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Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute [J. F. D., R. J., J. W.] and the Departments of Medical Microbiology and Immunology [J. F. D., J. W.], Biochemistry and Molecular Biology [R. J., J. W.], University of South Florida College of Medicine, Tampa, Florida 33612; Department of Cancer Research, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105 [A. J. K.]
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ABSTRACT |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Gab2 is a multisite docking protein recently cloned from
p210Bcr-Abl-transformed hematopoietic cells
(10)
. Gab2 is constitutively tyrosine phosphorylated and
associated with the protein tyrosine phosphatase SHP2 in
p210Bcr-Abl-transformed hematopoietic cells such
as human CML K562 cells. We found that a novel
pyrido[2,3-d]pyrimidine derivative (11)
,
PD180970 (see structure in Fig. 2A
), potently
inhibits Gab2 tyrosine phosphorylation in K562 cells. Further studies
showed that PD180970 is an inhibitor of the
p210Bcr-Abl tyrosine kinase and induces apoptosis
of K562 cells, whereas it has no detectable effect on Ph-negative HL60
human leukemic cells. These findings suggest that PD180970 is a
novel Abl tyrosine kinase inhibitor that can selectively affect
Bcr-Abl-positive leukemic cells.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Preparation of Anti-Gab2 Antibody.
A peptide (CEWTDVRQSSEPSKGAKL) containing the COOH-terminal
region of Gab2 (10)
was synthesized, conjugated with
keyhole limpet hemocyanin, and used to generate antisera against
Gab2 in rabbits. The antibody was affinity-purified using the antigen
peptide. Horseradish peroxidase-conjugated anti-Gab2 antibody was
prepared from the affinity-purified antibody using the EZ-Link Plus
Activated Peroxidase kit (Pierce).
Immunoprecipitation and Immunoblotting.
Cells (2 x 106
cells/sample) were collected by centrifugation (1000 x
g for 3 min at 4°C), and lysed in buffer A [25
mM Tris-HCl (pH 7.2), 150
mM NaCl, 25 mM NaF, 1
mM benzamidine, 1% Triton X-100, 1
mM
Na3VO4, 20
mM p-nitrophenyl phosphate, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl
fluoride]. Immunoprecipitations and the subsequent immunoblotting were
performed essentially as described (13)
using the
antibodies indicated in the figure legends.
In Vitro Immune-Complex Kinase Assay.
p210Bcr-Abl (2 x
106 cells/sample) was immunoprecipitated from
K562 cells using a monoclonal anti-Abl antibody. The immunoprecipitates
were washed three times with buffer A and once with buffer B [20
mM Tris-HCl (pH 7.2), 20 mM NaCl]. The
autophosphorylation reaction was carried out by incubating the
p210Bcr-Abl immune complex in 30 µl of reaction
buffer {10 mM Tris-HCl (pH 7.2), 2 mM
p-nitrophenyl phosphate, 4 mM
MgCl2, 2 mM
MnCl2, 10 µM ATP
containing 10 µCi of [
-32P]ATP}
containing the indicated concentrations of PD180970 for 15 min at
30°C. The reaction was stopped by addition of SDS-gel loading buffer
and heated at 95°C for 10 min. Proteins were resolved on 8%
SDS-polyacrylamide gels. Autophosphorylation of
p210Bcr-Abl was analyzed with a PhosphorImager
and also by autoradiography.
In Vitro Abl and Src Kinase Assays.
The activity of a purified bacterially expressed Abl
protein tyrosine kinase (Calbiochem) was assayed using a synthetic
peptide (EAIYAAPFAKKK) as substrate. The reaction was carried out at
30°C for 10 min in 40 µl of reaction mixture {50 mM
Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1
mM EDTA, 1 mM DTT, 0.015% Brij 35, 0.1 mg/ml
BSA, 10 µM ATP, 5 µCi of
[-32P]ATP (3000 Ci/mmol), 100
µM peptide substrate, and 2 ng of Abl kinase}
containing 1–100 nM PD180970 or DMSO (vehicle). The
reaction was stopped by spotting 35 µl of the reaction mixture onto
p81 phosphocellulose filter (2.5 cm2 and
immediately immersed in 2% phosphoric acid. The filters were washed
five times with 2% phosphoric acid, and radioactivity retained on the
filters was measured by liquid scintillation counting.
Src kinase activity was determined using a recombinant c-Src produced in Sf9 insect cells and a Src substrate peptide (KVEKIGEGTYGVVYK). The reaction was performed as above except that 6 units of c-Src and 150 µM Src substrate peptide were used in each reaction and the reaction was carried out at 30°C for 20 min.
Cell Viability, Nuclear Staining, PARP Cleavage, and Annexin V-PI
Binding Assays.
For determination of cell growth and viability, cells
(1.5 x 105) in triplicate were
incubated in 2 ml of RPMI 1640–10% FCS containing tyrosine kinase
inhibitors or DMSO (solvent for the inhibitors). The volume of the DMSO
was kept at 0.1% of the medium volume. At the indicated times, cells
were collected by centrifugation (1000 x g
for 3 min). Cell viability was determined by the trypan blue exclusion
assay (14)
. At least 200 cells were examined in each
sample.
For nuclear staining, samples (2 x 104 cells/each) were collected onto microscope glass slides by cytospin. The cells were mounted with mounting medium containing DAPI (Vector Laboratories) and examined with a fluorescence microscope.
To analyze PARP cleavage (6) , cells (1.5 x 106) were treated with PD180970 (0.5 µM) or DMSO for the indicated time. Cells were collected, washed once with cold PBS, and lysed in buffer C [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride] for 30 min at 4°C. Cell lysate supernatants (40 µg protein/each) were resolved on 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and analyzed by immunoblotting with an anti-PARP antibody.
Evaluation of apoptosis by the annexin V-PI binding assay (7) was performed using the Annexin V-FITC Apoptosis Detection Kit (PharMingen) according to the supplier’s instructions. At least 1 x 104 cells in each sample were analyzed. Control cells stained with annexin V-FITC or PI alone were used to compensate for the flow cytometric analysis. Annexin V and PI double-negative cells are defined as live cells; annexin V-positive, PI-negative cells are defined as early apoptotic cells; and annexin V and PI double-positive cells are defined as late apoptotic and necrotic cells.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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) and in other p210Bcr-Abl-transformed
hematopoietic cells (10)
, suggesting that Gab2 is involved
in p210Bcr-Abl-initiated cell signaling.
Incubation of K562 cells with PD180970 inhibited Gab2 tyrosine
phosphorylation with an IC50 of 80
nM (Fig. 1, A and C
).
PD180970 treatment also resulted in dissociation of the Gab2-SHP2
complex in K562 cells (data not shown). PD180970 was originally
identified as a Src tyrosine kinase inhibitor (11)
. To
assess the contribution of the Src tyrosine kinase inhibitor activity
of PD180970 on inhibition of Gab2 tyrosine phosphorylation, we tested
the effects of two Src family tyrosine kinase-specific inhibitors, PP1
and PP2 (12)
, on Gab2 tyrosine phosphorylation in K562
cells. In contrast to PD180970, PP1 (10 µM) and
PP2 (10 µM) had no apparent effects on Gab2
tyrosine phosphorylation and Gab2-SHP2 association in K562 cells (not
shown). These observations suggest that the inhibition of Gab2 tyrosine
phosphorylation by PD180970 in K562 cells is unlikely to be due to
inhibition of Src family tyrosine kinases.
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, shows that PD180970 inhibited tyrosine phosphorylation of
p210Bcr-Abl in a concentration-dependent manner
with an IC50 of 170 nM. The
bottom panel of Fig. 1B
shows that equal amounts
of p210Bcr-Abl protein were present in each
immunoprecipitate.
CrkL is a well-characterized
p210Bcr-Abl substrate (15
, 16)
and
is constitutively tyrosine-phosphorylated in K562 cells (Fig. 1D
). To confirm the inhibitory effect of PD180970 on
p210Bcr-Abl tyrosine kinase activity in K562
cells, CrkL was immunoprecipitated from K562 cells that were treated
for 12 h with various concentrations of PD180970 and analyzed by
immunoblotting with RC20H. As shown in Fig. 1, A and D
, PD180970 potently inhibited CrkL tyrosine phosphorylation
in K562 cells (IC50 = 80
nM). On the other hand, incubation of K562 cells
with PP1 and PP2 (10 µM for 12 h) did not
result in any change in CrkL tyrosine phosphorylation (data not shown).
Together, these data indicate that PD180970 can inhibit
p210Bcr-Abl tyrosine kinase activity in K562
cells.
PD180970 Inhibits p210Bcr-Abl Autophosphorylation and
Recombinant Abl Tyrosine Kinase Activity in Vitro.
To assess whether PD180970 can directly affect
p210Bcr-Abl tyrosine kinase activity,
p210Bcr-Abl was immunoprecipitated from K562
cells, and in vitro autophosphorylation of
p210Bcr-Abl was performed in the presence or
absence of PD180970. As shown in Fig. 2B
, PD180970 potently inhibited autophosphorylation of
p210Bcr-Abl in vitro. The
IC50 obtained from three independent experiments
for inhibition of p210Bcr-Abl autophosphorylation
by PD180970 was 5 nM.
To further confirm that PD180970 can directly inhibit Abl
tyrosine kinase activity, we determined the effect of PD180970 on the
kinase activity of purified recombinant Abl tyrosine kinase
(17)
. This Escherichia coli-expressed
recombinant protein is a truncated form of v-Abl (18)
that
contains an Abl tyrosine kinase domain identical to that of c-Abl. As
illustrated in Fig. 2C
, PD180970 inhibited the tyrosine
kinase activity of the recombinant Abl protein, with an
IC50 of 2.2 nM. A similar
IC50 value was obtained when the kinase assay was
performed with a 10-fold lower concentration of Abl protein (not
shown). These results demonstrate that PD180970 can directly and
potently inhibit Abl tyrosine kinase activity. Fig. 2C
also
shows that PD180970 inhibited c-Src tyrosine kinase activity with an
IC50 of 0.8 nM, confirming
that PD180970 is a potent inhibitor of c-Src tyrosine kinase
(11)
.
PD180970 Causes Apoptotic Cell Death of K562 Cells.
We next determined the effects of PD180970 on cell growth and
viability of K562 cells. The Bcr-Abl-negative human HL60 leukemic cell
line was used as a control. K562 cells and HL60 cells were incubated
with PD180970 (0.5 µM) or DMSO (solvent) for 1–4 days,
and cell numbers were counted each day after trypan blue staining. Fig. 3A
shows that the viable cell number of PD180970-treated K562
cells was below the initial plating cell number after day 1. Fig. 3B
shows that this was due to cell death. In striking
contrast to the effects on K562 cells, PD180970 had no apparent effects
on cell proliferation and viability of HL60 cells (Fig. 3
).
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, PP1 and PP2 (10 µM) had moderate
effects on K562 cell viability: 79–80%, 65–72%, and 38–55% of
K562 cells treated with PP1 and PP2 were still viable on days 2, 3, and
4, respectively. In comparison, only 34 ± 2%,
15 ± 2%, and 6 ± 3% of cells treated
with PD180970 (0.5 µM) remained viable on days
2, 3, and 4, respectively (Fig. 3B
). Thus, there were
considerable differences in the viability of K562 cells treated with
PD180970 and with the Src kinases inhibitor PP1 or PP2.
To determine whether PD180970 causes apoptosis of K562 cells, we
first examined the nuclei of K562 cells by fluorescence microscopy
after DAPI staining. As illustrated in Fig. 4A
, many PD180970-treated K562 cells had condensed and
fragmented nuclei, which is characteristic of apoptotic cells. We next
analyzed the PARP protein in K562 cells. It has been demonstrated
previously that the 116-kDa PARP is specifically cleaved into 85- and
25-kDa fragments by caspase-3 in cells, including K562 cells, that are
undergoing apoptosis (6)
. Fig. 4B
shows that
little 85-kDa PARP was present in mock (DMSO)-treated K562 cells,
whereas PD180970 treatment resulted in the apoptotic-specific cleavage
of PARP.
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, top panels). Incubation of K562
cells with PD180970 (0.5 µM) for 24 and 48
h increased annexin V-positive/PI-negative cells to 15.1
± 3.1% and 34.5 ± 7.4%, respectively (Fig. 4C
, bottom panels). A considerable increase in
annexin V-PI double-positive cells (22.5 ± 2.9%) was
also detected in K562 cells treated with PD180970 for 48 h.
Parallel experiments showed no change in annexin V-PI binding of HL60
cells treated with PD180970 (data not shown). These results demonstrate
that PD180970 induced apoptosis of K562 cells. |
Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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PD180970 inhibits p210Bcr-Abl autophosphorylation in vitro with an IC50 of 5 nM. In cellular tyrosine phosphorylation assays, PD180970 inhibits tyrosine phosphorylation of p210Bcr-Abl, with an IC50 of 170 nM, whereas it inhibits tyrosine phosphorylation of p210Bcr-Abl substrates Gab2 and CrkL, with an IC50 of 80 nM. The higher concentration required for inhibition of tyrosine kinase autophosphorylation than for inhibition of phosphorylation of exogenous substrates is a general feature of competitive inhibitors of protein tyrosine kinases (19) . In particular, this has been observed previously with the p210Bcr-Abl tyrosine kinase inhibitor CGP57148 (7) .
PD180970 was originally identified as a Src tyrosine kinase
inhibitor (11)
. Although the Src kinase inhibitory
activity of PD180970 may contribute to the effects of PD180970 on K562
cells to some degree, several lines of evidence suggest that the
effects of PD180970 on K562 cells is largely due to inhibition of the
p210Bcr-Abl tyrosine kinase. (a)
PD180970 inhibited (IC50 = 2.2
nM) recombinant Abl tyrosine kinase purified from
E. coli (Fig. 2C
). This result unequivocally
demonstrates that PD180970 is a potent inhibitor of the Abl tyrosine
kinase. (b) The Src tyrosine kinase inhibitors PP1 and PP2
(12)
had no apparent effect on tyrosine phosphorylation of
Gab2 and CrkL in K562 cells. (c) The effects of PP1 and PP2
on K562 cell viability were relatively small compared with that of
PD180970 (Fig. 3B
). The difference between the extent of
K562 cell death caused by P180970 and by PP1/PP2 is likely due to
inhibition of p210Bcr-Abl by PD180970 (Fig. 3, B and D
). This view is further supported by our
observations in 32Dcl3 murine myeloid cells. Src tyrosine kinase is
known to be involved in IL-3-stimulated 32Dcl3 proliferation
(20)
. We found that PD180970 (0.5
µM), PP1 (10 µM), and
PP2 (10 µM) cause a similar 50%
inhibition of 32Dcl3 cell proliferation, whereas all three compounds
have little effect on the IL-3 dependent cell viability (data not
shown). These results suggest that PD180970, PP1, and PP2 have similar
effects on Src-like kinases at the concentrations used in our studies.
It has been suggested that p210Bcr-Abl renders myeloid cells resistant to apoptosis (6) . Our finding that PD180970 causes apoptosis of K562 cells is consistent with this notion. Apoptosis of K562 cells and other Bcr-Abl positive cells induced by the Abl kinase inhibitor CGP57148 has also been reported (7) . PD180970 had no apparent effect on cell proliferation and viability of Bcr-Abl-negative HL60 leukemic cells. In the normal 32Dcl3 myeloid cells, PD180970 partially inhibited IL-3-dependent cell proliferation but had little effect on cell viability (data not shown). These findings are very important in terms of the development of novel CML therapeutic agents. Studies of the Abl tyrosine kinase inhibitor CGP57148 also demonstrate that inhibition of Abl tyrosine kinase activity causes cell death only in Bcr-Abl-positive leukemic cells (3 , 7) . Taken together, our results demonstrate that PD180970 is among the most potent p210Bcr-Abl tyrosine kinase inhibitors identified and represents another potential therapeutic agent for CML.
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Acknowledgments |
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FOOTNOTES |
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1 Supported in part by NIH Grants CA77467 (to
J. W.) and CA55652 (to R. J.). 
2 To whom requests for reprints should be
addressed, at Molecular Oncology Program, MRC3-East, H. Lee Moffitt
Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL
33612. Phone: (813) 979-6713; Fax: (813) 903-6817; E-mail: wu{at}moffitt.usf.edu
3 The abbreviations used are: Ph, Philadelphia
chromosome; CML, chronic myelogenous leukemia; Gab2,
Grb2-associated binder-2; PARP, poly(ADP-ribose) polymerase; PI,
propidium iodide; IL, interleukin.
Received 2/15/00. Accepted 5/ 1/00.
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 E. Joseloff, C. Cataisson, H. Aamodt, H. Ocheni, P. Blumberg, A. J. Kraker, and S. H. Yuspa Src Family Kinases Phosphorylate Protein Kinase C delta on Tyrosine Residues and Modify the Neoplastic Phenotype of Skin Keratinocytes J. Biol. Chem., March 29, 2002; 277(14): 12318 - 12323. [Abstract] [Full Text] [PDF]
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J. F. Dorsey, J. M. Cunnick, S. M. Mane, and J. Wu Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl+ K562 leukemic cells by Gab2 Blood, February 15, 2002; 99(4): 1388 - 1397. [Abstract] [Full Text] [PDF]
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B. M. F. Mow, J. Chandra, P. A. Svingen, C. G. Hallgren, E. Weisberg, T. J. Kottke, V. L. Narayanan, M. R. Litzow, J. D. Griffin, E. A. Sausville, et al. Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro Blood, January 15, 2002; 99(2): 664 - 671. [Abstract] [Full Text] [PDF]
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B. J. Druker, S. G. O'Brien, J. Cortes, and J. Radich Chronic Myelogenous Leukemia Hematology, January 1, 2002; 2002(1): 111 - 135. [Abstract] [Full Text]
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) and c-Src (
) proteins were
assayed by phosphorylation of peptide substrates in the presence of
various concentrations of PD180970. The results represent the average
of two experiments performed in duplicate; bars, SD. The
radioactivity in the control (100%) samples was 1,231,575
± 346,023 cpm for the v-Abl kinase assay and 322,232
± 92,185 cpm for the c-Src kinase assay.
) phosphotyrosine (PTyr;
B–D, top panels) or with antibodies
against Abl, Gab2, or CrkL (B–D, bottom
panels) to confirm equal amounts of protein in each
immunoprecipitate. Arrows indicate location of the
indicated proteins. A, average levels of tyrosine
phosphorylation of each protein from two independent experiments;
bars, SD.
