitrophenol produced using an HPLC
system with monitoring at 315 nm. For the HPLC, a
C18 column (3.5 lm; 4.6 · 150 mm) was used as the sta-
tionary phase, and 50% aqueous acetonitrile containing
We previously produced monoclonal autoantibodies that
react with DNA from an MRL-lpr/lpr mouse using
1
0
hybridoma technology. Monoclonal IgG was purified
from the culture supernatant of a hybridoma clone by
protein G affinity chromatography (Amersham Biosci-
ences) according to the manufacturer’s instructions. Fab
fragments were prepared by digestion of the purified
mAb G1-5 IgG with papain (Sigma), followed by purifica-
2
2
tion by protein G affinity chromatography as described.
.3. Cytotoxicity assay
Cell viability was measured using a Cell Counting Kit-8
4
0
.1% trifluoroacetic acid was used as the mobile phase.
Kinetic data were collected under conditions where less
than 1% of the substrate was converted to the product.
Background hydrolysis rates were measured in the ab-
sence of the Ab and under the same conditions used
for the catalytic assays.
(
(
CCK-8) according to the manufacturer’s instructions
Dojindo Molecular Technologies, Japan). Briefly,
4
1
00 ll of target cells (2 · 10 cells for suspension cul-
3
tured cells and 6 · 10 cells for adherent cells) in the
presence or absence of the Ab was added to each well
of a 96-well plate (Costar), and the plate was incubated
4.6. Western blotting and fluorescent caspase assays
for 24 h at 37 ꢁC in a humidified 5% CO atmosphere.
2
Following incubation, 10 ll Dojindo’s tetrazolium salt,
WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
Western blotting was performed as described previous-
2
4
ly. Abs against caspase-3 and poly (ADP-ribose) poly-
merase (PARP) were purchased from Stressgen and Cell
Signaling Technology, respectively. Cells were plated in
5
-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium
salt), was added to each well for 3 h at 37 ꢁC, and
amount of the produced water-soluble formazan dye
was measured at 450 nm with a microplate reader.
4
white 96-well plates at the density of 2 · 10 cells per well.
The cells were treated with mAb G1-5 or soluble tumor
necrosis factor-related apoptosis inducing ligand
(sTRAIL) for 24 h at 37 ꢁC, and caspase activity was mea-
sured using a tagged caspase substrate (rhodamine-
labeled DEVD peptide) according to the manufacturer’s
instructions (Apo-ONE Homogeneous Caspase 3/7 As-
say; Promega). The release of the fluorogenic moiety by
activated caspase was measured at 485 nm using a fluoro-
metric plate reader (LS55 luminescene spectrometer; Per-
kin Elmer). Treatments were performed in triplicate, and
the average and standard deviation (SD) were plotted.
4
.4. Analysis of DNA and oligonucleotide cleavage
Plasmid DNA (pUC18) or M13mp18 dsDNA was incu-
bated with G1-5 IgG or G1-5 Fab in 25 mM Tris–HCl
(
pH 7.5) containing 10 mM MgCl . After incubation at
2
3
7 ꢁC, DNA was separated by electrophoresis on a 1%
agarose gel and stained with 0.5 lg/ml ethidium bromide.
For analyzing cleavage of dsDNA and ssDNA further,
affinity-linked oligonucleotide nuclease assay was per-
2
3
0
formed as described previously. The 5 -digoxigenin-
0
4.7. Cell surface binding and intracellular localization of
mAb G1-5
and 3 -biotin-labeled single-stranded oligonucleotides
were obtained from Genotech. The double-stranded
oligonucleotides were produced by mixing equimolar
amounts of the two complementary single-stranded
Cell surface binding and entrance of mAb G1-5
into cells were analyzed in HL60 or L929 cells by flow

E.-J. Lee et al. / Bioorg. Med. Chem. 15 (2007) 2016–2023
2023
cytometry and fluorescence microscopy using FACS-
Vantage (Becton–Dickinson) and Axiovert 200 (ZEISS),
respectively. The mAb G1-5 and anti-mouse IgG were
labeled using either fluorescein-EX (Invitrogen) or rho-
damine Red TM-X (Molecular Probes). Detailed meth-
ods of indirect FITC labeling and direct rhodamine
7. Wentworth, P., Jr.; Liu, Y.; Wentworth, A. D.; Fan, P.;
Foley, M. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A.
1
998, 95, 5971–5975.
8
9
. Nguyen, H. T. T.; Jang, Y. J.; Jeong, S.; Yu, J. Biochem.
Biophy. Res. Commun. 2003, 311, 767–773.
. Dubrovskaya,V.V.;Andryushkova,A.S.;Kuznetsova,I.A.;
Toporkova, L. B.; Buneva, V. N.; Orlovskaya, I. A.; Nevin-
sky, G. A. Biochemistry(Moscow)2003, 68, 1081–1088.
2
5
labeling are described.
1
1
1
1
1
0. Park, J. S.; Kim, Y. T.; Lee, C. H.; Youn, J. K.; Jang, Y. J.
Korean J. Biol. Sci. 1998, 2, 371–375.
1. Alacon-Segovia, D.; Llorente, L.; Ruiz-Argielles, A.
Immunol. Today 1996, 17, 164.
2. Madaio, M. P.; Yanase, K. J. Autoimmun. 1998, 11, 535–
Acknowledgments
This work was supported by a grant from the Korea
Research Foundation (MOEHRD, Basic Research
Promotion Fund; KRF-2006-521-C00132) and
Grant 2006 from the Department of Medical Sciences,
Graduate School of Ajou University. Support was
also partly received from the KOSEF fund. E.L. is a
beneficiary of the Brain Korea Program (BK21
Korea).
5
38.
3. Zack, D. J.; Stempniak, M.; Wong, A. L.; Taylor, C.;
Weisbart, R. H. J. Immunol. 1996, 157, 2082–2088.
4. Reichlin, M. J. Autoimmun. 1998, 6, 76–78.
a
15. Foster, M. H.; Kieber-Emmons, T.; Ohliger, M.; Madaio,
M. P. Immunol. Res. 1994, 13, 186–206.
16. Adamus, G.; Machnicki, M.; Elerding, H.; Sugden, B.;
Blocker, Y. S.; Fox, D. A. J. Autoimmun. 1998, 11, 523–533.
1
1
1
7. Rivadeneyra-Espinoza, L.; Ruiz-Arguelles, A. J. Autoim-
mun. 2006, 26, 52–56.
8. Park, J. S.; Kim, Y. T.; Chung, H. Y.; Beak, K.; Jang, Y.
J. Mol. Cells 2001, 11, 55–63.
9. Ulrich, H. D.; Mundorff, E.; Santarsiero, B. D.; Driggers,
E. M.; Stevens, R. C.; Schultz, P. G. Nature 1997, 389,
271–275.
References and notes
1
. Jang, Y. J.; Stollar, B. D. Cell. Mol. Life Sci. 2003, 60,
09–320.
3
2
3
. Paul, S. Ann. N. Y. Acad. Sci. 1998, 865, 238–246.
. Kozyr, A. V.; Sashchenko, L. P.; Kolesnikov, A. V.;
Zelenova, N. A.; Khaidukov, S. V. Immunol. Lett. 2002,
20. Adamus, G. Autoimmun. Rev. 2003, 2, 63–68.
21. Ternynck, T.; Avrameas, A.; Ragimbeau, J.; Buttin, G.;
Avrameas, S. J. Autoimmun. 1998, 11, 511–521.
22. Jang, Y. J.; Lecerf, J.-M.; Stollar, B. D. Mol. Immunol.
1996, 33, 197–210.
23. Mouratou, B.; Rouyre, S.; Guesdon, J.-L. J. Immunol.
Methods 2002, 269, 147–155.
24. Jang, Y. J.; Park, K. S.; Chung, H. Y.; Kim, H. I. Cancer
Lett. 2003, 194, 107–117.
8
0, 41–47.
4. Tawfik, D. S.; Chap, R.; Green, B. S.; Sela, M.; Eshhar, Z.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2145–2149.
5. Yu, J.; Choi, S. Y.; Han, E. Y.; Hwang, J. S.; Seo, S. H.;
Park, H.; Youn, H. J. Mol. Cells 1996, 6, 411–415.
6. Yu, J.; Choi, S. Y.; Moon, K. D.; Chung, H. H.; Youn, H.
J.; Jeong, S.; Park, H.; Schultz, P. G. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 2880–2884.
25. Jeon, Y. E.; Seo, C. W.; Yu, E. S.; Lee, C. J.; Park, S. G.
Mol. Immunol. 2007, 44, 827–836.