Title: Role of Tyrosine Kinases in Induction of the c-junProto-oncogene in Irradiated B-lineage Lymphoid Cells
Abstract: Exposure of B-lineage lymphoid cells to ionizing radiation induces an elevation of c-jun proto-oncogene mRNA levels. This signal is abrogated by protein-tyrosine kinase (PTK) inhibitors, indicating that activation of an as yet unidentified PTK is mandatory for radiation-induced c-jun expression. Here, we provide experimental evidence that the cytoplasmic tyrosine kinases BTK, SYK, and LYN are not required for this signal. Lymphoma B-cells rendered deficient for LYN, SYK, or both by targeted gene disruption showed increased c-jun expression levels after radiation exposure, but the magnitude of the stimulation was lower than in wild-type cells. Thus, these PTKs may participate in the generation of an optimal signal. Notably, an inhibitor of JAK-3 (Janus family kinase-3) abrogated radiation-induced c-jun activation, prompting the hypothesis that a chicken homologue of JAK-3 may play a key role in initiation of the radiation-induced c-jun signal in B-lineage lymphoid cells. Exposure of B-lineage lymphoid cells to ionizing radiation induces an elevation of c-jun proto-oncogene mRNA levels. This signal is abrogated by protein-tyrosine kinase (PTK) inhibitors, indicating that activation of an as yet unidentified PTK is mandatory for radiation-induced c-jun expression. Here, we provide experimental evidence that the cytoplasmic tyrosine kinases BTK, SYK, and LYN are not required for this signal. Lymphoma B-cells rendered deficient for LYN, SYK, or both by targeted gene disruption showed increased c-jun expression levels after radiation exposure, but the magnitude of the stimulation was lower than in wild-type cells. Thus, these PTKs may participate in the generation of an optimal signal. Notably, an inhibitor of JAK-3 (Janus family kinase-3) abrogated radiation-induced c-jun activation, prompting the hypothesis that a chicken homologue of JAK-3 may play a key role in initiation of the radiation-induced c-jun signal in B-lineage lymphoid cells. The proto-oncogene c-jun is the cellular counterpart of the v-jun oncogene of avian sarcoma virus 17 (1Ryder K. Lau L.F. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1998; 85: 1487-1491Crossref Scopus (514) Google Scholar, 2Schutte J. Viallet J. Nau M. Segal S. Fedorko J. Minna J. Cell. 1989; 59: 987-997Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 3Neuberg M. Adamkiewicz J. Hunter J.B. Mueller R. Nature. 1989; 341: 589-590Crossref Scopus (89) Google Scholar, 4Mitchell P.J. Tjian R. Science. 1989; 245: 371-378Crossref PubMed Scopus (2195) Google Scholar, 5Bohmann D. Bos T.J. Admon T. Nishimura R. Vogt P.K. Tjian R. Science. 1998; 238: 1386-1392Crossref Scopus (953) Google Scholar). c-jun expression is activated in response to a diverse set of DNA-damaging agents including araC (6Kharbanda S.M. Sherman M.L. Kufe D.W. J. Clin. Invest. 1990; 86: 1517-1523Crossref PubMed Scopus (39) Google Scholar) UV radiation (7Rosette C. Karin M. 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Protein-tyrosine kinases (PTKs) 1The abbreviations used are: PTKs, protein-tyrosine kinases; Gy, gray(s); PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin. 1The abbreviations used are: PTKs, protein-tyrosine kinases; Gy, gray(s); PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin. play important roles in the initiation and maintenance of biochemical signal transduction cascades that affect proliferation and survival of B-lineage lymphoid cells (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar, 21Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar, 22Uckun F.M. Evans W.E. Forsyth C.J. Waddick K.G. Tuel-Ahlgren L. Chelstrom L.M. Burkhardt A. Bolen J. Myers D.E. Science. 1995; 267: 886-891Crossref PubMed Scopus (260) Google Scholar, 23Myers D.E. Jun X. 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Isselbacker K.J. Pilai S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10606-10609Crossref PubMed Scopus (168) Google Scholar, 30Jugloff L.S. Jongstra Bilen J. J. Immunol. 1997; 159: 1096-1106PubMed Google Scholar, 31Thomis D.S. Gurniak C.B. Tivol E. Sharpe A.H. Berg L.J. Science. 1995; 270: 794-797Crossref PubMed Scopus (473) Google Scholar, 32Nosaka T. Van Deursen J.M. Tripp R.A. Thierfelder W.E. Witthuhn B.A. McMickle A.P. Doherty P.C. Grosveld G.C. Ihle J.N. Science. 1995; 270: 800-802Crossref PubMed Scopus (571) Google Scholar). Oxidative stress has been shown to activate BTK (Brutons's tyrosine kinase), SYK, and Src family PTKs (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar, 24Tuel-Ahlgren L. Jun X. Waddick K.G. Jin J. Bolen J. Uckun F.M. Leuk. Lymphoma. 1996; 20: 417-426Crossref PubMed Scopus (7) Google Scholar, 25Qin S. Minami Y. Hibi M. Kurosaki T. Yamamura H. J. Biol. Chem. 1997; 272: 2098-2103Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). We have previously shown that PTK activation precedes and mandates radiation-induced activation of c-jun proto-oncogene expression in human B-lineage lymphoid cells (10Chae H.P. Jarvis L.J. Uckun F.M. Cancer Res. 1993; 53: 447-451PubMed Google Scholar). However, the identity of the PTK responsible for radiation-induced c-jun activation remains unknown. The purpose of the present study was to examine the potential involvement of BTK, SYK, and LYN in radiation-induced c-jun activation. To this end, we used DT-40 chicken lymphoma B-cell clones rendered deficient for these specific PTKs by targeted gene disruption. Our findings indicate that BTK plays no role in radiation-induced c-jun activation. Similarly, neither LYN nor SYK was required for activation of c-jun after radiation exposure, but our results suggest that their participation may influence the magnitude of the c-jun response. Notably, an inhibitor of JAK-3 (Janus family kinase-3) abrogated the radiation-induced c-jun activation, prompting the hypothesis that activation of a chicken JAK-3 homologue may be mandatory for induction of c-jun transcription after radiation exposure. The establishment and characterization of BTK-, SYK-, and LYN-deficient clones and reconstituted SYK-deficient cell lines of DT-40 chicken lymphoma B-cells were previously reported (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar,21Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar, 33Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (223) Google Scholar, 34Dibirdik I. Kristupaitis D. Kurosaki T. Tuel-Ahlgren L. Chu A. Pond D. Tuong D. Luben R. Uckun F.M. J. Biol. Chem. 1998; 273: 4035-4039Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The culture medium was RPMI 1640 (Life Technologies, Inc.) supplemented with 1% chicken serum (Sigma), 5% fetal bovine serum (Hyclone Laboratories, Logan, UT), and 1% penicillin/streptomycin (Life Technologies, Inc.). Cells (2 × 106/ml) were treated for 24 h at 37 °C with either 1) the PTK inhibitory isoflavone genistein (Calbiochem) at 111 μm(30 μg/ml) or 2) the JAK-3-specific PTK inhibitor 4-(3′-bromo-4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (C16H14Br(N3O3)), kindly provided by Dr. Xing-Ping Liu (Alexander and Parker Pharmaceutical Inc., Roseville, MN), at 270 μm (100 μg/ml) prior to radiation in order to assess the effects of these agents on radiation-induced c-jun activation. The precursor 4-chloro-6,7-dimethoxyquinazoline was prepared as shown Scheme FS1. In this procedure, 4,5-dimethoxy-2-nitrobenzoic acid (compound 1) was treated with thionyl chloride, which directly reacted with ammonia to give 4,5-dimethoxy-2-nitrobenzamide (compound 2) (35Nomoto F. Obase H. Takai H. Hirata T. Teranishi M. Nakamura J. Kubo K. Chem. Pharm. Bull. (Tokyo). 1990; 38: 1591-1595Crossref PubMed Scopus (23) Google Scholar). Compound 2 was reduced with sodium borohydrate and catalyzed by copper sulfate (36Thomas C.L. Catalytic Processes and Proven Catalysts. Academic Press, New York1970Google Scholar) to give 4,5-dimethoxy-2-aminobenzamide (compound 3), which was directly refluxed with formic acid to give 6,7-dimethoxyquinazoline-4(3H)-one (compound 4). Compound 4 was refluxed with phosphorus oxytrichloride to provide the key starting material (compound 5) with good yield (35Nomoto F. Obase H. Takai H. Hirata T. Teranishi M. Nakamura J. Kubo K. Chem. Pharm. Bull. (Tokyo). 1990; 38: 1591-1595Crossref PubMed Scopus (23) Google Scholar). More specifically, for the synthesis of 4,5-dimethoxy-2-nitrobenzamide (precursor compound 2), a suspension of 2 g (8.8 mmol) of 4,5-dimethoxy-2-nitrobenzoic acid (5Bohmann D. Bos T.J. Admon T. Nishimura R. Vogt P.K. Tjian R. Science. 1998; 238: 1386-1392Crossref Scopus (953) Google Scholar, 6Kharbanda S.M. Sherman M.L. Kufe D.W. J. Clin. Invest. 1990; 86: 1517-1523Crossref PubMed Scopus (39) Google Scholar) in 10 ml of SOCl2was stirred under reflux for 50 min. After cooling, the reaction mixture was poured into a mixture of 50 ml of concentrated NH4OH and 30 g of ice. The precipitated crystals were collected by filtration, washed with water, and dried to give 1.85 g of crude crystals. Recrystallization fromN,N-dimethylformamide yielded 1.76 g of pure product (88.5%). For the synthesis of 6,7-dimethoxyquinazoline-4(3H)-one (precursor compound 4), 400 mg of NaBH4 was added with stirring over 4 h to a solution of 1.58 g (7 mmol) of 4,5-dimethoxy-2-nitrobenzamide (compound 2) in MeOH containing a catalytic amount of CuSO4. The reaction mixture was poured into 200 ml of ice water with stirring to give 4,5-dimethoxy-2-aminobenzamide (compound 3), which was directly refluxed with 20 ml of HCOOH for 5 h. After removal of solvent, the residue was recrystallized fromN,N-dimethylformamide to give 1.18 g of pure crystals (81.5%), m.p. 295.0–297.0 °C. To synthesize 4-chloro-6,7-dimethoxyquinazoline (precursor compound 5),N,N-dimethylformamide (7 g, 90 mmol, 8.6 ml) was added dropwise over 20 min to a stirred solution of oxalyl chloride (11.42 g, 90 mmol, 8 ml) in 200 ml of 1,2-dichloroethane at 25 °C under N2, resulting in an exothermic gas evolution. When gas evolution ceased, 6,7-dimethoxyquinazoline-4(3H)-one (compound 4) (12.36 g, 60 mmol) was added with mechanical agitation, and the mixture was heated to reflux for 4 h and then cooled to 25 °C. The reaction mixture was quenched with dilute aqueous Na2HPO4 solution (0.5 m, 250 ml). The resulting mixture was stirred on an ice bath for 2 h, and the solid was collected, rinsed with water (2 × 50 ml), and dried at 50 °C under vacuum to give 11.2 g of product (75.0%), m.p. 259.0–263.0 °C. Compounds 1 and 2 were prepared through the condensation of 4-chloro-6,7-dimethoxyquinazoline with the substituted aniline as shown in Scheme FS2. More specifically, a mixture of 448 mg (2 mmol) of 4-chloro-6,7-dimethoxyquinazoline and 2.5 mmol of substituted anilines in 20 ml of alcohol (EtOH or MeOH) was heated to reflux. Heating was continued for 4–24 h; sufficient Et3N was added to basify the solution; and the solvent was then concentrated to give crude product, which was recrystallized fromN,N-dimethylformamide. During the characterization of compounds 1 and 2 and their precursors, melting points were obtained using a Fisher-Johns melting point apparatus and are uncorrected. 1H NMR spectra were recorded using a VAV-300 (Department of Chemistry, University of Minnesota) or Varian-300 spectrometer and in Me2SO-d6 or CDCl3solution. Chemical shifts are reported in parts/million with tetramethylsilane as an internal standard at 0 ppm. Coupling constants (J) are given in hertz, and the abbreviations s, d, t, q, and m refer to singlet, doublet, triplet, quartet, and multiplet, respectively. Infrared spectra were recorded on a Nicolet Protege 460-IR spectrometer. Mass spectroscopy data were recorded on a Finnigan MAT 95, VG 7070E-HF (Department of Chemistry, University of Minnesota), or HP 6890 spectrometer. UV spectra were recorded on a Beckman DU 7400 apparatus and using MeOH as the solvent. TLC was performed on a precoated silica gel plate (Silica Gel KGF, Whatman). Silica gel (200–400 mesh, Whatman) was used for all column chromatography separations. All chemicals were reagent-grade and were purchased from Aldrich or Sigma. Selected analytical data for synthesized 4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline were as follows: yield, 84.29%; m.p. 245.0- 248.0 °C; 1H NMR (Me2SO-d6) δ 11.21 (s, 1H, -NH), 9.70 (s, 1H, -OH), 8.74 (s, 1H, 2-H), 8.22 (s, 1H, 5-H), 7.40 (d, 2H, J = 8.9 Hz, 2′,6′-H), 7.29 (s, 1H, 8-H), 6.85 (d, 2H, J = 8.9 Hz, 3′,5′-H), 3.98 (s, 3H, -OCH3), and 3.97 (s, 3H, -OCH3); UV (MeOH) λmax (ε) 203.0, 222.0, 251.0, and 320.0 nm; IR (KBr)umax 3428, 2836, 1635, 1516, 1443, and 1234 cm−1; gas chromatography-mass spectrometry, m/z298 (M+ + 1, 100.00), 297 (M+, 26.56), 296 (M+ − 1, 12.46). Selected analytical data for synthesized 4-(3′-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline were as follows: yield, 89.90%; m.p. 233.0–233.5 °C; 1H NMR (Me2SO-d6) δ 10.08 (s, 1H, -NH), 9.38 (s, 1H, -OH), 8.40 (s, 1H, 2-H), 7.89 (d, 1H,J2′,5′ = 2.7 Hz, 2′-H), 7.75 (s, 1H, 5-H), 7.55 (dd, 1H, J5′,6′ = 9.0 Hz,J2′,6′ = 2.7 Hz, 6′-H), 7.14 (s, 1H, 8-H), 6.97 (d, 1H, J5′,6′ = 9.0 Hz, 5′-H), 3.92 (s, 3H, -OCHER), and 3.90 (s, 3H, -OCH3); UV (MeOH) λmax (ε) 203.0, 222.0, 250.0, and 335.0 nm; IR (KBr)umax 3431 (br), 2841, 1624, 1498, 1423, and 1244 cm−1; gas chromatography-mass spectrometry, m/z378 (M+ + 2, 90.68), 377 (M+ + 1, 37.49), 376 (M+, 100.00), 360 (M+, 3.63), 298 (18.86), 282 (6.65). Cells (2 × 106 /ml) in plastic tissue culture flasks were irradiated with 10–20 Gy at a dose rate of 4 Gy/min during log-phase growth and under aerobic conditions using a 137Cs irradiator (J. L. Shephard, Glendale, CA) as described previously (24Tuel-Ahlgren L. Jun X. Waddick K.G. Jin J. Bolen J. Uckun F.M. Leuk. Lymphoma. 1996; 20: 417-426Crossref PubMed Scopus (7) Google Scholar, 38Uckun F.M. Jaszcz W. Chandan Langlie M. Waddick K.G. Gajl Peczalska K. Song C.W. J. Clin. Invest. 1993; 91: 1044-1051Crossref PubMed Scopus (29) Google Scholar). In some experiments, cells were preincubated with PTK inhibitors for 24 h prior to irradiation. A 506-base pair c-jun probe was obtained by polymerase chain reaction (PCR) amplification of chicken genomic DNA. Primer sequences were determined based upon the sequence of chicken c-jun (GenBankTM accession code CHKJUN). Two primers (5′-ACTCTGCACC CAACTACAACGC-3′ and 5′-CTTCTACCGTCAGCTTTACGCG-3′) were used for amplification. Amplification was performed with a mixture of Taq polymerase and a proofreading polymerase (eLONGase/Taq polymerase plusPyrococcus sp. GB-D polymerase, Life Technologies, Inc.) on a thermocycler (Ericomp Delta II cycler) using a hot start. PCR products were subsequently cloned into the cloning vector PCR2.1 (Invitrogen, San Diego, CA). An insert of the proper size (506 base pairs) was identified as chicken c-jun by sequence analysis using Thermosequenase fluorescent labeled primer cycle sequencing kit (Amersham) and analyzed on an automated sequencer (ALF Express Sequencer, Amersham Pharmacia Biotech). A 538-base pair chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was generated by reverse transcription and subsequent PCR amplification from chicken RNA with the following primers: 5′-AGAGGTGCTGCCCAGAACATCATC-3′ and 5′-GTGGGGAGACAGAAGGGAACAGA-3′. A 413-base pair chicken β-actin probe was generated by reverse transcription-PCR amplification from chicken RNA with the following primers: 5′-GCCCTCTTCCAGCATCTTTCTT-3′ and 5′-TTTATGCGCATTTATGGGTT-3′. The amplified cDNAs were cloned into PCR2.1. Total RNA was extracted from ∼2.5 × 107 cells with Trizol reagent, a monophasic solution of phenol and guanidine isothiocyanate as described by Chomczynski and Sacchi (39Chomczynski P. Sacchi N. Biochemistry. 1987; 162: 156-159Google Scholar). Poly(A)+ RNA was isolated directly from 1–3 × 108 cells with an Invitrogen Fast Trak 2.0 mRNA isolation kit. In brief, cells were lysed in an SDS lysis buffer containing a proprietary mixture of proteases. The lysate was directly incubated with oligo(dT) for absorption and subsequent elution of poly(A)+ RNA. Two micrograms of poly(A)+ or 20 μg of total RNA were denatured in formaldehyde/formamide loading dye at 65 °C prior to loading onto a 1% agarose-formaldehyde denaturing gel. Transcript sizes were determined relative to RNA markers of 0.5–9 kilobases. The gels were stained with Radiant Red in H2O to check loading and integrity of RNA prior to transfer. The RNA was subsequently transferred to positively charged nylon membrane with 20× SSC transfer buffer (1× SSC = 0.15 m sodium chloride and 0.015m sodium citrate) by downward capillary transfer. The c-jun fragment was radiolabeled by random priming with [α-32P]dCTP (3000 Ci/mm; Amersham Pharmacia Biotech) (40Feinberg A.P. Vogelstein B.A. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16646) Google Scholar). Northern blots were hybridized overnight at 42 °C in prehybridization/hybridization solution (50% formamide with proprietary blocking and background reduction reagents; Ambion Inc., Austin, TX) for 16–24 h, and unbound probe was removed by washing to a final stringency of 0.1% SDS and 0.1× SSC (65 °C). The blots were analyzed both by autoradiography and using the Bio-Rad storage phosphor imager system for quantitative scanning. The blots were subsequently stripped in boiling 0.1% SDS and then rehybridized with a chicken GAPDH and/or chicken β-actin probe to normalize for loading differences. Exposure of human lymphoma B-cells to 10–20-Gy γ-rays results in enhanced c-junexpression with a maximum response at 1–2 h (10Chae H.P. Jarvis L.J. Uckun F.M. Cancer Res. 1993; 53: 447-451PubMed Google Scholar). We previously reported that, in DT-40 chicken lymphoma B-cells, ionizing radiation triggers biochemical and biological signals similar to those in human lymphoma B-cells (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar). To determine if DT-40 chicken lymphoma B-cells show a similar c-jun response to ionizing radiation, we irradiated DT-40 cells with 5, 10, 15, or 20 Gy and examined total RNA harvested from cells 2 or 4 h after radiation exposure for expression levels of 1.8-kilobase chicken c-jun transcripts by quantitative Northern blot analysis. As shown in Fig. 1 A, radiation exposure increased the level of c-jun transcripts in a dose- and time-dependent manner without significantly affecting the GAPDH transcript levels, with a maximum stimulation index (as determined by comparison of the c-jun/GAPDH ratios in non-irradiated versus irradiated cells) of 3.1, 4 h after 20 Gy. In seven additional independent experiments, the stimulation index for 20-Gy ionizing radiation at 2 h after radiation exposure ranged from 2.4 to 3.8 (mean ± S.E. = 2.9 ± 0.4). We next examined the role of PTK in radiation-induced activation of c-jun expression in chicken lymphoma B-cells since PTK inhibitors were shown to prevent radiation-induced c-junactivation in human lymphoma B-cells. As shown in Fig. 1 B, ionizing radiation did not significantly enhance c-jun expression levels in DT-40 cells treated with the PTK inhibitory isoflavone genistein (stimulation index = 1.1), indicating that activation of a PTK is required for radiation-induced c-jun expression in chicken lymphoma B-cells as well. These findings established DT-40 chicken lymphoma B-cells as a suitable model to further elucidate the molecular mechanism of radiation-induced c-jun activation. BTK is abundantly expressed in lymphoma B-cells, and its activation has been shown to be required for radiation-induced apoptosis of DT-40 cells (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar). DT-40 cells rendered BTK-deficient by targeted disruption of thebtk genes do not undergo apoptosis after radiation exposure. Therefore, we set out to determine if BTK could be the PTK responsible for radiation-induced c-jun activation as well, by comparing the levels of c-jun induction in BTK-deficient (BTK−) versus wild-type DT-40 cells. Contrary to our expectations, 20-Gy ionizing radiation did not fail to induce c-jun expression in BTK-deficient DT-40 cells in any of the three independent experiments performed. The stimulation indices ranged from 1.6 to 3.9 (mean ± S.E. = 2.4 ± 0.5) (Fig. 2 ). Thus, ionizing radiation-induced increases in c-jun transcript levels do not depend upon the presence of BTK. Since SYK is also abundantly expressed in DT-40 cells and is rapidly activated after ionizing radiation, we next examined if SYK might be the PTK responsible for radiation-induced increases in c-juntranscript levels. As shown in Fig. 3 A, 20-Gy ionizing radiation enhanced c-jun expression in SYK−DT-40 cells rendered SYK-deficient by targeted gene disruption even though the stimulation indices observed in five independent experiments were lower than those in wild-type cells (1.9 ± 0.2versus 2.9 ± 0.4; p < 0.01). Thus, SYK is not required for radiation-induced c-jun activation in DT-40 cells, but it may participate in generation of an optimal signal. DT-40 cells express high levels of LYN, but do not express other members of the Src PTK family, including BLK, HCK, SRC, FYN, and YES, at detectable levels (20Uckun F.M. Waddick K.G. Mahajan S. Jun X. Takata M. Bolen J. Kurosaki T. Science. 1996; 273: 1096-1100Crossref PubMed Scopus (166) Google Scholar, 33Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (223) Google Scholar, 41Takata M. Homma Y. Kurosaki T. J. Exp. Med. 1995; 182: 907-914Crossref PubMed Scopus (183) Google Scholar). Since it has previously been demonstrated that Src family PTKs are essential for UV-stimulated increases in c-jun expression, we postulated that the predominant Src family member, LYN, might mediate radiation-induced c-jun expression in DT-40 cells. To test this hypothesis, we examined the ability of ionizing radiation to activate c-junexpression in DT-40 cells rendered LYN-deficient by targeted gene disruption. LYN-deficient (LYN−) cells showed enhanced c-jun expression after irradiation; however, the stimulation indices were lower than those in wild-type DT-40 (Fig. 3 B ). Since LYN and SYK have been shown to cooperate in the generation of other signals in B-cells (21Kurosaki T. Curr. Opin. Immunol. 1997; 9: 309-318Crossref PubMed Scopus (181) Google Scholar), we examined the ability of ionizing radiation to induce c-junexpression in LYN−SYK− DT-40 cells, generated by targeted disruption of the syk gene in LYN−-deficient DT-40 cells. As shown in Fig. 3 B, LYN−SYK− DT-40 cells showed elevated c-jun transcript levels after irradiation, indicating that the c-jun response does not depend on either of these PTKs, either alone or in cooperation. Similar to SYK, LYN is not required for radiation-induced c-junactivation in DT-40 cells, but it may participate in generation of an optimal response. Interestingly, in four independent experiments, we observed higher base-line expression levels of c-jun in SYK−DT-40 cells than in wild-type DT-40 cells (range of 1.4–2.3-fold, mean ± S.E. = 1.6 ± 0.2-fold), suggesting that SYK may be involved in regulation of base-line c-jun levels. To further explore this possibility, we compared c-jun levels in SYK− cells with those in SYK− cells reconstituted with the wild-type or kinase domain mutant (K−) syk gene. We observed that reconstitution with wild-type syk reduced the higher base-line expression levels of c-jun in SYK− cells, whereas reconstitution with K− syk failed to reduce c-jun levels (data not shown). These results implicate SYK as a negative regulator of c-jun expression. This novel function of SYK seems to depend on its kinase domain. B-cell signal transduction events direct fundamental decisions regarding cell survival during periods of oxidative stress. A better understanding of the dynamic interplay between