Role of Tyrosine Kinases in Induction of the c-junProto-oncogene in Irradiated 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 (1)(2)(3)(4)(5). c-jun expression is activated in response to a diverse set of DNA-damaging agents including araC (6) UV radiation (7), topoisomerase II inhibitors (8), alkylating agents (9), and ionizing radiation (10). As an immediate-early response gene that is rapidly induced by pleiotropic signals, c-jun may have important regulatory functions for cell cycle progression, proliferation, and survival (11). c-jun encodes the nuclear DNA-binding protein JUN, which contains a leucine-zipper region involved in homo-and heterodimerization (1,3,11,12). JUN protein dimerizes with another JUN protein or the product of the c-fos gene and forms the activating protein-1 transcription factor (11,12). JUN-JUN homodimers and JUN-FOS heterodimers preferentially bind to a specific heptameric consensus sequence found in the promoter region of multiple growth regulatory genes (11)(12)(13). Alterations of c-jun proto-oncogene expression can therefore modulate the transcription of several growth regulators affecting cell proliferation and differentiation (11). c-jun plays a pivotal role in Ras-induced transformation and has also been implicated as a regulator of apoptosis when de novo protein synthesis is required (11,14,15). c-jun induction is required for ceramide-induced apoptosis and stress-induced apoptosis after UV exposure or other forms of DNA damage (16). This induc-tion is thought to be triggered by activation of JNK (JUN N-terminal kinase) (also known as stress-activated protein kinases), which leads to enhanced c-jun transcription (11,17,18) by phosphorylation of JUN at sites that increase its ability to activate transcription. Ectopic expression of a dominant-negative c-jun mutant lacking the N terminus or a dominant-negative JNK abolishes stress-induced apoptosis (15,19).
Protein-tyrosine kinases (PTKs) 1 play important roles in the initiation and maintenance of biochemical signal transduction cascades that affect proliferation and survival of B-lineage lymphoid cells (20 -32). Oxidative stress has been shown to activate BTK (Brutons's tyrosine kinase), SYK, and Src family PTKs (20,24,25). 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 (10). 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.
Chemical Synthesis and Characterization of the JAK-3 Inhibitors-The precursor 4-chloro-6,7-dimethoxyquinazoline was prepared as shown Scheme 1. 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) * This work was supported by a special grant from the Parker Hughes Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Compounds 1 and 2 were prepared through the condensation of 4-chloro-6,7-dimethoxyquinazoline with the substituted aniline as shown in Scheme 2. 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 Et 3 N was added to basify the solution; and the solvent was then concentrated to give crude product, which was recrystallized from N,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. 1 H NMR spectra were recorded using a VAV-300 (Department of Chemistry, University of Minnesota) or Varian-300 spectrometer and in Me 2 SO-d 6 or CDCl 3 solution. 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 reagentgrade and were purchased from Aldrich or Sigma.
c-jun Probe-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 (GenBank TM accession code CHKJUN). Two primers (5Ј-ACTCTGC-ACC CAACTACAACGC-3Ј and 5Ј-CTTCTACCGTCAGCTTTACGCG-3Ј) were used for amplification. Amplification was performed with a mixture of Taq polymerase and a proofreading polymerase (eLONGase/Taq polymerase plus Pyrococcus 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: 5Ј-AGAGGTGCTGCCC-AGAACATCATC-3Ј and 5Ј-GTGGGGAGACAGAAGGGAACAGA-3Ј. A 413-base pair chicken ␤-actin probe was generated by reverse transcription-PCR amplification from chicken RNA with the following primers: 5Ј-GCCCTCTTCCAGCATCTTTCTT-3Ј and 5Ј-TTTATGCGCATTT-ATGGGTT-3Ј. The amplified cDNAs were cloned into PCR2.1.
RNA Isolation and Northern Blot Hybridization Analysis-Total RNA was extracted from ϳ2.5 ϫ 10 7 cells with Trizol reagent, a monophasic solution of phenol and guanidine isothiocyanate as described by Chomczynski and Sacchi (39). Poly(A) ϩ RNA was isolated directly from 1-3 ϫ 10 8 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) ϩ RNA.
Two micrograms of poly(A) ϩ or 20 g 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 H 2 O to check loading and integrity of RNA prior to transfer. The RNA was subsequently transferred to positively charged nylon membrane with 20ϫ SSC transfer buffer (1ϫ SSC ϭ 0.15 M sodium chloride and 0.015 M sodium citrate) by downward capillary transfer. The c-jun fragment was radiolabeled by random priming with [␣-32 P]dCTP (3000 Ci/mM; Amersham Pharmacia Biotech) (40). 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 0.1ϫ 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 ␤-actin probe to normalize for loading differences. SCHEME 2. Preparation of compounds 1 and 2. SCHEME 1. Preparation of 4-chloro-6,7-dimethoxyquinazoline.

Exposure of DT-40 Chicken Lymphoma B-cells to Ionizing
Radiation Activates the c-jun Proto-oncogene-Exposure of human lymphoma B-cells to 10 -20-Gy ␥-rays results in enhanced c-jun expression with a maximum response at 1-2 h (10). 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 (20). 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 1.8kilobase chicken c-jun transcripts by quantitative Northern blot analysis. As shown in Fig. 1A, 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 Ϯ S.E. ϭ 2.9 Ϯ 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-jun activation in human lymphoma B-cells. As shown in Fig. 1B, ionizing radiation did not significantly enhance c-jun expression levels in DT-40 cells treated with the PTK inhibitory isoflavone genistein (stimulation index ϭ 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.

Cytoplasmic Protein-tyrosine Kinases BTK, LYN, and SYK Are Not Required for 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 (20). DT-40 cells rendered BTK-deficient by targeted disruption of the btk 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 Ϫ ) 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 Ϯ S.E. ϭ 2.4 Ϯ 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-jun transcript levels. As shown in Fig. 3A, 20-Gy ionizing radiation enhanced c-jun expression in SYK Ϫ 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 Ϯ 0.2 versus 2.9 Ϯ 0.4; p Ͻ 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 (20,33,41). 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-jun expression in DT-40 cells rendered LYN-deficient by targeted gene disruption. LYN-deficient (LYN Ϫ ) cells showed enhanced c-jun expression after irradiation; however, the stimulation indices were lower than those in wild-type DT-40 (Fig. 3B). Since LYN and SYK have been shown to cooperate in the generation of other signals in B-cells (21) 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.

Effects of a JAK-3 Inhibitor on Radiation-induced c-jun Activation in DT-40
Cells-B-cell signal transduction events direct fundamental decisions regarding cell survival during periods of oxidative stress. A better understanding of the dynamic interplay between B-cell signaling pathways is needed to determine how vital decisions are dictated during intracellular oxidation changes. STAT proteins (signal transducers and activators of transcription) are a family of DNA-binding proteins that were identified during a search for interferon-␣-or -␥stimulated gene transcription targets (42)(43)(44). There are presently seven STAT family members. The JAK family of cytoplasmic protein kinases was originally demonstrated to also function in interferon signaling and is now known to participate in a broad range of receptor-activated signal cascades (45). Different ligands and cell activators employ specific JAK and STAT family members (45)(46)(47). The basic model for STAT activation suggests that in unstimulated cells, latent forms of STAT proteins are predominantly localized within the cyto-plasm. Ligand binding induces STAT proteins to associate with intracellular phosphotyrosine residues of transmembrane receptors (45)(46)(47). Once STAT proteins are bound to receptors, receptor-associated JAK kinases phosphorylate the STAT proteins. STAT proteins then dimerize through specific reciprocal SH2-phosphotyrosine interactions and may form complexes with other DNA-binding proteins (45)(46)(47). STAT complexes translocate to the nucleus and interact with DNA response elements to enhance transcription of target genes. Signaling events regulating apoptotic responses have been shown to utilize STAT proteins (48 -51). Notably, a recent study demonstrated JAK activation by tyrosine phosphorylation in cells that are exposed to reactive oxygen intermediates, which in turn leads to tyrosine phosphorylation and activation of STAT-1, STAT-3, and STAT-6 (52).
After establishing that LYN, BTK, and SYK kinases are not required for radiation-induced c-jun activation, we set out to determine if c-jun activation is functionally linked to the JAK-STAT pathway. To this end, we examined the effects of a JAK-3 inhibitory novel quinazoline derivative on c-jun expression levels in irradiated DT-40 cells. To identify a potent JAK-3-specific inhibitor, the effects of two novel quinazoline derivatives on the enzymatic activity of JAK-1, JAK-2, and JAK-3 were examined using Sf21 cells that were infected with baculovirus expression vectors for these kinases, as described previously (37) (Fig. 4). Infected cells were harvested; JAK proteins were immunoprecipitated with appropriate antibodies (anti-JAK-1: HR-785, catalog no. sc-277, affinity-purified rabbit polyclonal IgG, 0.1 mg/ml, Santa Cruz Biotechnology; anti-JAK-2: (C-20)-G, catalog no. sc-294-G, affinity-purified goat polyclonal IgG, 0.2 mg/ml, Santa Cruz Biotechnology; and anti-JAK-3: C-21, catalog no. sc-513, affinity-purified rabbit polyclonal IgG, 0.2 mg/ml, Santa Cruz Biotechnology); and kinase assays were performed following a 1-h exposure of the immunoprecipitated JAK proteins to the quinazoline compounds, as described in detail elsewhere (20,(22)(23)(24). As shown in Fig. 4B, both compounds inhibited JAK-3 (panels B.3 and B.4), but not JAK-1 (panel B.1) or JAK-2 (panel B.2). Electrophoretic mobility shift assays were performed to examine the effects of both compounds on cytokine-induced STAT activation. Specifically, 32Dc11/IL2R␤ cells (a gift from James Ihle, St. Jude Children's Research Hospital Memphis, TN) were exposed at 8 ϫ 10 6 /ml in RPMI 1640 medium supplemented with fetal bovine serum to compound 1 or compound 2 at a final concentration of 10 g/ml in 1% Me 2 SO for 1 h and subsequently stimulated with IL-2 or IL-3 as indicated. Cells were collected after 15 min and resus-pended in lysis buffer (100 mM Tris-HCl, pH 8.0, 0.5% Nonidet P-40, 10% glycerol, 100 mM EDTA, 0.1 mM NaVO 3 , 50 mM NaF, 150 mM NaCl, 1 mM dithiothreitol, 3 g/ml aprotinin, 2 g/ml pepstatin A, 1 g/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride). Lysates were precleared by centrifugation for 30 min. Cell extracts (ϳ10 g) were incubated with 2 g of poly(dI-dC) for 30 min, followed by a 30-min incubation with 1 ng of polynucleotide kinase 32 P-labeled double-stranded DNA oligonucleotide representing the IRF-1 STAT DNA-binding sequence (Santa Cruz Biotechnology). Samples were resolved by nondenaturing polyacrylamide gel electrophoresis and visualized by autoradiography. As shown in Fig. 4C, both compounds inhibited the JAK-3-dependent STAT activation after stimulation with IL-2, but they did not affect the JAK-1/JAK-2-dependent STAT activation after stimulation with IL-3. Compound 2 was selected for further experiments designed to examine the effects of JAK-3 inhibition on radiation-induced c-jun activation.
homologue of JAK-3 may be the PTK responsible for radiationinduced c-jun activation. Since JAK-3 knockout mice are B-celldeficient (31,32), conclusive evidence for the role of JAK-3 in radiation-induced c-jun activation must await the development of JAK-3-deficient DT-40 clones.
JAK-3 maps to human chromosome 19p12-13.1 (53)(54)(55)(56). A cluster of genes encoding proto-oncogenes and transcription factors is also located near this region. JAK-3 expression has been demonstrated in mature B-cells as well as B-cell precursors (57,58). JAK-3 has also been detected in leukemic B-cell precursors and lymphoma B-cells (57,59). The physiological roles for JAK-3 have been borne out through targeted gene disruption studies in mice, the genetic analysis of patients with severe combined immunodeficiency, and biochemical studies of JAK-3 in cell lines (45)(46)(47). A wide range of stimuli result in JAK-3 activation in B-cells, including interleukin-7 and interleukin-4 (58,60,61). The B-cell marker CD40 constitutively associates with JAK-3, and ligation of CD40 results in JAK-3 activation, which has been shown to be mandatory for CD40mediated gene expression (62). JAK-3 constitutive activity has been observed in v-abl-transformed pre-B-cells, and coimmunoprecipitations have shown that v-abl physically associates with JAK-3, implicating JAK-3 in v-abl-induced cellular transformation (63).
Cross-talk between the JAK-STAT pathway and other signaling cascades may take place though JAK-3. The insulin receptor substrate proteins IRS1 and IRS2 have been shown to be tyrosine-phosphorylated by JAK-3 in response to IL-2, IL-4, IL-7, and IL-15 (64 -66). The IRS proteins are then able to associate with the p85 regulatory subunit of phosphatidylinositol 3-kinase, which participates in multiple signaling pathways. Another possible mechanism for cross-talk is through JAK-3 interactions with Sam68, which is associated with multiple regulatory proteins including Src family PTK, Cbl, SHP1, phospholipase C␥, and p85 (67,68).
In summary, this study confirms and extends studies that demonstrated that ionizing radiation induces c-jun expression, and this signal is triggered by activation of PTK. Our findings demonstrate that BTK, SYK, and LYN are not the primary PTKs responsible for the initiation of this signal. After ruling out the requirement for these PTKs in c-jun induction, we sought an alternative mechanism for this effect of ionizing radiation using a compound with specific inhibitory effects on JAK-3. Our observation that this inhibitor is capable of abrogating the c-jun signal implicates JAK proteins as important regulators of radiation-induced c-jun activation. Future studies should explore the cross-talk between JNK-c-jun and JAK-STAT signal transduction pathways in irradiated B-lineage lymphoid cells.