[CANCER RESEARCH S3. 5104-5107,
November 1. 1993]
Advances in Brief
Elevated Production of Active Oxygen in Bloom's Syndrome Cell Lines1
Thomas N¡cotera,2Kuldip Thusu, and Paresh Dandona
Biophysics Department,
¡K.T., P. D.¡
Roswell Part Cancer Institute, Buffala, New York 14263 ¡T.N./, and Department
Abstract
is considered to reflect the level of oxidative stress (9). An elevated
rate of formation of endogenously generated Superoxide radicals acted
on by an increased concentration of SOD is expected to result in high
levels of hydrogen peroxide. Without a parallel induction of the catalase and peroxidase activities that would remove the hydrogen per
oxide formed, considerable cellular damage is likely to occur.
Neutrophils possess a bactericidal activity attributable, in part, to
their ability to generate oxygen radicals including Superoxide, hydro
gen peroxide, and hydroxyl radicals (10). Superoxide is produced
primarily through the activation of a plasma membrane-bound
NADPH oxidase system (11). The contribution that Superoxide and its
dismutation products make to the bactericidal capacity of competent
cells is demonstrated by the susceptibility to infection of patients with
chronic granulomatous disease, in whom there is present a defect in
respiratory burst oxidases (12). Other cell types, including EBV-transformed B-lymphoblastoid cell lines, can also undergo an oxidative
Based on our previous evidence indicating that the elevated sister chroiii.itiil exchange that characterizes Bloom syndrome (BS) cells may arise in
response to elevated production of active oxygen, we have quantitated the
levels of active oxygen in two control, two BS and one BS revertant cell
lines, l HUHN.¡l-,l.
p, ini, ni chemiluminescence was used as a measure of
active oxygen production following treatment of the cells with the calcium
ionophorc A23187 or the chemotactic tripeptide iV-formylmethionylleucylphenylalanine. A peptide factor present in plasma was required for prim
ing the cells to undergo the oxidative response. As determined with
A23187, active oxygen production was elevated in BS cell lines by 48.6%
above control. Using A'-formylmethionylleucylphenylalanine,
active oxy
gen production was found to be increased by 250-314%. Chemilumines
cence was inhibited in a dose-dependent manner by diphenylene iodonium, which specifically binds to and inhibits membrane-associated
NAUPH oxidase activity. This compound inhibited oxygen radical pro
duction nearly 3 times more effectively in control cells than in BS cells. The
capacity to produce elevated levels of oxygen radicals may contribute to
the spontaneous chromosomal instability of BS cells and to the associated
high incidence of neoplasia in individuals with BS.
burst (13), although at a much lower rate than neutrophils. The func
tion of the oxidative burst in these cells is not presently clear (13).
In this study we assessed the capacity of various BS cell lines to
produce active oxygen following activation with either of two ago
nists, the calcium ionophore A23187 and the chemotactic tripeptide
fMLP. We also examined the participation of NADPH oxidase in the
oxidative burst occurring in these cells.
Introduction
BS3 is a rare autosomal recessive disorder that exhibits numerous
clinical features including sun sensitivity, growth retardation, and
immunodeficiency leading to infections of the respiratory tract (1).
The most prominent feature is a marked predisposition towards the
development of multiple types of cancer at a considerably earlier age
than that of the general population (1). BS cells exhibit a slow rate of
DNA replication (2), a high rate of spontaneous mutagenesis (3),
spontaneous somatic recombination (4), and chromosomal alterations
(5). Elevated SCEs are useful for identifying the disorder. Numerous
proposals have been advanced to explain the spontaneous chromo
somal damage observed in BS. It has been suggested that oxygen
radicals may cause the spontaneous chromosomal damage observed in
BS cells cither through their excessive production (6) or through a
deficiency in their removal (7). Indirect evidence that Superoxide
radicals are overproduced in BS cells has been provided previously.
Thus, in all six BS cell lines studied we found Superoxide dismutase
activity to be elevated over that of controls (7). That finding that has
now been confirmed by another laboratory (8). Further, a-tocopherol.
an inhibitor of lipid peroxidation, has been shown to inhibit the
formation of SCEs in BS cells in a dose-dependent manner (7); finally,
SCEs have been generated in control lymphoblastoid cell lines when
the superoxide-generating compound, paraquat, was administered at a
concentration that induces SOD activity (7). Since SOD activity mir
rors the cellular oxidative state in these cells, high dismutase activity
Received 8/30/93; accepted 9/17/93.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 V.S.C. Section 1734 solely to indicate this fact.
1This work was aided by Institutional Grant IN-54-29 of the American Cancer Society
and by a grant from the Buffalo Medical Foundation.
2 To whom requests for reprints should be addressed, at Biophysics Department.
Roswell Park Memorial Institute. Elm and Carlton Streets, Buffalo. NY 14263.
3 The abbreviations used are: BS. Bloom's syndrome: SCE, sister chromatid exchange;
SOD, Superoxide dismutase; fMLP, W-formylmethionylleucinylphenylalanine;
phenylene iodonium; EBV. Epstein-Barr virus.
DPI, di
of Medicine, Millard Filmare Hospitals, Buffalo, New York 14209
Materials and Methods
Cell Lines. The EBV-transformed B-lymphoblastoid BS cell line GM3403,
control B-lymphohlastoid cell lines GM3299 and GM1953, and BS revenants
GM4408 were obtained from the Human Mutant Cell Repository (Camden,
NJ). The lymphoblastoid cell line HG1525 was a gift from Dr. James German
(New York Blood Center, New York, NY).
Plasma Preparation. Fresh blood drawn from normal individuals prior to
each assay was allowed to stand for 2 h, after which it was centrifuged for 10
min at 180 X g. The plasma layer was removed by pipeting and was again
centrifuged at 400 x g for 10 min. The supernatant was once again removed,
leaving behind any remaining cells pelleted to the bottom of the tube.
Chemiluminescence.
Cells were grown in RPMI 1640 (GIBCO, Grand
Island, NY) containing penicillin, streptomycin, and 10% fetal calf serum
(Hyclone, Logan, UT). Log phase cells were adjusted to a concentration of
4 X 10s cells/ml in fresh media. Aliquots (0.5 ml) of this cell suspension were
placed into a cuvet, to which 50 /tl of fresh centrifuged plasma and 15 /¿Iof
luminol (10 mm in 0.1 Mborate buffer) were added. The cell preparation was
incubated for 2 min and 1.0 to 5.0 fil of A23187 or fMLP (dissolved in
dimethyl sulfoxide) was added. Incubation proceeded for the length of time
required for each agonist to effect the maximal level of chemiluminescence.
With the calcium ionophore A23187 (0.5-8.0 /AM)maximum output was at
tained in 6 min, and with fMLP (2.5-600 I\M)3 min were required. The assays
were conducted at 37°C with continuous stirring. For studies using the
NADPH oxidase inhibitor DPI (17.5-210 HM) (obtained from Dr. O. T. G.
Jones, University of Bristol, Bristol, United Kingdom), the cells were preincubated for 2 min with inhibitor prior to the addition of agonist. Apparent
kinetic constants (Vm and Kr) for DPI on chemiluminescence were calculated
using the non-linear least squares computer program NFIT (Island Products,
Galveston, TX). Nondenatured and heat-denatured proteinase K (30 /ig)
(Boeringer Mannheim. Indianapolis, IN) was preincubated with plasma in the
assay. SOD (10 jig) and catalase (20 and 40 ¡ig)were also tested for inhibition
of chemiluminescence both individually and in combination. Peak chemilu-
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ELEVATED
minescence was obtained by monitoring continuously
Model 800 Lumi-Aggregometer (Havertown, PA).
PRODUCTION
OF ACTIVE
IN BS CELL
LINES
Table 2 Active oxygen species released in fMLP stimulated B-lvmpltohlastoid
determined hy SOU and catalase treatment
with a Chrono-Log
cells as
Active oxygen species released following the activation of cells are characterized using
the luminol-dependent chemiluminescence protocol in "Materials and Methods." SOD
Results
Plasma Requirement. BS, BS revenant, or control EBV-transformed B-lytnphoblastoid cell lines did not generate an oxidative burst
without first being exposed to a small quantity of fresh plasma ob
tained from normal individuals (Table 1). This result is in contrast to
the finding that under similar conditions EBV-transformed B-cells
required only agonist for expression of oxidase activity (13). Lym
phocytes isolated from whole blood were also incapable of undergo
ing an oxidative burst without addition of plasma (data not shown).
The level of the oxidative burst obtained varied with the source of
plasma, but the difference between the oxidative response of BS and
control cells remained constant.
Characterization of the Oxidase-stimulating Plasma Compo
nent. Plasma obtained from freshly centrifuged blood demonstrated
little or no capacity to stimulate NADPH oxidase activity when added
to cells alone or in combination with agonist (Table 1). Allowing the
blood to stand at ambient temperature for 2 h prior to centrifugation
induced full oxidase activity (Table 1). Activated plasma stored in the
cold for 2 days or heated at 60°Cfor 10 min lost its oxidase activity.
Treatment of the plasma with 30 jug of proteinase K for 10 min also
resulted in the loss of all oxidase activity (Table 1). These experiments
indicate that the plasma component required for the activation of
NADPH oxidase is a protein, which is either released from blood cells
or processed in the plasma during standing at room temperature.
SOD and Catatase Inhibition of Chemiluminescence. Luminoldependent chemiluminescence offers a highly sensitive means of de
tecting active oxygen, although it is not specific towards any one
species of reactive oxygen. SOD (10 fj.g) inhibited 81% of the che
miluminescence, whereas catalase (40 jug) inhibited only 47% of the
activity (Table 2). A catalase concentration (20 fig) that, by itself, was
essentially inactive, complemented SOD in eliminating all chemilu
minescence. These data indicate that Superoxide is the major species
of active oxygen generated and that hydrogen peroxide may subse
quently be formed, likely through the dismutation of the Superoxide
radical.
Stimulation of Oxidase Activity by A23187 and fMLP. Activa
tion of luminol-dependent chemiluminescence in response to the cal
cium ionophore A23187 demonstrated a 48.6% increase in oxidative
activity in two separate experiments using one BS and one control
B-lymphoblastoid cell line (Fig. \,A and B). In order to compare their
priming effect, plasma from two separate donors was used. Although
the level of chemiluminescence attained differed slightly between the
two experiments (Fig. 1, A and B), the response was consistently
higher in BS cells. A sigmoidal dose-response curve is suggested for
the cells stimulated with A23187.
Activation of all four B-cell lines by fMLP using plasma from the
same donor consistently yielded an approximately 3-fold higher level
of chemiluminescence in BS cell lines as compared to control cell
Table 1 Oxidase-stimulating activity of plasma in B-lympfioblastoid cells stimulated
with fMLP under various incubation conditions
The oxidase-stimulating plasma component was assayed using the luminol-dependent
chemiluminescence protocol described in "Materials and Methods." Fresh blood was
preincubated at 37°Clor 2 h and centrifuged and the plasma was removed. Isolated plasma
was further treated as noted and added to the assay mixture
fMLP
OXYGEN
Plasma
Treatment
+
+
+
2h preincubation
No incubation
2h prcincubation
2 h prcincubation
Preincubation at 60°Cfor 10 min
Preincubation with proteinase K'1
•¿f
+
' Activity is 4.25 mV/2 X IO5 cells.
' Plasma was prctrcutcd for 10 min with 30 fig of proteinase K.
Activity
(10 fig) and catalase (20 and 40 fig) scavenge Superoxide and hydrogen peroxide,
respectively. Each value given is the average of triplicate determinations.
Treatment
IO fig SOD"
20 fig catalase''
40 /j.g catalase
10 fig SOD + 20 >ig catalase
" 3100 units/mg protein of SOD were used.
'' 1500 units/mg protein of catalase were used.
Inhibition (%) ±SE
81.5 ±11.5
5.5 ±13.2
47.4 ±11.5
UK)
lines (Fig. 1C). A repeat experiment using a different source of plasma
showed a 2.5-fold increase in the maximal chemiluminescence in the
BS cell line GM3403 as compared to control cell line GM3299 (Fig.
ID). The BS cell line HG1525, when compared to control cell line
GM1953, also yielded an approximately 3-fold increase in active
oxygen following activation by fMLP and another source of plasma
(data not shown). Thus, the plasma is not responsible for the differ
ential oxidative response displayed by BS and control cells. Activation
of NADPH oxidase by fMLP generated a hyperbolic curve in contrast
to A23187, which generated a sigmoidal curve. These results indicate
that these two compounds activate NADPH oxidase by different
mechanisms.
Oxidase Activity of a Revertant BS Cell Line. A B-lymphoblas
toid cell line of BS origin, GM4408, is typical of BS cells that exhibit
low SCE levels and are considered to be revertan! cells. It has previ
ously been demonstrated that this cell line also contains normal SOD
(7) activity. We find (Fig. Iff) that normal levels of active oxygen are
produced in this cell line following stimulation by fMLP.
Inhibition of Oxidase Activity by DPI. Evidence for participation
of plasma membrane-bound NADPH oxidase in the oxidative burst
that occurred after stimulation of cells was provided by the fact that
the enzyme was inhibited by DPI (14). Control cells were inhibited
nearly 3-fold more effectively than were BS cells, controls exhibiting
an apparent K, of 40.1 nin and BS cells. 111.3 nM (Fig. IF). This
differential inhibition by DPI indicates a potentially aberrant protein
present in the BS cells.
Discussion
Numerous DNA repair and processing defects have been proposed
to explain the chromosomal instability associated with BS cells (15).
Early studies reported relatively normal rates of DNA repair (16-20)
in response to various types of photo- and chemically induced DNA
damage. Recent studies report a defective uracil-DNA glycosylase
enzyme (15, 21) and reduced DNA ligase I activity (22). However, a
defect in the encoding glycosylase gene has not been reported and the
DNA ligase I gene is normal and is normally transcribed (22). From
the early studies emerged the proposal that an endogenous mutagcnic
factor produced in BS cells is the cause of the chromosomal damage
(23). A small molecular weight clastogenic factor isolated from the
media of BS cells or from the plasma of BS patients was reported to
have caused chromosomal damage in normal cells (6). Addition of
SOD reduced the level of chromosomal damage induced by the clas
togenic factor (6). It was suggested thereby, that a deficiency in the
removal of oxygen radicals plays a role in the spontaneous chromo
somal damage observed in BS cells. However, several lines of indirect
evidence have been advanced to support the hypothesis that sponta
neous oxidative stress in BS cells rather than a deficiency in the
removal of active oxygen is responsible for the observed chromo
somal damage (7).
In the present study, luminol-dependent chemiluminescence was
used as a measure of oxygen radical production following the activa
tion of the cells with several agonists. The calcium ionophore pro-
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ELEVATI-.!)
PRODI'CI
ION OÃ- ACTIVI
OXYCil-N
IN US < I•!.[.LINES
0123456789
Fig. 1. Extent of chcmilumincscence (CL) ob
tained in response to treatment with calcium ionophore A23187 using (A ) BS cells GM3403 (•)and
control cells GM3299 (O) and plasma from donor
I and (B) BS cells HG1525 (•) control cells
GM1953 (O) and plasma from donor 2. Extent of
chemilumincscence obtained in response to treat
ment with fMLP for (C) BS cells HG1525 (•)and
GM3403 (•)and control cells GM1953 (O) and
GM3299 (CD)using plasma from donor 3. (D) exlent of chemiluminesccnce response to fMLP of BS
cells HG1525 (•)and control cells GM1953 (O)
using plasma from donor 4. Each point is the av
erage of triplicate determinations ±SE (bars). (£)
Extent of chemiluminescence obtained in BS cells
GM1525 (•)and control cells GM4408 (O) in
response to fMLP and plasma from donor 5. Each
point is the average of duplicate determinations ±
SE. (F) Inhibition of luminol-dependenl chemilu
minescence by DPI in the BS cell line GM3403 (•)
and in the control cell line GM3291) (O) in response
to fMLP and plasma from donor 6. Each point is the
average of duplicate determinations ±SE.
10 20 30 40 50 60 7C
x
N
V
PU
O
1
Calcium
0
lonophore
A231B7
10
(xlf/M)
20 30 40 50
fMLP (xlO°M)
60
1.4 r
IO
o
fr?
X
N
O
IP
fi
'2
U
a
u
CL,
0.0<
o
10
20
fMLP
30
40
(xl08M)
duced a 48.6% increase in oxygen radicals and fMLP produced a
250-314% increase. In addition, the tumor promoter TPA was also
shown to cause excessive production of oxygen radicals in BS cells
(data not shown). In every case, BS cells produced significantly
greater quantities of oxygen radicals than did control cells. It has
previously been reported that activation of NADPH oxidase activity in
EBV-transformed B-lymphoblastoid cell lines is not elicited by fMLP
(13). The reason for the difference between our results and that of
others is not clear. However, the major difference in our finding is the
requirement for plasma; only when the cells are activated do they
respond to a variety of agonist. Exploitation of these contrasting
observations may lead to a clearer understanding of the activation
process.
Participation of NADPH oxidase in the oxidative burst is strongly
implicated by the dose-dependent inhibition of chemiluminescence
with nanomolar concentrations of DPI. DPI has been previously
shown to inhibit NADPH oxidase activity in both neutrophils (24) and
in B-cell lines (25) through covalent binding to a M, 45,000 FAD-
50
60
50
100
150
200
250
DPI (nM)
containing subunit of NADPH oxidase. The altered inhibition kinetics
observed suggest a possible aberrant oxidase peptide. In contrast to
chronic granulomatous disease, in which there exists a defect in one
of several subunits of NADPH oxidase that results in reduced activity
(12), we observe an increased activity in BS cells.
The suggested production of oxygen radicals at the plasma mem
brane is consistent with the hypothesis that SCE formation may be
mediated by lipid peroxidation. This hypothesis is supported by the
observation that SCE formation is inhibited by the addition of a-tocopherol, a potent inhibitor of lipid peroxidation. We have preliminary
data indicating that phospholipase A2 activity is elevated in two BS
cell lines and that this activity is decreased to levels present in control
cell lines by treatment with 10~4 M a-tocopherol.4 Phospholipase A2
cleaves fatty acids including arachidonic acid from the C-2 position of
phospholipids. Arachidonic acid is a well-known activator of NADPH
oxidase (13, 26). Furthermore, several protease inhibitors have been
4 Unpublished observations.
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ELEVATED
PRODUCTION
OÃ- ACTIVE
shown to decrease SCE formation in BS cells by approximately 50%
(27). Protease/protease inhibitor complexes are known to regulate
NADPH oxidase-dependent Superoxide production in neutrophils
(28). However, the mechanism for altered regulation of NADPH oxi
dase activity in BS cells remains to be established.
In conclusion, the data presented in this paper represent the first
direct demonstration of an increase in oxygen radical production in
lymphoblastoid cell lines from Bloom's syndrome. This increase in
oxygen radical production supports the long debated notion that an
endogenous mutagenic process takes place and contributes to the
observed chromosomal damage that is characteristic of Bloom's syn
drome cells rather from than a defect in DNA repair processing.
OXYGEN
IN BS CELL
LINES
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12. Dinaucr. M. M.. and Orkin. S. H. Chronic granulomatous disease. Annu. Rev. Med..
43: 117-124. 1992.
13. Jones, 0. T. G., Hancock, J. T.. and Henderson. I.. M. Oxygen radical production by
transformed B lymphocytes. Mol. Aspects Med., 12: 87-92, 1991.
14. Hancock, J. T.. and Jones. O. T. G. The inhibition by diphenylcneiodonium and its
analogues of Superoxide generation by macrophages. Biochem. J., 242: 103-107,
1987.
15. Nicolera. T. M. Molecular and biochemical aspects of Bloom's syndrome. Cancer
Genet. Cytogenet.. 53: 1-13. 1991.
16. Ahmed. F. E., and Setlow, R. B. Excision repair in ataxia telangiectasia. Fanconi's
anemia. Cockayne syndrome and Bloom's syndrome after treatment with ultraviolet
radiation and Ã-V-acctoxy-2-acetylaminofluorene. Biochim. Biophys. Acta. 52/: 805817, 1978.
17. Vincent, R. A., Hays, M. D.. and Johnson. R. C. Single-stranded DNA breakage and
repair in Bloom's syndrome cells. In: P. C. Hanawalt, E. C. Friedberg, and C. F. Fox
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Acknowledgments
The authors wish to thank Dr. Alexander Bloch tor careful reading of the
manuscript.
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Elevated Production of Active Oxygen in Bloom's Syndrome
Cell Lines
Thomas Nicotera, Kuldip Thusu and Paresh Dandona
Cancer Res 1993;53:5104-5107.
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