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.
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INTRODUCTION |
The proto-oncogene c-jun is the cellular counterpart of
the v-jun oncogene of avian sarcoma virus 17 (1-5).
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 (11-13). 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 induction 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.
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MATERIALS AND METHODS |
Cell Lines--
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 (20, 21, 33, 34). 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.).
Use of PTK Inhibitors--
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.
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) (35). Compound 2 was
reduced with sodium borohydrate and catalyzed by copper sulfate (36) 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 (35).
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 (5, 6) in 10 ml of SOCl2
was 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 from
N,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 from
N,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 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 Et3N
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. 1H NMR spectra were recorded
using a VAV-300 (Department of Chemistry, University of Minnesota) or
Varian-300 spectrometer and in
Me2SO-d6 or CDCl3
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 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)
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)
max (
) 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/z
298 (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)
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)
max (
) 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/z
378 (M+ + 2, 90.68), 377 (M+ + 1, 37.49), 376 (M+, 100.00), 360 (M+, 3.63), 298 (18.86), 282 (6.65).
Irradiation of Cells--
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 (24, 38). In some experiments,
cells were preincubated with PTK inhibitors for 24 h prior to
irradiation.
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 (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 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: 5'-AGAGGTGCTGCCCAGAACATCATC-3' and
5'-GTGGGGAGACAGAAGGGAACAGA-3'. A 413-base pair chicken
-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.
RNA Isolation and Northern Blot Hybridization
Analysis--
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 (39). 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 0.015 M sodium citrate) by downward capillary transfer. The
c-jun fragment was radiolabeled by random priming with
[
-32P]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 0.1× 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
-actin probe to normalize for loading
differences.
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RESULTS AND DISCUSSION |
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 1.8-kilobase 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 ± S.E. = 2.9 ± 0.4).

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Fig. 1.
Radiation-induced c-jun
activation in wild-type DT-40 lymphoma B-cells.
A, dose response for
induction of c-jun
mRNA. DT-40 chicken cells were irradiated at the indicated doses
(0, 10, 15, and 20 Gy). Total RNA was extracted after a 2- or 4-h
post-irradiation time period. Twenty micrograms of RNA were loaded on a
Northern gel and transferred by capillary blotting to a nylon membrane.
The Northern blot was hybridized with a 32P-labeled chicken
c-jun probe (upper panel) or a chicken
GAPDH probe (lower panel). The inset
shows the values for the c-jun/GAPDH transcript expression
ratios as determined with the Bio-Rad storage phosphor imager and
corresponding stimulation index (SI) values. B,
effect of the PTK inhibitor
genistein on induction of
c-jun mRNA. Cells were treated with 30 µg/ml genistein for 24 h at 37 °C prior to exposure to 20-Gy
ionizing radiation. c-jun expression levels were determined
as described for A. Kb, kilobases.
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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 = 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
) 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.

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Fig. 2.
Radiation-induced activation of c-jun
in BTK DT-40 cells. Shown are two
representative experiments (A and B) illustrating
the induction of c-jun mRNA expression by ionizing
radiation in wild-type (WT) and BTK DT-40
cells. Poly(A)+ RNA was isolated from non-irradiated cells
as well as irradiated cells (20 Gy, with a 2-h post-radiation recovery
period). Northern blots of 2 µg of poly(A)+ were
hybridized with the c-jun probe (upper panel),
-actin probe (middle panel in A only), and
GAPDH probe (lower panel). The inset
below each panel shows the relative expression of c-jun
normalized for RNA load (c-jun/GAPDH ratio) and the
stimulation index (SI; -fold induction over non-irradiated
controls). Kb, kilobases.
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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
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 ± 0.2 versus 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.

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Fig. 3.
Induction of c-jun mRNA
expression by ionizing radiation in wild-type and mutant DT-40 cell
lines. DT-40, BTK DT-40, and SYK DT-40
cells (shown in A) as well as LYN DT-40 and
LYN SYK DT-40 cells (shown in B)
were irradiated with 20 Gy, and poly(A)+ RNA (in
A) or total RNA (in B) was harvested after a 2-h
recovery period. RNA from non-irradiated cells was used as a control.
Northern blots containing 2 µg of poly(A)+ (in
A) or 20 µg of total RNA (in B) from each cell
line were hybridized with both a 32P-labeled
c-jun probe (upper panel) and a GAPDH
probe (lower panel). The insets below
the panels show the relative expression of c-jun normalized
for RNA loading (c-jun/GAPDH ratios) as well as the
stimulation index (SI; -fold induction over non-irradiated
controls). WT, wild type; Kb, kilobases.
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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
) 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), we
examined the ability of ionizing radiation to induce c-jun expression in LYN
SYK
DT-40 cells, generated
by targeted disruption of the syk gene in
LYN
-deficient DT-40 cells. As shown in Fig.
3B, 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-jun
activation 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.
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 (42-44). 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
(45-47). The basic model for STAT activation suggests that in
unstimulated cells, latent forms of STAT proteins are predominantly
localized within the cytoplasm. Ligand binding induces STAT proteins to
associate with intracellular phosphotyrosine residues of transmembrane
receptors (45-47). 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
(45-47). 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, 22-24). 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 × 106/ml in RPMI 1640 medium supplemented with fetal bovine
serum to compound 1 or compound 2 at a final concentration of 10 µg/ml in 1% Me2SO for 1 h and subsequently
stimulated with IL-2 or IL-3 as indicated. Cells were collected after
15 min and resuspended in lysis buffer (100 mM Tris-HCl, pH
8.0, 0.5% Nonidet P-40, 10% glycerol, 100 mM EDTA, 0.1 mM NaVO3, 50 mM NaF, 150 mM NaCl, 1 mM dithiothreitol, 3 µg/ml
aprotinin, 2 µg/ml pepstatin A, 1 µg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride). Lysates were precleared
by centrifugation for 30 min. Cell extracts (~10 µg) were incubated
with 2 µg of poly(dI-dC) for 30 min, followed by a 30-min incubation
with 1 ng of polynucleotide kinase 32P-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.

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Fig. 4.
JAK-3 inhibitors. A,
structures of JAK-3 inhibitors. B, specificity of JAK-3
inhibitors. Sf21 cells infected with baculovirus expression
vectors for JAK-1, JAK-2, or JAK-3 were subjected to
immunoprecipitation with anti-JAK antibodies. JAK-1 (panel
B.1), JAK-2 (panel B.2), and JAK-3
(panels B.3 and B.4, which illustrate
results from two independent experiments) immune complexes were treated
with 1% Me2SO (vehicle control (CON)), compound
1, or compound 2 for 1 h prior to hot kinase assays, as described
(20, 22). Both compounds inhibited JAK-3 when used at 10 µg/ml,
whereas they did not inhibit JAK-1 or JAK-2 even at 75 µg/ml.
C, electrophoretic mobility shift assays of 32Dc22/IL2R
cells. Compound 1(100 µg/ml) and Compound 2 (100 µg/ml) inhibited
IL-2-triggered JAK-3-dependent STAT activation, but not
IL-3-triggered JAK-1/JAK-2-dependent STAT activation in
32Dc11/IL2R cells.
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As shown in Fig. 5, ionizing
radiation failed to induce c-jun expression in DT-40 cells
treated with the JAK-3 inhibitor. These findings provide circumstantial
evidence that a chicken homologue of JAK-3 may be the PTK responsible
for radiation-induced c-jun activation. Since JAK-3 knockout
mice are B-cell-deficient (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.

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Fig. 5.
Effects of a JAK-3 inhibitor on c-jun
induction in irradiated DT-40 cells. Cells were treated with
the quinazoline derivative
4-(3'-bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (100 µg/ml) for 24 h at 37 °C prior to exposure to 20-Gy ionizing
radiation. c-jun expression levels were determined as
outlined in the legend to Figs. 1-3. SI, stimulation index;
Kb, kilobases.
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JAK-3 maps to human chromosome 19p12-13.1 (53-56). 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 (45-47). 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 CD40-mediated 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.
We thank Shanna Karas for cell culture
assistance and David Pond and Nicole Janosek for technical assistance,
Bruce Witthuhn for the JAK expression vectors and EMSAs, and Sandeep
Mahajan for kinase assays. We also thank Drs. Alice Chu and Julie
Gelderloos for helpful discussions and a critical review of the
manuscript.