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From www.bloodjournal.org by guest on June 17, 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 17, 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 17, 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 17, 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 17, 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 17, 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 17, 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 17, 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. 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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 17, 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. 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