From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Human
Neutrophil-Mediated
By Alan
Lichtenstein,
Lysis
Miriam
Seelig,
Because
of recent
questions
concerning
the sensitivity
of
human
tumor
cells to neutrophil-induced
oxidative
injury,
we studied
six freshly
obtained
human
ovarian
cancer
(OC)
specimens.
Stimulation
of neutrophils
(PMNs)
by phorbol
myristate
acetate
(PMA)
did not result
in OC cytolysis
during
the first nine hours of incubation.
However,
three of
six specimens
were significantly
lysed by stimulated
PMNs
when
assay
length
was
increased
to 1 8 hours. Cytotoxicity
was mediated
by PMN production
of reactive
oxidative
intermediates
(ROls). Presentation
of ROls to OC targets
as
preformed
or enzymatically
generated
molecules
in cell-
M
OST
INVESTIGATIONS
responses
to tumor
growth
killer
lymphocytes
and
macrophages.
port
has
accumulated
(PMNs)
in the anti-tumor
readily
lyse continuously
gets
in vitro,
obtained
there
cells
potential
tance
sis,
role
The
mechanism
100-fold
cell
than
maximally
Since
the
tinuously
freshly
shown
for
about
the
an inherent
induced
lysis
from
2
of murine
of
affect
been
(LD)
to
above
targets
long-term
culture,
explanted
human
targets.
These
human
PMNs
could
(for human
tumors)
were
that
performed
may
have
in the current
ovarian
with
become
study,
cancer
con-
assays.4
In
mented
the role
other investigators
between
OC
intraperitoneal
relative
addition,
oxidative
lysis
previous
cells
of human
when
has
in murine
a temporal
tumor
short-term
PMN
assays
OC,5 and
correlation
influx
The
cells
docu-
results
used
Lysis
was mediated
mesh
or
Reagents.
(St
Louis):
The
following
EDTA,
myristate
(PMA),
xanthine,
Dimethylsulfoxide
Louis).
fern,
by
and
to 9
oxygen
Fresh
serous
Blood.
PMA
mesylate
was stored
OC specimens.
or
mucinous
was
obtained
was
in ethanol
All
primary
adenocarcinomas.
Vol 74, No 2 (August
1). 1989:
a gift
from
catalase,
urea,
(XO),
and
fetal
Cells
were
thus freed
through
procedure
significant.
RBCs
no. 100
was repeated
obtained
and
manner
to
interface,
solution
RBCs
gradient.
(formed
centrifuged
were
until
in this
Ficoll-Hypaque
on the gradient
sedimented
Ficoll
calf
serum
(FCS,
GIBCO,
1% nonessential
at
found
The
with
500
50%,
g for
100%
the
in
the
bottom.
Tumor
and
epithelial
cells
the
ing
The
monolayer
removed
times
30
Ficoll
cells
were
were
present
with
(CGD)
were
VA
of
1%
sodium
and
harvested
0.25%
of
by tapping
PMNs
X-linked
studied.
Wadsworth
minutes
Human
the
cells were
tumor
NY),
1% L-glutamine,
trypsin
incubation,
the flasks
1%
by overlayand
the
sharply.
0.02%
cells
were
They
were
and resuspended.
cells.
patients
ten
Island,
acids,
a solution
to
the plastic
from
three
with
five
After
Grand
amino
penicillin-streptomycin.
form
Approval
Patients
and
volunteers
for
research
from
were
and
Both patients
with CGD
b. PMNs
were
as
from
Boards
informed
protected.
donors
UCLA
these
blood
their
and
studies.
samples
privacy
were
would
have nondetectable
described,7
two
disease
the
for
that
that
previously
and
granulomatous
obtained
Review
purposes
isolated
normal
of chronic
was
Institutional
obtained
the
Medical
nology
Division
Center,
and
of
and
be
cytochrome
and
Cancer
Submitted
RBCs
were
by
Oncology,
29. 1988;
accepted
US Public
Health
RR865;
Gynecologic
and
the
VA
Wadsworth
of Microbiology
and
UCLA
Center,
Medical
Immuand
Center.
August
Supported
CA12800,
Hematology/Oncology.
the Departments
Gynecological
the Jonsson
from
Ciba-Geigy
specimens
cells
were
were
Address
Concern
reprint
Medical
Los Angeles,
March
24, 1989.
Service
Grants
No. CA37184,
Oncology
Group
Grant
No.
Foundation;
(St
(Suf-
requests
Center,
CA,
The publication
sulfate.
and
Research
funds
of
the
charge
payment.
“advertisement”
to Alan
691/WI
I 1H.
Lichtenstein,
Wilshire
MD,
and
VA
Wads-
Sawtelle
Blvds,
90073.
costs
ofthis
article
This
article
must
with
18 U.S.C.
section
were defrayed
therefore
be
1734
solely
in part
hereby
by page
marked
to indicate
this
fact.
at -20#{176}Cat 10’mol/L.
pp 805-809
by passage
same
a discontinuous
at the 80%
pyruvate,
worth
methimazole,
ferrous
Mallinckrodt
Tumor
tumors
Cells
50% interface.
After harvest,
the tumor cells were washed
three times in HBSS.
The cells were then maintained
as monolayer
cultures
on plastic in RPMI
1640 media supplemented
with 10%
Sigma
phorbol
from
tumor
Solid
needles.
VA.
purchased
thiourea,
oxidase
fragments
The
cell suspension
was overlayered
80%, and 100% Ficoll-Hypaque)
minutes.
Peritoneal
lymphocytes
fraction,
tumors.
with
washed
twice in Hanks’
balanced
salt
cells were separated
from contaminating
and
The tumor
From
(three
by reactive
(SOD),
n-mannitol,
xanthine
(DMSO)
Deferoxamine
NY).
were
dismutase
no longer
lymphocytes
solid
apart
confirm
METHODS
reagents
superoxide
acetate
aminotriazole,
AND
or
screens.
was
pooled
(HBSS).
fluid
from the tissue
cell release
cells.
and teased
stainless-steel
were
tumor
& Stratton, Inc.
a scalpel
CA1360-13;
MATERIALS
Zighelboim
human
peritoneal
with
were isolated
to PMN-induced
are
Cells
after
hours).
However,
a significant
tumor-lytic
effect on three of
six fresh OC targets
was detected
when assay durations
were
increased
to I 8 hours.
intermediates
(ROts).
either
Effector
as a more
research
cytoreduction
and
therapy
in humans.6
resistance
against
minced
we evaluated
(OC)
of tumor-lytic
PMNs
have demonstrated
tumor
(IP)
cells
washed
altered
and Jacob
1989 by Grune
at the
target
for PMNs.
OC cells
are well suited
for
culture
and use as targets
in cell-mediated
cyto-
toxicity
e
EDTA.
investigations
freshly
tor
PMNs.
mmol/L,
Cancer
free systems
duplicated
the enhanced
lysis at 1 8 hours (as
compared
with six hours).
Since addition
of catalase
at
three
or six hours did not inhibit enhanced
lysis at 1 8 hours
(achieved
by PMNs or in cell-free
systems),
it appears
that
an initial ROl-mediated
lethal event occurs early. but longer
incubations
are required
for the event to become
manifested
as cell death.
These
data
suggest
that
shorter
assays may underestimate
the potential
of PMNs as effec-
isolated
of nine
20
tumor
whether
of H202
resis-
by
Berek,
cytoly-
to oxidative
50%
ranged
those
during
the
sup-
stimulated.
passaged
relevant
short-term
less
doubt
injury
causing
types
higher
can
cells
of target
data
raised
the question
generate
lytic concentrations
when
tumor
of H20,
target
they
as has
by demonstrating
human
concentration
human
.
of PMNs
major
that
patients
and macrophages.2
contributed
further
of nonleukemic
the
In contrast,
Jonathan
from
of
participation
by neutrophils
process.
Although
PMNs
can
cultured
leukemia-lymphoma
tar-
from
lymphocytes’
recent
report3
cell-mediated
on the role
for
is no evidence
tumor
activated
A
on human
have focused
of Ovarian
either
isolated
©
I 989
by Grune
& Stratton,
Inc.
0006-4971/89/7402-0012$3.00/0
805
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LICHTENSTEIN
806
removed
by hypotonic
water lysis. Purity of PMN suspensions
was
>97% and viability
was >96%.
Chromium
release
assay.
Tumor targets were labeled by incubating
10 cells with 50 Ci chromium-Si
for one hour at 37#{176}C.
After being washed three times, targets (10k in 0.1 mL) were mixed
with PMN effectors
(at different
effector/target
E:T ratios),
H2O2,
xanthine
and
XO or H2O, and EDTA-chelated
ferrous
sulfate
(ferrous
sulfate chelated
by a threefold
molar excess of EDTA)
in
0.1 mL in microtiter
plates.
PMA (10
mol/L)
or inhibitors/
scavengers
were added
in appropriate
concentrations.
Samples
were
run in quadruplicate.
Plates were then centrifuged
briefly
(1,200
rpm, three minutes)
to enhance
cell contact.
After incubation
at
37#{176}C
in a 5% CO2 incubator
for varying
durations,
plates were
centrifuged
(1,200
rpm, ten minutes)
and 0.1 mL supernatant
was
removed
and counted
in a ‘y-counter.
Percentage
of specific
lysis was
determined
as follows:
(cpm experimental
group - cpm minimal
release/cpm
maximal
release
cpm minimal
release)
x 100.
Minimal
release was determined
by incubating
targets
alone with
PMA,
and maximal
release
was determined
by adding
HC1 to a
concentration
of I mol/L.
Maximal
release was always >90% of
incorporated
counts.
Minimal
release is described
in the Results
section.
The LD
for H202 concentrations
was calculated
as previously described.3
The assays
were performed
in RPMI
media
supplemented
with 5% FCS. The media and serum were specifically
selected
for low endotoxin
levels, and endotoxin
contamination
was
not detected
by the Limulus
assay (<0.125
eu/mL).
Statistics.
The t test was used for all P value determinations.
PMN
lysis
of
OC
explanted
high
OC
PMA
targets
targets
spontaneous
counts),
two
release
after
targets.
PMNs
were
in endotoxin-free
media
and
for varying
durations.
Eight
mixed
freshly
were
Peripheral
studied
chromium
targets
could
18-hour
culture
six
targets
fresh
demonstrated
(>40%
be tested.
from
<30%
(80%
of experiments),
were used to compute
the data
blood
initially.
release
not
the
Because
of incorporated
The
other
of the
spontaneous
six was
usually
and only these
experiments
shown in Fig I . None of these
>
nine
three
10%
PMN
lysis
during
the
hours
of the
detected
too high
lysis.
significant
to four
cells.
for one
hours
lysis of OC
PMA-stimulated
the
or
PMA-stimulated
lysis
and
of
348)
was
release
was
of targets
(six
did not elicit
pretreatment
followed
to 18
significant
of targets
by washing
did
not
cells
when
either
unstimulated
PMNs
were
used
as effector
PMN
lysis
of OC
correlated
with E:T ratio until a plateau
50:1 . Cytolysis
was suppressed
at higher
of tumor
cells or tumor
cell fragments
cells
at
I 8 hours
was reached
at 25 to
ratios.
Phagocytosis
was never observed
microscopically.
proliferation
In vitro
maintained
for
of the
However,
nos. 204 and
intermittently
thawed
cells
to
were
lysis
was
and
and
cryopreserved
In
in
sensitivity
to
to that
of the
comparable
usually
we could
to cryopreserve;
was impossible.
cultures.
proliferated
release
was
time,
this
initially
reinitiate
successfully
spontaneous
During
of no. 348 cells
of this
target
222
thawed
OC cells
explanted
2 to 3 months.
only
not grow suffIcient
numbers
thus,
further
investigation
cases,
all
vitro,
and
their
PMA-stimulated
initially
cultured
cells.
Mediators
patients
of
with
of no.
204
by
On two
lysis.
CGD
concurrently
were
occasions,
available
PMNs
for testing.
PMA-stimulated
assayed
PMNs
normal
control
stimulated
(<3%
lysis
PMNs
from
at all
E:T
two
CGD
ratios).
from
obtained
patients
Likewise,
two
Specific
donors
and 22% ± 3% at E:T ratios of 25:1 (mean
plicate
samples
following
18 hrs of incubation).
lysis
from
was
I 5%
two
I
±
were
lysis
ineffective
of no.
222
donors
PMNs
cells
was
were
inhibitors/
of ROIs
(at least
three
experiments
for each
In all cases, the added
reagents
in concentrations
were
not toxic
to targets
when
heat-inactivated
targets.
When
was added
assay,
the
H,O,-induced
at three
added
alone.
catalase)
the same
or six hours
As shown,
markedly
concentration
after
detected
lysis at 1 8 hours
was
events
resulting
in enhanced
initiation
inhibof
of the
unaltered.
Thus,
lysis are corn-
pleted in the first three hours of the assay. SOD had no effect
on no. 204 but inhibited
lysis of no. 222. Heat-inactivated
SOD
Neither
had no effect
on
did myeloperoxidase
and aminotriazole
dase-catalyzed
known
HAs
Fig
ratio
1
-
.
25:1.
Time
course
of
OF
PMN-induced
ASSAY
lysis
222
experiments,
of
OC
cells;
E:T
lysis of no. 222
(MPO)
inhibitors
any effect,
of PMNs
of hydroxyl
significantly
but
the
have
reactions
scavengers
thiourea)
no.
%
SD of quadruIn contrast,
±
at 18 hours
by control
PMNs
from two healthy
19% ± 1.5% and 26% ± 2.3%,
whereas
CGD
ineffective
(lysis <4% at all E:T ratios).
Figure
2 summarizes
experiments
utilizing
catalase
#348
incubation
with
PMA
222,
Spontaneous
alone (no PMNs)
Furthermore,
increase
PMNs
substantial
204,
at either
3, 6, 9, or 18 hours,
targets
in the absence
of PMA did not result in
In addition,
catalase
(but not
ited lysis of both
C,)
Co
However,
(nos.
hours)
with PMA
chromium
release.
shown
#204
incubation.
specimens
after
1 8-hour
incubations.
to test lysis after
1 8 hours.
scavengers
inhibitor).
#222
of
six
In all experiments
cultured
with PMNs
PMN
RESULTS
stimulated
with
with chromated
first
ET AL
had
urea
this
agent
ble
of scavenging
effect
(mannitol,
PMN
cytotoxicity
on
demonstrated
is biochemically
hydroxyl
indicating
that
are not critical.
radicals
inhibited
little
no.
204.
no significant
related
(not shown).
methimazole
to thiourea
radicals,8
these
peroxiSeveral
DMSO,
against
In
concurrent
effect.
but
control
Since
is incapaassays
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PMN
LYSIS
OF OVARIAN
CANCER
CELLS
807
100
Fig 2.
Effect
of ROl scavengers
on lysis of OC
cells. Data are percentage
of inhibition.
mean
± SD
of at least three
experiments
for each
inhibitor.
Inhibition
determined
from lysis at an E:T ratio of
25:1 . Inhibitor
concentrations:
catalase
5,000
UI
ml, SOD 200 UImL, methimazole
1 mmol/L,
aminotriazole
25 mmolIL,
mannitol
50 mmollL,
DMSO
750 mmolIL,
thiourea
50 mmol/L,
urea 50 mmoll
1, and deferoxamine
1 mmol/L.
Open
bars:
no.
204.
Hatched bars: no. 222. SignifIcant
(P < .05)
depression
of lysis
of no. 204 by catalase;
significant depression
of lysis of no. 222 by catalase.
SOD,
mannitol,
DMSO,
thiourea,
and
deferox-
75
z
0
cn
50
z
25
CATALASE
HI
SOD
METHIMA-
CATALASE
AMINO
ZOLE
MANNITOL
support
a role
for these
Furthermore,
significantly
the
lysis
inhibited
of I mmol/L
were
of lysis
(-35%
inhibition).
was
prevent
formation
Fenton
reaction.
had
most
also
By
with
of an inhibitory
the
tion
derived
Pretreatment
quent
from
the
PMN
lysis,
with
whereas
of the
lysis
in tumor
lysis
was
exposed
and
no.
in
deferoxamine
would
mmol/L)
by the
of no.
iron-catalyzed
saturated
(Table
I).
with
Table
rather
than
from
in I 8-hour
to preformed
222
was
systems.
A
exposed
to
enzymatically
than
more
tenfold
Table
1
.
lnhibitio
in the
2), there
to 0.025
was
U/mL
to
these
concentrations
no
addition,
In
lysis
(data
was
XO
or
used alone
at these
thiourea,
methimazole,
no. 204
H,02
86%)
the
was
at
xanthine
enhanced
longer
present
lysis
assay.
As
concentraIn contrast,
after
of XO.
heat-inactivated
xanthine
of
18
(1
I 0% with enzyme
after
six hours.
<
(46%
not
or six hours
detected
adding
with
with
I 8 hours
In all
cases,
catalase,
abrogated
lysis.
not
to targets
when
toxic
concentrations,
and
and aminotriazole
DMSO,
mannitol,
had no effect
on
not shown).
Table
of no.
204
2.
Oxidative
Lysis
in Cell-Free
Systems
Lysis
to
(%)
x0 Concentration
Target No. 204
Incubation
Time (h)
shifted
to the
indicated
a
sensitivity
Catalase
6
-
18
-
18
+
18
Hl
0.005
5±1
9±4
46±6t
86±7t
4±2
41
(U/mL)
0.01
0.025
6±1
9±2
St
±
82
±
8±2
5
84
±
Lysis
(%)
Use of Deferoxamine
30±2
B. Inassay
12
iron saturated
Target
(%)
25
None
5
1
19±2.8
0.9
±
8
±
No.
222
Incubation
Time
(h)
Reagents
Added
Inhibitors
11
±
1.2
H,O2+FeSO4
-
1
5
±
1
18
H2O,
-
6
1±1
0
C.
In assay,
19
±
1.9
13
±
1.1
9
±
0.6
18
FeSO4
D.
PretreatmentofPMNs
27
±
3
15
±
2
7
±
1.3
18
H,O2
+
FeSO4
E. Pretreatmentoftargets
18
±
1.8
12
±
0.9
6
±
1
18
H,O,
+
FeSO4
Catalase
3
±
1
18
H,O,
+
FeSO4
DMSO
8
±
4t
18
H202
+
FeSO4
Mannitol
7
±
3t*
with
designated
PMA-stimulated
1 8 hours
mmol/L
were
preincubated
three
times,
for
PMA-stimulated
Percentage
with
saturated
with
and then
E, no. 222
are mean
no. 222
targets
incubated
for
and percentage
of lysis assayed. In oup
B, deferoxamine
was present during the entire assay. In ‘oup C. deferoxamine
had been previously
mmol/L
and chromated
PMNs
±
identical
cells
were
three
with
iron as described.’7
deferoxamine
added
1 mmol/L
to targets
chromated,
hours,
three
in the presence
preincubated
washed
for
In oup
three
times,
with
and
hours,
of PMA.
1
Cytotoxicity
PMNs
D.
of lysis determined
H,O,
(10
mol/L)
added
at 5,000
added
1
to
results.
samples.
of 25,
This experiment
5, and
1 : 1 . Data
was repeated
run
mannitol
was added
Specific
lysis,
from
U/mL;
was
targets
U/mL)
St
mixed
for no. 204
for no. 222
added
±
at 750
cells.
with
cells or
Catalase
mmol/L,
and
at 50 mmol/L.
mean
six-hour
±
SD of three
heat-inactivated;
P
control,
tDifferent
from
corresponding
(catalase,
DMSO,
or mannitol),
§Hl,
chromated
to 0.025
DM50
24
-
(0. 1 7 mmol/L)
FeSO4
±
3±2
-
and XO (0.005
was
tDifferent
at E:T ratios
assay
(1 mmol/L)
washed
deferoxamine
then
xanthine
In group
PMNs.
SD of quadruplicate
8t
n of Lysis by Deferoxamine
Lysis
A.
demonstrated
18-hour
addition
generated
but was significantly
of 1.2 x l0
mol/L
increase
also
lysis
of XO,
lysis
hydroxyl
radicals
in cell-free
systems.
Lysis of no. 204 by
increasing
concentrations
of reagent
grade
H2O2 revelaed
an
ID50 of 7.3 x l0
mol/L
when the assay duration
was six
hours.
When
the assay was prolonged
to I 8 hours,
the shape
of the curve was identical
left. The resulting
LD50
was
the
at three
concentrations
incubation
increase
assays
was observed
when
or enzymatically
generated
of catalase
significant
had
similar
H20,
but
DEFEROXA-
However,
on the enhanced
was generated
by
to different
catalase,
during
no effect
PMNs.
subse-
of PMNs
concentration
204
present
by H202.
lysis
genera-
inhibited
pretreatment
U/mL)
abrogated
shown
(Table
tions of 0.005
I also
radical
deferoxamine
cell-free
same
UREA
USED
(5,000
the assay had
hours.
When
effect.
Oxidative
Catalase
deferoxamine
for hydroxyl
targets
H,O,.
assay completely
depression
mmol/L
effect
required
222
also
Concentrations
deferoxamine
iron
of no. 222
cells.
significant
radicals
In contrast,
less
222
but
iron,
of hydroxyl
of no.
deferoxamine
0.5
chelating
that
was
of no.
observed
in lysis
chelator
effective,
suggests
iron
moieties
iron
TNIOUAEA
MINE
AGENTS
further
DMSO
TRIAZOLE
amine.
cells.
I
I
enzyme
P
<
to five experiments.
.05.
<
control
.05.
boiled
performed
for 20 minutes.
without
inhibitor
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
LICHTENSTEIN
808
To the no. 222 target,
we added
sulfate (0.17 mmol/L)
and reagent
L). The ferrous
were not toxic
EDTA-chelated
grade
H202
ferrous
mol/
(10
sulfate
and H202
in the concentrations
used
when used individually
(Table
2). However,
the combination
were exposed
achieved
lysis of 24%
for 18 hours
(Table
2).
when no. 222 targets
In contrast,
only 1%
radical
tory effects
nonspecific.
tion
aminotriazole
DMSO
products
and
lysis
(Table
no effect
significantly
aminotriazole
shown).
In addition,
(not
present
from
added
shown).
at three
Phorbol
time
0 prevented
or six hours
pretreated
mol/L
for
exhibit
increased
H202
mannitol
whereas
2),
one to four
sensitivities
assays
experiments.
mol/L
for
followed
of H202
by
and
U/mL)
no effect
when
targets
mol/L),
no.
not
grade
sulfate.
222
to oxidants
in three
(10k
222).
by the combinammol/L)
(0.17
targets
were
PMNs
as
generated
ROIs.
remained
resistant
specimens
resistance
well
In
as to
our
after
have
continuously
and
sensitive
documented
cultured
the
hema-
study,
were
sensitive
of human
tumor
phenomenon.
Whether
depended
targets
to
the
or
three
of
PMN
lysis.
cells may not
effective
lysis
of three
explanted
reports
have studied
cytolysis
with
incubations
suggest
PMNs
PMN-induced
of six hours
was
comparable
macrophages4
and
to that
IL-2-activated
pended
on production
of ROIs.
Although
experiments
with
was
oxidative
in
cytotoxins
is not entirely
using
inhibitors
ity
ROl
is
problem
a
limitation
with
the
nature,
clear.
CGD
PMNs
the
identity
We
investigated
or scavengers,
but their
of this approach.
This
no.
222
target
and
this
the rate
inhibitory
of formation
to lysis of no.
indeed
prevented
required
iron
itself.
This
iron was
lysis by chelating
was derived
from
is similar
the
iron,
no.
to other
studies
in
for the oxidative
lysis
required
1), why
mol/L
reagent-grade
possible
is in the
ferrous
was
exogenous
explanation
ferric
state
iron
PMNs
(used
it
in Table
of superoxide
to
react
The
data
be unable
by
for lysis
system
by lO
(Table
it can serve as a substrate
radicals
(Fenton
reaction).
as shown
secretion
required
in the cell-free
2)?
is that the required
target-derived
(Fe3)
and must be first reduced
(Fe2)
before
of hydroxyl
in formation
iron
H202
which
subsequently
in Table
itself
with
reduce
achieve
reduces
2 suggest
to
1) can
the
Fe3
H202
that
this
to Fe2,
(Haber-Weiss
the no. 222
ferric
to
for H2O2
Stimu-
iron
target
critical
for
cytolysis.
Thus, when exposed
to reagent-grade
H202 alone
(Table
2, line 2), it is not injured
unless
an exogenous
source
of ferrous
iron
is also present
(Table
our cytotoxicity
sensitivity
of targets
to ROIs
well as to stimulated
PMNs.
2, line 4).
assay to 1 8 hours increased
the
presented
in cell-free
systems
as
These
data suggest
that detec-
such
must
redox
injury
for
we
as the glutathione
be depleted
before
that
successful
between
ROIs
and
as
de-
interaction
that
issue
hydroxyl
granule
by
which
possibility
proteins.
We
between
H202
occurs
is that
absence
in
against
ROIs,
cycle or target
cell catalase,
occurs. A second possibility
is
disruption
peptide
documented
such
defensins
of primary
H202-mediated
that
events
of protection
previously
and
an
release of such granule
components
or a
of action
might
explain
the successful
only
with
longer
incubations.
A third
a rapid
target-dependent
result
is unknown.
sensitivity
defenses
lysis depends
on a synergistic
interaction
and products
of nonoxidative
metabolism
such
granules.’9
A slower
slower
mechanism
lysis
ultimate
lack of specificis a particular
putative
This
or should
of increased
the cellular
indicated
the
of SOD.
effect
ROIs.
The mechanism
One possibility
is that
Lysis
of
MPO
longer
most
by monocytes
lymphocytes.’2
effects
by
by the
tion of PMN-mediated
lysis at I 8 hours
(in contrast
to 3, 6,
or 9 hours)
is due to the increased
susceptibility
of targets
to
or ROI-mediated
or less. Our
data
achieved
mediated
222
little
the
the inhibi-
estab-
these
assays
may underestimate
the potential
in human
tumor
control.
The degree
of OC lysis
detected
lysis
cells
the
have
either
Prolonging
of our
tumor
the
(Table
may
Thus,
complete
be a generalized
levels,’6
of no.
with
should
by
against
of hepatocytes’7
and endothelial
cells.’8
However,
if targetderived
iron is sufficient
for oxidative
lysis by intact
PMNs
allowing
reaction).
enzymatically
(O’Donnell-Tormey
et al used
continuously
cultured
lished
lines),
a peculiar
sensitivity
of OC cells, or the
duration
of our cytotoxicity
assay is unclear.
Certainly,
other
be inconsistent
A
of inhibition
effect
killing
1).
to inhibit
by increasing
to lytic
a PMN-cytotoxic
However,
(Table
2).
through
six OC targets
but three other
incubations,
on our use of freshly
masked
222 tumor
target
which
target-derived
lated
resistance
of nonheto PMA-stimulated
preformed
1 8-hour
(Fig
and
inhibi-
to mitigate
system,
concentrations
system.
would
One
iron
a marked
targets
have
If deferoxamine
Table
1 suggests
during
separate
topoietic
human
tumor
lines, this study is the first indication
that these
effectors
can kill freshly
obtained
human
tumor
cells. In contrast
to our results,
a recent
report
by O’Donnell-
human
may
MPO
DMSO
ability
the lack
H2O2-halide-MPO
H202
used
appears
increase
lysis by accelerating
In contrast,
SOD was quite
did
reagent
‘
Tormey
et a13 demonstrated
matopoietic
human
tumor
of the
accumulated
Although
methimazole
actually
of H202.
washing)
either
DISCUSSION
to lyse
reactions.
or
was
deferoxamine’s
by
2).
Although
previous
ability
of human
PMNs
MPO-catalyzed
the
chelator
documented
inhibitor
ferrous
222
iron-saturated
report’5
tors
we used,
H2O2
production’3
to targets.’4
The
enzyme
sulfate
whereas
recent
and
binding
system
not be lysed
could
ferrous
concentrations
of the assay
(not
no. 222 targets
(10
The
LD
for H202
was comparable
no. 204 and 3.4 x 1O
mol/L
for
no.
(l0
(Table
initiation
to lysis
(5,000
had
no. 204 and
hours
of EDTA-chelated
H202
after
but
of nos. 204 and 222
were directly
compared
no. 204
However,
tion
catalase
lysis
sensitivity
or the combination
The
1 8-hour
inhibited
the detected
and methimazole
had
the
of the iron-chelator
deferoxamine
may also be
Of concern
is the only modest
reversal
of inhibi-
when
was present
after a six-hour
incubation.
Cytotoxicity
at
18 hours
was probably
due to hydroxyl
radicals
since (a) it
was completely
dependent
on the presence
of iron and, (b)
lysis
At
scavengers.
can also depress
superoxide
thiourea
can prevent
PMN
ET AL
of
occur
plasma
when
catalase
insult
slowly
membrane
is added
is followed
and
by
eventually
integrity.
at three
The
or six
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
PMN
LYSIS
OF
OVARIAN
CANCER
CELLS
809
hours to stimulated
PMNs
or to cell-free
ROts indicates
that
the lethal
lytic events
are completed
early,
providing
support
can inhibit
only -50%
of lysis (Fig 2). These inhibitors
would
have less of an effect on H2O2 which could then, by itself, lyse
for this
With
no. 222.
204
and
mol/L,
latter
hypothesis.
reagent-grade
222
were
the sensitivities
H202,
comparable
respectively).
(LD
of l0
only
In contrast,
ble to lysis by lower concentrations
iron. This suggests
that both targets
generated
hydroxyl
explain
by
PMNs
but
only
DMSO,
mannitol,
and
no. 222
3.4
was
of nos.
no.
222
can
be
H2O2 sensitivities
of the hydroxyl
suscepti-
and the chelator
lysed
Two
questions
responses.
major
in antitumor
x iO
by
might
radical
unclear.
PMA,
remain
First,
to other
generalized
of H2O2 combined
with
can be lysed by H2O2
radicals.
The comparable
why optimal
concentrations
scavengers
to lysis
tumor
concerning
whether
of
types
the role of PMNs
our results
can be
nonovarian
Second,
since
our studies
were
which
is an optimal
PMN
stimulant,
physiologic
stimulators
quantities
of
unknown.
These
can
ROIs
to
trigger
achieve
production
lysis
of
are currently
questions
origin
performed
whether
is
with
more
of sufficient
these
under
targets
is
investigation.
deferoxamine
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1989 74: 805-809
Human neutrophil-mediated lysis of ovarian cancer cells
A Lichtenstein, M Seelig, J Berek and J Zighelboim
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