Download Reactive Oxygen Intermediates Activate NF-KB in a

From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Reactive Oxygen Intermediates Activate NF-KBin a Tyrosine KinaseDependent Mechanism and in Combination With Vanadate Activate
the ~ 5 6 ’ and
‘ ~ ~ 5 9 ‘ Tyrosine
~”
Kinases in Human Lymphocytes
By Gary L. Schieven, Jean M. Kirihara, Dorothea E. Myers, Jeffrey A. Ledbetter, and Fatih M. Uckun
We have previously observed that ionizing radiation induces tyrosine phosphorylation in human B-lymphocyte
precursors by stimulation of unidentified tyrosine kinases
and this phosphorylation is substantially augmented by
vanadate. Ionizing radiation generates reactive oxygen intermediates (ROI). Because HzO, is a potent ROI generator
that readily crosses the plasma membrane, we used H,O,
to examine the effects of ROI on signal transduction. We
now provide evidence that the tyrosine kinase inhibitor
herbimycin A and the free radical scavenger N-acetyl-cysteine inhibit both radiation-inducedand H,O,-induced activation of NF-KB,indicatingthat activation triggered by ROI
is dependent on tyrosine kinase activity. H,Oz was found
to stimulate Ins-I ,4,5-P, production in a tyrosine kinase-
dependent manner and to induce calcium signals that were
greatly augmented by vanadate. The synergistic induction
of tyrosine phosphorylation by H,Oz plus vanadate included physiologically relevant proteins such as PLCyl . Although treatment of cells with H,Oz alone did not affect
the activity of src family kinases, treatment with H,O, plus
vanadate led to activation of the p56Ickand p59W”tyrosine
kinases. The combined inhibition of phosphatasesand activation of kinases provides a potent mechanism for the
synergistic effects of HzOzplus vanadate. Inductionof tyrosine phosphorylation by ROI may thus lead to many of the
pleiotropic effects of ROI in lymphoid cells, including
downstream activation of PLCyl and NF-KB.
0 1993 by The American Society of Hematology.
R
sis of bound and solvent water in the cell producing OH
radicals, hydrated electrons, and H202. Additional H,Oz is
generated in irradiated cells by the dismutation of superoxide anions that are produced by the action of hydrated electrons on oxygen molecule^.^ H202 can be converted into
highly active ROI.’ Compelling evidence indicates that a
cascade of cytoplasmic events is initiated when cells are irradiated, leading to activation of NF-KB’ and the protein kinase C (PKC)-dependent induction of c-jun expres~ion.~~’
Tyrosine phosphorylation is an early response to stimulation via sIg in B lymphocytes* and via CD3/Ti and accessory molecules in T lymphocyte^.','^ Tyrosine phosphorylation is essential for both T-cell” and B-cell” activation, as
shown by the use of tyrosine kinase inhibitors. The src family tyrosine kinases have been found to play key roles in this
signal transduction process in T and B cell^.'^^'^ We have
recently shown that ionizing radiation induces tyrosine
phosphorylation in human B-lymphocyte precursors by
stimulation of unidentified tyrosine-specific protein kin a s e ~ . The
’ ~ tyrosine phosphorylation induced by ionizing
radiation was greatly increased when cells were pretreated
with vanadate.” More recently, we observed in human lymphocyte precursors that activation of NF-KBby irradiation
with y-rays is abrogated by the tyrosine kinase inhibitor
herbimycin A.I6 Thus, tyrosine phosphorylation is an important and mandatory proximal step in the radiation-induced activation of NF-KB.The mechanism by which ionizing radiation triggers tyrosine kinases and activates NF-KB
in human lymphoid cells is as yet unknown. The present
study provides evidence that radiation-induced activation
of NF-KBis triggered by ROI. We also report that the synergistic effects of ROI and vanadate on tyrosine phosphorylation are due to kinase activation in addition to phosphatase
inhibition, leading to phosphorylation ofphysiologically relevant substrates and downstream signaling events such as
calcium mobilization.
EACTIVE OXYGEN intermediates (ROI) have been
implicated in a variety of clinical conditions, including rheumatoid arthritis and other autoimmune diseases, as
well as radiation injury.’ NF-KB is an inducible transcription factor that is involved in the regulation of a series of
target genes.2 A large number of agents have been shown to
activate NF-KB,including protein synthesis inhibitors, mitogens, calcium ionophores, and viruses (by the action of
viral transactivator proteins or double-stranded RNA intermediates).’ Notably, the KBelements in regulatory domains
of target genes serve as response elements for oxidant stress
triggered by ROI. Extensive studies by Schreck et ai3 have
shown that the induction of NF-KBin Jurkat T cells, mouse
fibroblasts, and mouse pre-B cells after stimulation with tumor necrosis factor, interleukin- 1, double-stranded RNA,
lipopolysaccharide, lectin, phorbol esters, calcium ionophores, or cycloheximide is dependent on ROI and can
therefore be effectively inhibited by the radical scavengers
N-acetyl-L-cysteine (NAC) and PDTC (a pyrrolidone derivative of dithiocarbamate). Ionizing radiation causes radioly-
From the Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA;and the Tumor Immunology Laboratory, Section
of Cancer and Leukemia Biology, Department of Therapeutic Radiology-Radiation Oncology, and the Bone Marrow Transplant Program, University of Minnesota Health Sciences Center, Minneapolis, MN.
Submitted August 19. 1992; accepted April 14, 1993.
Supported in part by Bristol-Myers Squibb and in part by US
Public Health Service Grants No. R29 CA-42111, ROI CA-42633,
and ROI CA-51425 from the National Cancer Institute, DHHS.
F.M. U. is a Scholar of the Leukemia Society of America. This is
publication no. 91 from the Tumor Immunology Laboratory, University of Minnesota.
Address reprint requests to Gary L. Schieven, PhD, Bristol-Myers
Squibb Pharmaceutical Research Institue, 3005 First Ave, Seattle.
WA 98121.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/8204-0031$3.00/0
1212
MATERIALS AND METHODS
Cells. In our studies, we used the pre-preB-cell line REH, preB-cell line NALM-6, the early B/Burkitt’s lymphoma cell lines
Daudi and Ramos, as well as a CD3’ subclone (CEM.6)” of the
6l00d. VOI 82, NO 4 (August 15). 1993: pp 1212-1220
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1213
REACTIVE OXYGEN INTERMEDIATE INDUCED SIGNALS
pre-T-cell line CEM. The radiation sensitivityofthese cell lines was
detailed in a previous report.18
Irradiation and treatments of cells. Cells (5 X 105/mL)in plastic
tissue culture flasks were irradiated with 500 to 2,000 cGy at a dose
rate of 100 cGy/min during log phase and under aerobic conditions
using a ”’Cs irradiator (Model Mark I; JL Shephard and Assoc,
Glendale, CA), as previously described.” In parallel experiments,
cells were treated with 3 to 9 mmol/L H202(Sigma, St Louis MO),
IO0 ymol/L sodium orthovanadate (Fisher Scientific, Pittsburgh,
PA), or both for 30 minutes at 37°C. For experimentsinvolving the
use of kinase inhibitors before irradiation or H202treatment, cells
were incubated for I hour at 37°C with (1) phosphate-buffered
saline (PBS), (2) the tyrosine kinase inhibitor genistein (370 ymol/
L; ICN Biomedical, Costa Mesa, CA), (3) the PKC inhibitor
I-(5-isoquinolinylsufonyl)-2-methylpiperazine(30 pmol/L H7;
GIBCO-BRL, Grand Island, NY), or for 24 hours at 37°C with (4)
the potent tyrosine kinase inhibitor herbimycin A (12 pmol/L;
In some
GIBCO-BRL), using previously described protoc01s.l~~~~
experiments, cells were preincubated with 20 mmol/L NAC for 1
hour according to the treatment protocol reported by Schrecket al.’
For the measurement of [Ca2+]i,cells were used immediately after
addition of stimulating agents. For IgM cross-linking, cells were
treated with 10pg/mL F(ab’), fragment goat antihuman IgM (Jackson ImmunoResearch Labs, West Grove, PA) for 1 minute. Crosslinking of CD3 alone or in conjunction with CD4 was achieved by
the use of biotinylated monoclonal antibodies (MoAbs) against
CD3 ((319-4) and CD4 ((317-2) followed by treatment with avidin,
as previously described.”
Nuclear extraction and electrophoretic mobility shift assays. Four hours after radiation or H202treatment, nuclear proteins were extracted according to previously published procedures.21,22Gel shift assays were performed as de~cribed.~’
Fifty-nanogram amounts of a double-stranded oligonucleotide
containing a tandem repeat ofthe 1 1-bpconsensus sequence for the
NF-KBDNA binding site (GGGGACTTTCC;obtained in kit form
from GIBCO-BRL) were end-labeled using [Y-’~P]ATPand T4
polynucleotide kinase according to the recommendations of the
manufacturer. One nanogram of the radiolabeled oligonucleotide
(400,000 cpm) was incubated with 10 yg nuclear protein for 20
minutes at room temperature in 25 mmol/L Tris-HCL, pH 7.6, 1
yg poly dI/dC (Boehringer Mannheim, Indianapolis, IN), 5 mmol/
L MgC12, 0.5 mmol/L EDTA, 1 mmol/L DTT, and 10%(vol/vol)
glycerol. Competition studies with unlabeled NF-KBprobe were
performed by preincubating the nuclear protein for 15 minutes on
ice with a 500-fold excess of unlabeled oligonucleotide before the
addition of the 32Pend-labeled NF-KBprobe. Controls used 500fold excess of unlabeled AP- 1 and NF- 1 oligonucleotide probes for
competition. DNA-protein complexes in the reaction mixture were
analyzed by polyacrylamide gel electrophoresis, using a 4.5% running gel under nondenaturing conditions, in 0.25X TBE buffer (25
mmol/L Tris, pH 8.2, 22.5 mmol/L borate, 0.25 mmol/L EDTA).
The gels were pre-run at 150 V for 2 hours at 4°C before the samples
were loaded and electrophoresed for an additional 3 hours at 150 V.
Gels were dried overnight and exposed to Kodak XAR 5 X-ray film
using intensifying screens at -70°C.
Immunoprecipitations, immunoblots. and kinase assays. Cells
were lysed on ice with NP-40 lysis buffer (50 mmol/L Tris, pH 8,
150 mmol/L NaCI, 1% NP-40, 100 ymol/L sodium orthovanadate,
100 pmol/L sodium molybdate, 8 pg/mL aprotinin, 5 pg/mL leupeptin, 500 pmol/L phenylmethysulfonyl fluoride [PMSF]) and
centrifuged at 13,OOOg to remove insoluble material. Immunoprecipitation of PLC-y1 was performed as previously described.24Immunoprecipitation of ras GTPase-activating protein (GAP) was
performed with rabbit antisera to human GAP (Upstate Biotechnology, Lake Placid, NY) and immunoprecipitation of ~ 3 4 ‘ ~was
’~
performed with an MoAb to human ~ 3 4 * ”(Upstate
~
Biotechnology). Immunoprecipitation of the {chain of the T-cell receptor was
performed using antkcantibody kindly provided by J. Sancho and
C. Terhorst (Beth Israel Hospital, Boston, MA).25Immune complexes were collected on protein A-Sepharose beads (Repligen,
Cambridge MA), washed four times with NP-40 lysis buffer and
once with PBS, and then subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) followed by immunoblotting. Immunoblots with anti-PLCy 1 were performed as previously described.24 Antiphosphotyrosine immunoblotting was
performed as previously described,” using affinity-purified rabbit
polyclonal antibodies.26Immune complex tyrosine kinase assays
were performed using the exogenous substrateenolase as previously
de~cribed.~’
Samples were immunoprecipitated with antisera prepared against unique amino acid sequences of src family tyrosine
kinases,28which were the kind gift of Dr Joseph Bolen (BristolMyers Squibb Pharmaceutical Research Institute, Princeton, NJ).
Measurement of [Cd’] and inositol 1,4,5-trisphosphate (Ins1,4,5-P,). [Ca2+]iresponses were measured using indo- I (Molecular Probes, Eugene, OR) and a model 50HH/2 150 flow cytometer
(Ortho, Westwood, MA), as previously de~cribed.~’
The histograms
were analyzed by programs that calculate the mean indo- 1 violet/
blue fluorescence ratio versus time. There are 100 data points on
the X (time) axis of all flow cytometric data. Ins-1,4,5-P3levels were
measured using a highly specific D-myo-inositol 1,4,5-[’H]trisphosphate assay system (Amersham Corp, Arlington Heights,
IL), as previously described.” This assay is based on the competition between unlabeled Ins-1,4,5-P, and a fixed quantity of a high
specific activity tritiated Ins-l.4,5-P3 tracer (3H-Ins-l,4,5-P3)for a
limited number of binding sites on a specific and sensitive bovine
adrenal binding protein preparation.
RESULTS
The role of reactive oxygen intermediates in radiation-induced activation of NF-KBin human lymphoid cells. We
used electrophoretic mobility shift assays (EMSA)to examine the effects of the ROI-scavenger NAC and tyrosine kinase inhibitors on (1) radiation-induced activation of NFKBand (2) ROI-induced activation of NF-KBin Ramos and
Daudi cells. After passive diffusion through the plasma
membrane, H,02 can be converted into highly reactive ROI
such as the superoxide anion and the hydroxyl radical.’
Therefore, H202was used as a potent ROI generator to examine the effects of ROI on NF-KB.Doses of radiation and
H202were chosen so as to show that strong signals could be
clearly inhibited by NAC and tyrosine kinase inhibitors.
The doses used were based on previous reports that estaband radiation6,’for such
lished optimum doses of H20231332
signals. One microgram of poly dI-dC was used to eliminate
nonspecific binding. Notably, ionizing radiation with 500 to
2,000 cGy y-rays stimulated a &specific DNA binding activity, as reflected by a marked increase in intensity of a
shifted band that was observed when the NF-KBprobe was
incubated with nuclear extracts from Ramos cells (Fig 1A).
This retarded band was eliminated by competition with a
500-fold molar excess of unlabeled NF-KBoligonucleotide,
confirming the specificity of the DNA-protein interactions
(Fig 1A). By comparison, a 500-fold excess of unlabeled
NF-I control oligonucleotidedid not compete with the binding of NF-KBprobe to the retarded band (data not shown).
Notably, the tyrosine kinase inhibitors genistein and herbimycin markedly inhibited NF-KBactivation, in accordance
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1214
A 2*
f,o
*Q&
SCHIEVEN ET AL
9
cp
&&e",
Go+ %,o* 9 8 8
o + @ $
$ ,."$ f
& & & & &
8 8 8 8 8
1
t
B
- I N _ _
with our recent studies.I6 As shown in Fig IA, NAC substantially inhibited the formation of DNA-NF-KB protein
complexes in irradiated Ramos cells. This unique observation indicates that ROI generation is a mandatory step in
radiation-induced activation of NF-KB.We next compared
the ability of 1,000 cGy y-rays and 9 mmol/L H202to activate NF-KBin Ramos cells. As shown in Fig 1B, H202was as
effective asionizing radiation in inducing NF-KB DNA binding activity, and NAC prevented the activation of NF-KBin
irradiated or H202-treatedRamos cells. Notably, both genistein and herbimycin were able to abrogate the H202-induced activation of NF-KB,whereas H7 was not (Fig IB).
Thus, ROI-mediated activation of NF-KB is triggered primarily by PTK activation and does not depend on PKC.
Our results suggest that reactive oxygen intermediates
might thus induce other kinases such that the activity of
PKC is not essential for NF-KBactivation. Similar results
have been obtained with Daudi cells (F. Uckun, unpublished results). Activation of NF-KBwas also observed at the
lower concentration of H 2 0 2 of 300 pmol/L (Fig 2). However, the combination of vanadate plus H202was not significantly more effective than H202alone in inducing NF-KB
activation (Fig 2 ) . indicating that H202alone delivers a sufficiently strong signal.
Hvdrogen pero,ridepiits vanadate strongly induce [Ca2']i
Jirx and inositol I,4.5-trispliosplia~e
proditciion. We next
examined the ability of the ROI generator H 2 0 2alone and
in combination with the phosphotyrosine phosphatase inhibitor vanadate to stimulate calcium signaling. H202gave
a small [Caz']i signal in Ramos cells, whereas vanadate
alone gave no signal (Fig 3). However, H 2 0 2 plus vanadate
gave a very strong signal. The signal generated by H 2 0 2plus
Ramos Cells
H202
(0.3mM)
H2O2(O.3mM)
+voo
H202
(1mM)
H202(ImM)
+V04
<
>
I
Fig 1. Tyrosine kinase-dependentactivation of NF-ICB
DNA binding activity in Ramos cells after exposure to ionizing radiation or
H202.EMSA were performed as described in Materials and Methods. (A) Ramos cells were irradiated with 5 to 20 Gy y-rays. In
parallel experiments, Ramos cells were pretreated at 37°C with 20
mmol/L NAC for 1 hour, 370 pmol/L genistein for 1 hour (GEN), or
1 2 pmol/L herbimycin (HERB)for 2 4 hours before ionizing radiation.
Radiation-inducedDNA-protein complexes reflecting activation of
NF-&-specific DNA binding activity are indicated by the arrow.
Competition (COMP) studies were performed with excess unlabeled probe. (B) Ramos cells were either irradiated with 10 Gy yrays in the presence or absence of 20 mmol/L NAC or treated with
9 mmol/L H2O2.In parallel experiments, cells were pretreated with
370 pmol/L genistein for 1 hour, 12 pmol/L herbimycin for 2 4 hour,
30 pmol/L H7 for 1 hour, or 20 mmol/L NAC for 1 hour before treatment with 9 mmol/L H,02.
Fig 2. Effects of vanadate and varying concentrations of H202
on NF-rB activation. Cells were treated with H202and 100 pmol/L
vanadate before EMSA performed as described in Materials and
Methods. DNA-protein complexes reflecting activation of NF-rBspecific DNA binding activity are indicated by the arrow.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1215
REACTIVE OXYGEN INTERMEDIATE INDUCED SIGNALS
duced by ROI treatment is therefore independent of the
T-cell receptor (TCR).
To investigate the basis for the ROI-induced [Ca2+]isignal in lymphocytes, we examined Ins-1,4,5-P3 levels in
Ramos cells after treatment with H z 0 2and vanadate (Table
1). Treatment with H20zresulted in rapid, substantial, but
transient Ins- 1,4,5-P, production, whereas vanadate alone
had little effect. The combination of H,O, plus vanadate
accelerated and increased Ins- 1,4,5-P, production. The levels of Ins-1,4,5-P3 were thus in good agreement with the
[Ca2']i signals observed in Ramos cells. Similar results were
obtained with other B-cell lines including Daudi (early B),
NALM-6 (pre-B), REH (pre-pre-B), and FL8.2 (pro-B)
(data not shown), showing that the effect occurs in cells at
different stages of development. These results are in accordance with previous reports that the combination of H202
and vanadate stimulated polyphosphoinositide breakdown
in a variety of other cell lines.3' As shown in Table 1, the
induction of Ins-1,4,5-P3by ROI was blocked by the tyrosine kinase inhibitor herbimycin A, but not by the PKC
inhibitor H7. Therefore, stimulation of Ins- 1,4,5-P, production by H,O, plus vanadate is dependent on tyrosine kinase
activity.
-H
......v
----H+V
J'
I
0
2
I
I
4
6
TIME (min)
8
I
Fig 3. [Ca2']i signals in Ramos cells in response to treatment
with 1 mmol/L HzOZ(H) and 100 pmol/L orthovanadate (V).
vanadate also gave a strong signal in CEM cells (Fig 4A) and
was observed in the presence of EGTA, showing that Ca"
was released from internal stores as well as entered cells
from the outside. The magnitude of the calcium signal was
significantlygreater than that observed after antibody crosslinking of CD3 with CD4 (Fig 4B), which is one of the strongest biologic stimulations for [Ca2']i signals in CEM cells."
Treatment with H20z alone gave a small but significant
[Caz+]isignal, whereas vanadate alone had no effect (Fig
4C). Sequential treatment with the compounds in either
order was as effective as the use of both together in generating a [Caz']i signal (Fig 4D). Downmodulation of CD3 by
antibody treatment had no effect on ROI-induced [Ca2']i
signaling (data not shown), although signaling via CD2 was
inhibited, as previously reported.24The [Ca2']i signal in-
Hydrogen peroxide and vanadate act synergistically to induce tyrosine phosphorylation of physiologically relevant
substrates in T- and B-cell lines. We had previously reported that ionizing radiation induced tyrosine phosphorylation, which was greatly augmented by ana ad ate.'^ The
combination of H,Oz plus vanadate has been reported to
synergistically induce tyrosine phosphorylation in insulinresponsive rat a d i p o c y t e ~ . While
~ ' ~ ~ ~the present study was
under review, a similar effect was described for T cells,33but
no substrates were identified. The current finding that the
ROI generator HZO2induced transient Ins- 1,4,5-P, and
[Ca2']i signals that were augmented and stabilized by vanadate led us to examine the effects ofthese reagents on physio-
4
F
1
1
IC -ti
......V
-.0
c
e
-T
-0
0
.-C
v
...... .................
3
2
t& ..........
-..
.*
1
...........
L-L-J
.......CD3 x CD4
(u
0
.....
Fig 4. [Ca2']i signals in CEM cells in responseto
treatment with 9 mmol/L H202(H) and 100 pmol/L
orthovanadate (V). (A) Signal in presence of 10
mmol/L EGTA. (B) Comparison to signal generated
by cross-linking CD3 and CD4. (C) Separate treatment with Hz02 and vanadate. (D) Sequential
treatment with H202and vanadate.
0
............
................
I
1
I
I
2
4
6
8
10
Ll2LL
-J
6
0
Time (min)
2
4
8 1 0
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
SCHIEVEN ET AL
1216
Table 1. Induction of Ins-1 ,4,5-P3 by H202and Vanadate
Ins-1.4,5-P3 (pmol/lO' RAMOS cells)
Time
3.8 f 0.3
1.7f 1.1
2.6 f 1.2
3.2 +_ 1.3
3.4 f 1.7
3.7 f 1.0
2.4 f 1.4
2.4 +_ 0.9
0
10 s
15 s
30 s
45 s
1 min
3 min
5 min
3.8 f 0.3
7.2 f 0.2
26.1 f 2.5
49.7f 12.7
61.7f 12.8
117.926.7
36.2f 12.2
14.1 f0.9
+ vo.
HlOZ
+ VO, + HERB
+ VO, + H7
3.8 f 0.3
33.0f 1.5
53.2f 6.3
118.8 f 8.5
192.8f 4.7
182.1 f 6.4
27.9 f 6.7
11.92 1.5
3.8 2 0.3
3.4f 0.1
7.4f 1.1
8.4f 5.2
10.0f 3.6
8.6f 3.4
6.0f 1.8
4.3 f 0.5
3.8 f 0.3
26.3 f 1.4
51.7 f 2.9
102.9f 4.0
175.1 f 2.1
161.9f 7.6
23.7f 5.4
7.4 2.1
H101
H102
*
vo.
3.8 f 0.3
4.0f 0.5
6.2f 0.5
10.3 f 0.9
16.8 f 1.4
13.4 f 0.3
4.1 20.3
2.7f 1.4
Cells were treated with 3 mmol/L H,02, 100pmol/L vanadate (VO,), Herbimycin A (HERB),and H7 as described in the Materials and Methods section
for the times indicated and were then assayed for lns-l.4,5-P3.
logically relevant substrates. The pattern of tyrosine phosplus vanadate
phorylation induced by treatment with H202
was compared with that resulting from biologic stimulations. As shown in Fig SA, treatment of CEM cells with
H 2 0 2plus vanadate induced tyrosine phosphorylation of
many proteins with molecular weights similar to those of
proteins in which phosphorylation was induced by crosslinking CD3 alone or cross-linking CD3 and CD4. However, the level of tyrosine phosphorylation was approximately IO times greater for many of the proteins after
chemical stimulation. Similarly, in Ramos cells (Fig 5B),
chemical stimulation induced tyrosine phosphorylation of
many proteins with molecular weights comparable to those
observed after sIgM cross-linking, but at much higher levels.
A
Stim:
k Da
219-
B
CEM
0)
Ramos
Stim:
C
0
z
kDa
219-
100676742.7-
42.7
224-
224
1
Fig 5. Antiphosphotyrosine immunoblot comparison of tyrosine
phosphorylation induced by chemical and biologic stimulations. (A)
CEM cells (5 X IOE)were stimulated by cross-linking CD3 or CD3
and CD4 for 1 minute using biotinylated antibodies, or with 9
mmol/L H202plus 1 0 0 rmol/L vanadate (HV) for 30 minutes. An
HV sample from 5 X 10' cells (0.1X ) was also prepared. (B)
Ramos cells (5 X 10') were stimulated with 6 mmol/L H202alone
(H) or in combination with 1 0 0 rmol/L vanadate (HV) for 30 minUtes or for anti-lgM stimulation cells were stimulated for 1 minute.
For both cell types, treatment with H,02 plus vanadate also
induced phosphorylation of proteins not readily observed
after biologic stimulation.
PLCyl was observed to be phosphorylated on tyrosine
after treatment of cells with H202
plus vanadate for 2 minutes (Fig 6A). Under these conditions, several PLCy I-associated proteins, including pp35/36, which associate via the
SH2 domain of PLCy 1 ,34 were also tyrosine phosphorylated. The anti-PLCy l Western blot showed that, although
equal amounts of PLCy 1 protein were recovered from cells
treated with H202or vanadate alone as from untreated cells,
less was recovered from cells treated with H202and vanadate together. Therefore, the fraction of PLCy I phosphorylated on tyrosine is greater than what is initially apparent
from Fig 6A. Vanadate was required to stabilize and augment the ROI signal before this phosphorylation could be
detected. The tyrosine kinasedependent induction of Ins1,4,S-P, by ROI and the ROI-induced (Ca2']i signals may
thus be attributed to the tyrosine phosphorylation of PLCy I
in these cells. As shown in Fig 6B, GAP and associated proteins, including species appearing to be the GAP-associated
proteins p I 903' and ~ 6 2 were
, ~ tyrosine
~
phosphorylated
after treatment with H 2 0 2plus vanadate. The {subunit of
the TCR was also phosphorylated (Fig 6C). Although our
data indicate that ROI induction of [Ca2+]isignals does not
require surface expression of the TCR, a TCR component is
acted upon under these conditions. In contrast to many
other proteins, the tyrosine phosphorylation state of p34OdC2
remained unchanged (Fig 6B). Thus, not all proteins that
can be phosphorylated on tyrosine are affected.
The combination ofhydrogenperoxide plus vanadate activatesp56Ickandp5VYntyrosine kinases. The induction of
tyrosine phosphorylation by H20zplus vanadate in T cells
has been ascribed to inhibition of phosphatase^.^^ However,
the strong phosphorylation of physiologically relevant substrates led us to examine effects on kinase activity as well.
The activity of pS6ICkfrom Ramos cells (Fig 7) and both
pS6lCkand Ps9'" from CEM
(Fig 8A) was
stimulated by treatment of the cells with H202PIUS vanadate. In addition to showing increased activity toward the
exogenous substrate enolase after treatment with H20z
plus
vanadate, pS6ICkhad a characteristic shift to a lower mobility- This shift in ~56"' mobility has been specifically observed early in lymphocyte activation and after treatment
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1217
REACTIVE OXYGEN INTERMEDIATE INDUCED SIGNALS
A
GAP and p3qcdc2I P
Anti-p-tyr Blot
-Anti-PLC
Blot
:&!-
Anti-p-tyr
Blot
OHVHV
0 HVHV
-Anti-
Zeta I P
Anti-p-tyr Blot
Anti-
Ip:
GAP p34cdc2
Stlm 0
kDa
HV
0
HV
219
219-
1
Stim 0
kDa
100-
HV
67-
PLCy1-
GAP-
IOC
42.7-
100
67.
6i
Fig 6. Tyrosine phospholylation of specific proteins after treatment of cells with 9 mmol/L H202
(H), 100 pmol/L orthovanadate (V), both (HV), or no
treatment (0).as detected by immunoprecipitation
followed by immunoblotting. (A) Phosphorylation
of PLCrl and associated proteins. (B) Phosphorylation of GAP and ~ 3 4 ' ~ '(C)
. Phosphorylation of
chain.
C
6
PLCYl I P
-*
27.442.7
zeto-
pp35'36'
18-
274
with phorbol mynstate acetate (PMA).37 The amount of
p561Ckthat could be detected by immunoblot analysis was
significantly reduced by treatment with H202plus vanadate
(Fig 8C). The increase in specific activity of p56"' is thus
greater than what is apparent in Fig 8A.
In contrast, treatment with H202or vanadate alone did
not stimulate the activity of these kinases. This finding that
ROI alone did not activate ~56"' or ~59""is consistent with
our previous study showing that ionizing radiation did not
substantially increase the activity of these kinases.I5 As
shown in Fig 7, ~56"' activity did not increase after irradiation in the presence or absence of vanadate. The lack of
~ 5 6 ' ~activation
'
after irradiation in the presence of vanadate may be explained by the radiation generating lower
amounts of ROI relative to treatment of cells with 9 mmol/
p56ICk Immune Complex
Kinase Assay
RAMOS Cells
I
I
if;;
Enolase-
Fig 7. Immune complex kinase assay of ~
5 after
6 treatment
~
of Ramos cells with y-radiation, 9 mmol/L H202,and 100 pmol/L
vanadate.
L H202.The activity of ~ 6 was2not ~affected
~ by H202or
vanadate (Fig 8A). To determine whether H 2 0 2plus vanadate acted directly on the responsive kinases, immunoprecipitates were treated directly with 9 mmol/L H202
plus 100
pmol/L vanadate for 30 minutes before the kinase reaction
(Fig 8B). Chemical treatment reduced the activity of ~ 5 6 " '
and ~ 5 9 ' ~ "in, terms of autophosphorylation as well as phosphorylation of enolase. Although the exact concentrations
of H202and vanadate in treated cells are unknown, these
results suggest that the increase in tyrosine kinase activity
observed in treated cells is not due to a direct reaction of the
enzymes with H202plus vanadate.
DISCUSSION
The biochemical mechanisms responsible for NF-KBinduction have been the focus of extensive research. Brach et
a!' have recently shown that ionizing radiation induces expression and binding activity of NF-KB in KG-I myeloid
leukemia cells. Schreck et a13 recently provided evidence
that diverse agents activate NF-KBthrough a common mechanism involving the synthesis of ROI. Herbimycin A has
been found to block interleukin- 1 -induced NF-KBactivation in human lymphoid cells.38Recently, we found that
radiation-induced activation of NF-KBis triggered by stimulation of tyrosine-specific kinases and can be abrogated by
the tyrosine kinase inhibitors herbimycin and genistein.I6
Taken together, these observations prompted the hypotheses that the biochemical signals induced by ionizing radiation are dependent on the generation of ROI and that many
of the pleiotropic effects of ROI on human lymphoid cells
are triggered by stimulation of tyrosine-specific protein kinases. We tested this hypothesis by determining whether
NAC and herbimycin could inhibit strong signals induced
by substantial doses of H 2 0 2 and radiation previously reported to be 0ptima1.6*'*~'*~*
Furthermore. we examined cell
responses within a short time oftreatment. Although 20 C y
of gamma irradiation will kill over 90% of Ramos cells, this
does not occur for 3 to 7 days and no change in trypan blue
dye exclusion occurs in the first 24 to 48 hours (F. Uckun,
unpublished results). The short exposures of Ramos cells to
H202reported here result in no change in viability even 48
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
SCHIEVEN ET AL
1218
A
Kinase Assays Following Cell Treatment
Ick
Treatment: 0 H V HV
t-3
Yes
fYn
I
t
f
I
0 H VHV
0 H VHV
-4
-w
Enolase-
B
-Ick
Treatment:
c
Assay Following
Treatment of Kinase
0
HV
Anti-p56Ick
Blot
fYn
0
HV
Treatment: 0
or
-
0
II)
hours after treatment. The ability of NAC and herbimycin
to inhibit the signals from such substantial dosages shows
the essential role of ROI and tyrosine kinase activation. Our
findings that lower doses of H,Oz (300 pmol/L) and radiation (5 Gy) also gave similar results shows that the effects are
not unique to high doses. NAC alone does not depress tyrosine phosphorylation in unstimulated cells (F. Uckun, unpublished results), indicating that it is acting only as a free
radical scavenger and not as a kinase inhibitor. The inhibitor studies thus indicate that activation of NF-KBby radiation was dependent on ROI generation and, furthermore,
activation of NF-KBby R01 was dependent on tyrosine kinase activity.
The augmentation of tyrosine phosphorylation when
cells were treated with H,O, plus vanadate was similar to
the effect previously observed when vanadate-treated cells
were irradiated,15 indicating a common mechanism. The
ROI-induced tyrosine phosphorylation in lymphoid cells
led to downstream events including NF-KBactivation, InsI ,4,5-P, generation, and [Ca2']i signals. Additional signal
transduction pathways such as those involving ras and GAP
may also be affected. Thus, ROI induction of tyrosine phosphorylation may account for many of the pleiotropic effects
of ROI in lymphoid cells, whether generated chemically or
by ionizing radiation. This hypothesis is further supported
by the recent finding that the mammalian UV response in
HeLa cells leading to c-jzin induction is triggered by src kinases and inhibited by elevation of intracellular glutathione
levels." The identification of the particular ROI species involved in the kinase activation, as has been performed by
spin trapping precursors of thymine damage in X-irradiated
DNA,40could provide further insights into the mechanisms
involved.
--
HV
.*nmJzl-rT
Fig 8. Immune complex kinase assays after
treatment of CEM cells with 9 mmol/L H,O, (H),
100 Mmol/L vanadate (V), both (HV), or no treatment (0).(A) Cells were treated for 30 minutes before lysis and kinase assay. Autoradiography for
~
6 assay
2 is ~a fivefold longer exposure than for
p56'* and p59'*". (B)Immunoprecipitates of p56'*
and p59" were treated before kinase assay. (C)
lmmunoblot of ~ 5 6 " 'after treatment of cells.
The level of tyrosine phosphorylation in cells is the result
of a dynamic equilibrium between the opposing activities of
tyrosine kinases and phosphotyrosine phosphatases!' H,Oz
and vanadate are potent phosphotyrosine phosphatase inhibitors, which, when used together, strongly induced tyrosine phosphorylation in insulin-responsive cells such as Fa0
cells due to activation of the insulin receptor's tyrosine kinase a~tivity.~'.~'
lt has been shown that the potent phosphotyrosine phosphatase inhibitor phenylarsine oxide
(PAO) induces tyrosine phosphorylation in T cells by inhibition of phosphatases without kinase activation," and the
effects of H,02 and vanadate on lymphocytes has similarly
been believed to be only due to phosphatase inhibiti~n.~'
However, we have now found that the combination of two
potent phosphatase inhibitors, H,O, and vanadate, stimulates tyrosine kinases as well. This combination of kinase
activation and phosphatase inhibition provides a mechanism for the hyperinduction of tyrosine phosphorylation in
T- and B-cell lines. We propose that the triggering of an
ROI-sensitive tyrosine kinase constitutes the initial signal,
followed by stabilization of the resultant tyrosine phosphorylation due to inhibition of phosphatase activity. The signal
is then further amplified by the activation of src family kinases such as p56ICkand ~59"".The continued inhibition of
phosphatase activity would then maintain the signal at high
levels.
Notably, two kinases known to be involved in T-cell signaling, ~56"' and p59"", were activated, whereas ~ 6 2 ~ = ,
which has not been reported to be involved in T-cell signaling, was not affected. Furthermore, direct reaction of H,02
plus vanadate did not activate the kinases. Taken together,
these results suggest that regulatory elements of the signal
transduction pathways mediate the effects ofchemical treat-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
REACTIVE OXYGEN INTERMEDIATE INDUCED SIGNALS
1219
4. Wardman P: Principles of radiation chemistry, in Steel GG,
ment on these kinases. The activation of the kinases by
Adams GE (eds): The Biological Basis of Radiotherapy. New York,
H 2 0 z plus vanadate does not appear to be a function of
NY, Elsevier, 1983, p 51
phosphatase inhibition alone because P A 0 has not been
5. Brach MA, Hass R, Sherman ML, Gunji H, Weichselbaum R,
found to activate kinases." CD45 phosphotyrosine phosKufe
D: Ionizing radiation induces expression and binding activity
phatase is required for TCR- and CD2-mediated activation
ofthe nuclear factor KB.J Clin Invest 88:691, 1991
of protein tyrosine kinases.45 In contrast to these biologic
6. Sherman ML, Datta R, Hallahan DE, Weichselbaum RR,
receptor-mediated stimulations, the ability of a combinaKufe D W Ionizing radiation regulates expression of the c-jun protion of two potent phosphatase inhibitors to activate tyrotooncogene. Proc Natl Acad Sci USA 875663, 1990
sine kinases shows that the requirement for phosphatase
7. Hallahan DE, Sukhatme VP, Sherman JL, Virudachalam S,
activity can be circumvented.
Kufe D, Weichselbaum RR: Protein kinase C mediates x-ray inducThe combination of an ROI generator plus vanadate led
ibility of nuclear signal transducers EGRl and JUN. Proc Natl
to high levels of tyrosine phosphorylation of physiologically
Acad Sci USA 88:2156, 1991
8. Gold MR, Law DA, DeFranco A L Stimulation of protein
relevant proteins. This phosphorylation showed evidence of
phosphorylation by the B-lymphocyte antigen receptor.
specificity in that tyrosine phosphorylation of ~ 3 4 was
~ ~ ~tyrosine
'
Nature 3452310, 1990
not affected. Because phosphorylation of p34*" is regu9. June CH, Fletcher MC, Ledbetter JA, Samelson L E Increases
lated in response to incompletely replicated D N A t 6 the
in
tyrosine phosphorylation are detectable before phospholipase C
chemical treatment may not have affected that particular
activation after T cell receptor stimulation. J Immunol 144:I59 1,
signal transduction pathway. The effects of H202plus vanaI990
date offer a method we are currently investigating to pro10. Ledbetter JA, Schieven GL, Kuebelbeck VM, Uckun FM:
duce substantial quantities of tyrosine phosphorylated subAccessory receptors regulate coupling of the T-cell receptor comstrates for purification and identification.
plex to tyrosine kinase activation and mobilization of cytoplasmic
The effects of ROI on lymphoid cell signal transduction
calcium in T-lineage acute lymphoblastic leukemia. Blood
may have important consequences for a variety of disease
77:1271, 1991
states. H,02 is well suited as a messenger because it can
I I . June CH, Fletcher MC, Ledbetter JA, Schieven GL, Siegel
JN, Phillips AF, Samelson LE: Inhibition of tyrosine phosphorylafreely diffuse across cell membranes and in healthy individtion prevents T-cell receptor-mediated signal transduction. Proc
uals the concentration of H202is normally quite low.' Thus,
Natl Acad Sci USA 87:7722, 1990
the elevation of H,O, upon viral infection or inflammation
12. Lane PJL, LedbetterJA, McConnell FM, Draves K, Deans J,
could act as a significant signal. For cells already activated,
Schieven
GL, Clark EA: The role of tyrosine phosphorylation in
this signal might be expected to amplify their response by
signal transduction through surface Ig in human B cells. J Immunol
boosting tyrosine phosphorylation, NF-KB activation, and
146:715, 1991
calcium signaling. However, for resting T cells, ROI could
13. Siegel JN, Egerton M, Phillips AF, Samelson LE: Multiple
induce a calcium signal under nonmitogenic conditions
signal transduction pathways activated through the T cell receptor
that is potentially sufficient to induce a state of nonresponfor antigen. Semin Immunol 3:325, 1991
siveness, as has been observed for calcium signals induced
14. Burkhardt AL, Brunswick M, Bolen JB, Mond JJ: Anti-imby modulation of CD3 or by pulsing with calcium ionomunoglobulin stimulation of B lymphocytes activates src-related
p h o r e ~ . ~ It
',~
has
~ been proposed that many childhood acute
protein tyrosine kinases. Proc Natl Acad Sci USA 88:7410, 1991
15. Uckun FM, Tuel-Ahlgren L, Song CW, Waddick K, Myers
lymphoblastic leukemias arise from a combination of initial
DE, Kirihara J, LedbetterJA, SchievenGL: Ionizing radiation stimmutations followed by a later infection.49 The ROI generulates unidentified tyrosine-specific protein kinases in human Bated during the course of infection may play an important
role in this process, because ROI are tumor p r ~ m o t e r s . ~ ' lymphocyte precursors triggering apoptosis and clonogenic cell
death. Proc Natl Acad Sci USA 89:9005, 1992
Signals induced by ROI in lymphoid cells with accumulated
16. Uckun FM, Schieven GL, Tuel-Ahlgren LM, Dibirdik I,
mutations could conceivably tip the biochemical balance
Myers DE, Ledbetter JA, Song CW: Tyrosine phosphorylation is a
between oncogenic and tumor suppressor proteins in favor
mandatory proximal step in radiation-induced activation of the
of leukemogenesis. The replication of human immunodefiprotein kinase C signaling pathway in human B-lymphocyte preciency virus- 1 has been reported to be induced by ROI and
cursors. Proc Natl Acad Sci USA 90:252, 1993
inhibited by NAC3s5' via regulation of NF-KBa ~ t i v a t i o n . ~ ~ 17. Gilliland LK, Grossmann A, Rabiovitch PS, Ledbetter JA:
Our results suggest that ROI-induced tyrosine phosphorylaComposite signal transduction in T cell activation: Enhancement,
inhibition, and desensitization, in Cambier JC (ed): Ligands, Retion is likely to play a n essential role in this process. ROI-inceptors and Signal Transduction in Regulation of Lymphocyte
duced signals in lymphoid cells thus offer several avenues
Function. Washington, DC, American Society of Microbiology,
for future investigation.
1993, p 32 1
18. Uckun FM, Mitchell JB, Obuz V, Park CH, Waddick K,
REFERENCES
Friedman N, Oubaha L, Min WS, Song C W Radiation sensitivity
of human B-lineage lymphoid precursor cells. Int J Radiat Oncol
1. Halliwell B, GutteridgeJM: Free Radicalsin Biology and MedBiol Phys 21:1553, 1991
icine. Oxford, UK, Clarendon, 1989
19. Uckun FM, Gillis S, Souza L, Song CW: Effects of recombi2. Bauerle PA: The inducibletranscrition activatorNF-KB:Regunant growth factors on radiation survival of human bone marrow
lation by distinct protein subunits. Biochim Biophys Acta 1072:63,
progenitor cells. Int J Radiat Oncol Biol Phys 16:415, 1989
1991
20. Uckun FM, Schieven GL, Dibirdik 1, Chandan-Langlie M,
3. Schreck R, Reiber P, Bauerle PA: Reactive oxygen intermeTuel-Ahlgren L, Ledbetter JA: Stimulation of protein tyrosine
diates as apparently widely used messengers in the activation of the
phosphorylation, phosphoinositide turnover, and multiple previNF-KBtranscription factor and HIV-1. EMBO J 10:2247, 1991
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1220
ously unidentified serine/threonine-specific protein kinases by the
pan-B-cell receptor CD40/Bp5O at discrete developmental stages of
human B-cell ontogeny. J Biol Chem 266: 17478, 199 I
21. Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase I1 in a soluble extract from
isolated mammalian nuclei. Nucleic Acids Res 1I: 1475, 1983
22. Osborn L, Kunkel S, Nabel GJ: Tumor necrosis factor LY and
interleukin I stimulate the human immunodeficiency virus enhancer by the activation of the nuclear factor KB.Proc Natl Acad
Sci USA 86:2336, 1989
23. Singh H, Sen R, Baltimore D, Sharp PA: A nuclear factor
that binds to a conserved sequence motif in transcriptional control
elements of immunoglobulin genes. Nature 3 19:154, 1986
24. Kanner SB, Damle NK, Blake J, Aruffo A, Ledbetter JA:
CD2/LFA3 ligation induces phospholipase Cy I tyrosine phosphorylation and regulates CD3 signaling. J lmmunol 148:2023, 1992
25. Sancho J, Ledbetter JA, Choi MS, Kanner SB, Deans JP,
Terhorst C: CD3-.( surface expression is required for CD4-p56"'mediated upregulation of T cell antigen receptor-CD3 signaling in
T cells. J Biol Chem 267:787 I , I992
26. Kamps MP, Sefton BM: Identification of multiple novel
polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B
oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305, I988
27. Schieven GL, Kallestad JC, Brown TJ, Ledbetter JA, Linsley
PS: Oncostatin M induces tyrosine phosphorylation in endothelial
cells and activation of ~ 6 2 tyrosine
~ ' ~ kinase. J Immunol 149:1676,
1992
28. Eisenman E, Bolen JB: Src-related tyrosine protein kinases
as signaling components in hematopoietic cells. Cancer Cells 2:303,
1990
29. Rabinovitch PS, June CH, Grossman A, Ledbetter JA: Heterogeneity among T cells in intracellular free calcium response after
mitogen stimulation with PHA or anti-CD3. Simultaneous use of
indo- 1 and immunofluorescence with flow cytometry. J Immunol
137:952, 1986
30. Uckun FM, Dibirdik I, Smith R, Tuel-Ahlgren L, ChandanLanglie M, Schieven GL, Waddick KG, Hanson M, Ledbetter JA:
Interleukin 7 receptor ligation stimulates tyrosine phosphorylation,
inositol phopholipid turnover, and clonal proliferation of human
B-cell precursors. Proc Natl Acad Sci USA 88:3589, 1991
3 1. Zick Y, Sagi-Eisenberg R A combination of H,O, and vanadate concomitantly stimulates protein tyrosine phosphorylation
and polyphosphoinositide breakdown in different cell lines. Biochemistry 29: 10240, 1990
32. Heffetz D, Bushkin I, Dror R, Zick Y: The insulinomimetic
agents H,02 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Bioi Chem 265:2896, 1990
33. OShea JJ, McVicar DW, Bailey TL, Bums C, Smyth MJ:
Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation. Proc Natl
Acad Sci USA 89: 10306, 1992
34. Gilliland LK, Schieven GL, Noms N, Kanner SB, Aruffo A,
Ledbetter JA: Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLCyl SH2 domains. J Biol Chem
267: 136 IO, 1992
35. Settleman J, Narasimhan V, Foster LC, Weinberg RA: Molecular cloning of cDNAs encoding the GAP-associated protein
SCHIEVEN ET AL
p190: Implications for a signaling pathway from ras to the nucleus.
Cell 69539, 1992
36. Wong G, Muller 0, Clark R, Conroy L, Moran MF, Polakis
P, McCormick F: Molecular cloning and nucleic acid bindingproperties of the GAP-associated tyrosine phosphoprotein p62. Cell
69:551, 1992
37. Marth JD, Lewis DB, Cooke MP, Mellins ED, Gearn ME,
Samelson LE, Wilson CB, Miller AD, Perlmutter R D Lymphocyte
activation provokes modification of a lymphocyte-specific protein
tyrosine kinase (~56'"~).
J Immunol 142:2430, 1989
38. Iwasaki T, Uehara Y, Graves L, Rachie N, Bomsztyk K
Herbimycin A blocks IL- 1-induced NF-KBDNA-binding activity
in lymphoid cell lines. FEBS Lett 298:240, 1992
39. Devary Y, Gottlieb RA, Smeal T, Karin M: The mammalian
ultraviolet response is triggered by activation of src tyrosine kinases.
Cell 71:1081, 1992
40. Kuwabara M, Inanami 0,Endoh D, Sat0 F: Spin trapping of
precursors of thymine damage in X-irradiated DNA. Biochemistry
26:2458, 1987
41. Hunter T Protein-tyrosine phosphatases: The other side of
the coin. Cell 58:435, 1989
42. Kadota S, Fantus IG, Deragon G, Guyda HJ, Hersh B,
Posner B: Peroxide(s) of vanadium: A novel and potent insulin-mimetic agent which activates the insulin receptor kinase. Biochem
Biophys Res Commun 147:259, 1987
43. Fantus IG, Kadota S, Deragon G, Foster B, Posner B: Pervanadate [peroxide(s) of vanadate] mimics insulin action in rat adipocytes via activation of the insulin receptor tyrosine kinase. Biochemistry 28:8864, 1989
44. Garcia-Morales P, Minami Y, Luong E, Klausner RD, Samelson LE: Tyrosine phosphorylation in T cells is regulated by
phosphatase activity: Studies with phenylarsine oxide. Proc Natl
Acad Sci USA 87:9255, 1990
45. Koretzky GA, Picus J, Schultz T, Weiss A: Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc Natl Acad Sci USA 88:2037. 199 1
46. Smythe C, Newport JW: Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine
kinase that phosphorylates ~ 3 4 ~ ' ' Cell
. 68:787, 1992
47. Davis LS, Wacholtz MC, Lipsky PE: The induction o f T cell
unresponsiveness by rapidly modulating CD3. J Immunol
142:1084, 1989
48. Jenkins MK, Pardoll DM, Miuguchi J, Chused TM,
Schwartz RM: Molecular events in the induction of a nonresponsive state in interleukin 2-producing helper T-lymphocyte clones.
Proc Natl Acad Sci USA 845409, 1987
49. Greaves MF: Models of childhood acute lymphoblastic leukemia. Leukemia 5:819, 1991
50. Cerutti PA: Prooxidant states and tumor promotion. Science
227:375, 1985
51. Roederer M, Staal FJ, Raju PA, Ela SW, Herzenberg LA,
Herzenberg L: Cytokine-stimulated human immunodeficiency
virus replication is inhibited by N-acetyl-L-cysteine. Proc Natl
Acad Sci USA 87:4884, 1990
52. Staal FJ, Roederer M, Herzenberg LA: Intracellular thiols
regulate activation of nuclear factor kappa B and transcription of
human immunodeficiency virus. Proc Natl Acad Sci USA 87:9943,
1990
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1993 82: 1212-1220
Reactive oxygen intermediates activate NF-kappa B in a tyrosine
kinase- dependent mechanism and in combination with vanadate
activate the p56lck and p59fyn tyrosine kinases in human lymphocytes
GL Schieven, JM Kirihara, DE Myers, JA Ledbetter and FM Uckun
Updated information and services can be found at:
http://www.bloodjournal.org/content/82/4/1212.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.