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NEOPLASIA
From the Department of Adult Oncology, Dana Farber
Cancer Institute; the Department of Medicine/Division of Hematology,
Brigham and Women's Hospital; and the Department of Medicine, Harvard
Medical School, Boston, MA.
The tyrosine kinase inhibitor STI571 inhibits BCR/ABL and induces
hematologic remission in most patients with chronic myeloid leukemia.
In addition to BCR/ABL, STI571 also inhibits v-Abl, TEL/ABL, the native platelet-derived growth factor (PDGF) Small molecule drugs that can selectively inhibit
tyrosine kinases are likely to be of benefit in a number of neoplastic
diseases. Although tyrosine kinase inhibitors have been studied for
many years, they often have had little specificity and thus were
unlikely to be suitable for clinical applications. Recently, more
selective tyrosine kinase inhibitors have been developed, including
STI571 (formerly CGP57148B; Novartis Pharmaceuticals, Basel,
Switzerland).1,2 STI571 has been shown to inhibit c-ABL,
ABL oncogenes, c-KIT, and the platelet-derived growth factor (PDGF) ARG is an ABL-related
gene with an overall structure (SH3-SH2-kinase domain)
similar to that of ABL.5 ARG is also highly related to ABL
at an amino acid sequence level in the SH3, SH2, and kinase domains
(89%, 90%, and 93% identity, respectively), though they are less
than 30% identical in the C-terminus.6 Like ABL, ARG is a
widely expressed tyrosine kinase typically detected on Western blot as
a series of bands at 135 to 150 kd.7 The function of ARG
is not well understood, but it is important for the proper development
of the nervous system and for brain function in the adult
mouse.8
The adenosine triphosphate (ATP) binding site of ARG is similar to the
ATP binding site of ABL (Figure 1),
suggesting the possibility that STI571 could inhibit ARG kinase
activity. However, the structural features that predict for sensitivity
to STI571 have not yet been defined, and there are a number of amino
acid differences between ABL and ARG in the kinase domain. To determine whether STI571 inhibits ARG, an assay for ARG kinase activity was
devised. There are no signaling pathways currently known to activate
ARG, and no ARG substrates have been described in intact cells.
Recently, the t(1;12)(q25, p13) chromosomal translocation in patients
with acute leukemia has been reported to result in fusion of the
TEL and ARG genes.9,10 TEL fusion
with other tyrosine kinases occurs in acute and chronic leukemias,
including TEL/PDGF
Cell lines and cell culture
Cell viability determination
Annexin-propidium iodide staining Cell viability and apoptosis in STI571-treated cells were assessed using the Annexin-V-Fluos Staining Kit (Boehringer Mannheim, Indianapolis, IN). Briefly, 1 × 106 cells cultured in the presence or absence of STI571 were washed once with 1× phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 5 minutes. Cell pellets were resuspended in 100 µL 20% Annexin-V-fluorescein labeling reagent and 20% propidium iodide (PI) in Hepes buffer. Cells were incubated for 15 minutes at room temperature, diluted in 0.8 mL Hepes buffer, and analyzed by flow cytometry. In addition, as controls, cells were stained with Annexin-V-fluorescein labeling reagent alone or PI alone or were left unstained.Antibodies Anti-pTyr monoclonal antibody (mAb) #4G10 was provided by Dr Brian Druker (University of Oregon Health Sciences Center, Portland) and was diluted 1:2500 for immunoblot. Monoclonal anti-actin (clone AC-15; Sigma) was diluted 1:1000 for immunoblot. Rabbit polyclonal anti-ARG, directed against the SH2 and SH3 domains of ARG, was a gift from Dr Anthony Koleske (Yale University, New Haven, CT) and diluted 1:1000 for immunoblot. Anti-ARG, goat polyclonal antibody (pAb) clone c-20, directed against the carboxy terminus of ARG, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used for immunoprecipitation. Anti-ABL (clone 3F12), which is directed against the SH2 domain of c-ABL, was developed by Dr Ravi Salgia and Dr James Griffin (Dana Farber Cancer Institute, Boston, MA) and was used at 1:500 for immunoblotting. Anti-PAC1, goat pAb clone c-20, targeted against a peptide mapping at the carboxy terminus of protein tyrosine phosphatase PAC1 of human origin, was obtained from Santa Cruz Biotechnology and used for immunoprecipitation.Immunoblotting and immunoprecipitation Cells were lysed in lysis buffer containing 0.02 M Tris, pH 8.0, 0.15 M NaCl, 10% glycerol, 1% NP-40 (wt/vol), 0.1 M NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 40 µg/mL leupeptin, and 20 µg/mL aprotinin. Cell lysates were incubated on ice for 25 minutes, with vortexing every 5 minutes, and then centrifuged at 12 000g for 15 minutes. Protein yields were determined in the supernatants by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were then loaded onto gel lanes or used for immunoprecipitation experiments. For immunoprecipitation, cell lysates were incubated with anti-ARG antibody (clone C-20) or anti-PAC-1 antibody (clone C-20) and protein G Sepharose overnight at 4°C. As a control, cell lysates were also incubated with protein G Sepharose beads alone. After incubation, immune complexes were washed twice with lysis buffer, twice with 1× PBS, and were dissolved in Laemmeli's sample buffer by boiling for 5 minutes. For immunoprecipitation and immunoblotting, immune complexes and whole cell lysates, respectively, were resolved on a sodium dodecyl sulfate (SDS)-7.5% polyacrylamide gel. Protein was then electrophoretically transferred to a Protran nitrocellulose transfer and immobilization membrane (Schleicher and Schuell, Dassel, Germany). The membrane was blocked for either 1 hour at 25°C or overnight at 4°C with 5% nonfat dry milk in 1× TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) and then probed for 2 hours at 25°C or overnight at 4°C with antibody in 1× TBST buffer (10 mM Tris-HCl, pH 8.0,150 mM NaCl, 0.05% Tween 20). After 3 washes with 1× TBST, membranes were incubated for 1 hour at 25°C with anti-mouse immunoglobulin, horseradish peroxidase-linked whole antibody (from sheep) (Amersham Life Science, Arlington Heights, IL) or anti-rabbit immunoglobulin, and horseradish peroxidase-linked whole antibody (from donkey) (Amersham Life Science). The membrane was washed 5× for 5 minutes/wash in 1× TBST buffer, and bound antibodies were detected with enhanced luminol and oxidizing reagent as specified by the manufacturer (NEN Life Science Products, Boston, MA). Filters were stripped with stripping buffer (2% SDS, 0.0625 M Tris, pH 6.8, and 0.7% 2-mercaptoethanol) for 30 minutes at 50°C before they were probed with additional antibodies.
Comparison of the amino acid sequence around the ATP binding site
of ABL, ARG, KIT, PDGF  R, and TEL/JAK2 is presented in Figure 1A. Amino acid
sequences of human ABL, ARG, KIT, PDGFR, and JAK2 were obtained from
GenBank (gi:2 144 425, 6 382 062, 125 472, 66 816, and
7 446 414, respectively). Approximately 135 amino acids from c-ABL
were aligned with corresponding sequences from the other kinases using
the ClustalW Program (http://www2.ebi.ac.uk/clustalw/) (Figure 1B),
starting 10 amino acids N-terminal of the consensus ATP binding site
(G-X-G-X-F/Y-G-X-V-X). KIT and PDGFR have inserts of approximately
100 amino acids that are lacking in ABL, ARG, or JAK2. ABL, KIT, and
PDGFR are known to be inhibited by STI571 at submicromolar
concentrations in vivo, whereas JAK2 is more than 100-fold less
sensitive. Over this span, there were 9 positions in which the amino
acids matched between ABL, ARG, KIT, and PDGFR, but not in JAK2 (ABL
positions R239, K274, E281, L301, L302, T306, T315, N358, and A366).
Expression of TEL/ARG increases tyrosine phosphorylation of cellular proteins in Ba/F3 cells Cell lines expressing TEL/ARG, TEL/ABL, or BCR/ABL were generated as described in "Materials and methods," and the overall tyrosine phosphorylation of cellular proteins was evaluated by immunoblotting with an anti-phosphotyrosine monoclonal antibody (Figure 2). Compared to Ba/F3 cells, each of the TEL-kinase fusion protein-transfected cell lines showed elevated tyrosine phosphorylation of multiple cellular proteins.
Inhibition of TEL/ARG autophosphorylation by STI571 Because TEL/ARG increased cellular tyrosine phosphorylation and most tyrosine kinase oncogenes are autophosphorylated, tyrosine phosphorylation of TEL/ARG was examined. TEL/ARG was immunoprecipitated from TEL/ARG-transfected Ba/F3 cells using a purified goat polyclonal ARG antibody (clone C-20) directed against the C-terminus of ARG (Figure 3). Immunoblotting with an anti-phosphotyrosine antibody revealed tyrosine phosphorylation of TEL/ARG. Exposure of cells to 1 µM STI571 for 24 hours before lysis resulted in a reduction of cellular tyrosine phosphorylation in general and of TEL/ARG specifically. Immunoblotting with a rabbit anti-ARG antibody directed against the SH2 and SH3 domains of ARG revealed similar levels of TEL/ARG protein in untreated and STI571-treated cells (Figure 3, lower panel). To confirm that the approximately 170-kd phosphorylated band observed after ARG immunoprecipitation was, indeed, specific for TEL/ARG, immunoprecipitation was performed with a purified goat polyclonal antibody directed against the phosphatase PAC1 (Figure 3). No band sizes comparable to that of TEL/ARG were detected, suggesting that ARG antibody-precipitated bands were specific. These results suggest that autophosphorylation of TEL/ARG is inhibited by STI571.
Comparison of the effects of STI571 on cellular tyrosine
phosphorylation in Ba/F3 cells expressing BCR/ABL, TEL/ABL,
TEL/ARG, TEL/PDGF R at concentrations of
STI571 < 1 µM, and there was no inhibition in cells expressing
TEL/JAK2 or untransfected Ba/F3 cells (Figure 4C). Two independent
TEL/JAK2 Ba/F3 lines were tested for responsiveness to STI571 in terms
of tyrosine phosphorylation and both yielded similar results (data
shown for only one of the 2 cell lines in Figure 4C). The
IC50 (50% inhibitory concentration) was approximately 0.5 µM STI571 for each of the sensitive cell lines, though
TEL/PDGFR-Ba/F3 cells were slightly more sensitive to the
kinase inhibitor.
Comparison of the effects of STI571 on cellular proliferation and
viability in Ba/F3 cells expressing BCR/ABL, TEL/ABL, TEL/ARG,
TEL/PDGF R-Ba/F3 cells were slightly more sensitive. Concentrations
of up to 1 µM STI571 did not inhibit the proliferation of
untransformed Ba/F3 cells growing in IL-3 or of TEL/JAK2-Ba/F3 cells.
Two independent TEL/JAK2 Ba/F3 lines were tested for responsiveness to
STI571 in terms of cell proliferation, and both yielded similar results
(data shown for only one of the 2 cell lines in Figure 5).
To determine whether the decrease in cell number observed in Figure 5
was due to the induction of apoptosis, Annexin V-PI staining was
performed on TEL/ARG, TEL/ABL, TEL/PDGF
The use of small molecule drugs to inhibit specific oncogenes in
leukemia promises to improve outcome and reduce side effects compared
to traditional chemotherapy. Tyrosine kinase oncogenes are likely to be
excellent drug targets for several reasons. First, tyrosine kinases are
frequently activated by chromosomal translocations in leukemia. In
addition to ABL, tyrosine kinases known to be activated in leukemias
include KIT, FLT3, JAK2, PDGF STI571 is believed to interact with the ATP binding site of kinases and
to block the binding of ATP.2 The characteristics that
distinguish sensitive from resistant kinases are partially understood.
Based on a crystal structure of the catalytic domain of ABL complexed
to a variant of STI571, initial drug binding requires that the kinase
be in an "inactive" conformation in which the "activation loop"
is unphosphorylated.30 The inactive conformation of the
loop is, contrary to the active conformation, diverse among protein
kinases.30 One factor possibly contributing to the
specificity of STI571 for ABL was reported to be the preservation of an
ion pair between lysine and glutamine residue side chains in the
inactive conformation of ABL that appears to contribute to
drug-substrate interaction. Inactive conformations of SRC tyrosine
kinases are characterized by a disrupted ion pair.30
Threonine residue 315 (T315) was also suggested to be necessary for the
interaction of ABL interaction with STI571.30 In
accordance with this, T315 in ABL is conserved in STI571 substrates
ARG, PDGF The studies shown here indicate the c-ARG kinase is sensitive to STI571 at a level comparable to that of c-ABL, at least when activated as part of the TEL/ARG oncogene. Direct testing of c-ARG for sensitivity to STI571 in an intact cell will require discovery of a normal signaling pathway that activates ARG. However, because the TEL/ARG fusion protein would not be expected to alter the structure of the ARG kinase domain, it is highly likely that c-ARG and TEL/ARG will be equally sensitive to STI571. The high homology between the SH2 domain and the kinase domain of ARG and ABL suggests that both proteins may share common substrates. Indeed, an in vitro study showed that both ABL and ARG catalyze the tyrosine phosphorylation of the C-terminal repeat domain of RNA polymerase II.31 In another study, the substrate preferences for c-ABL and c-ARG were compared using synthetic peptides, and considerable similarity in substrate selection was reported.32 In contrast, BCR/ABL and TEL/ABL were more promiscuous and phosphorylated a number of peptides that are poor substrates for either ABL or ARG.32 In general, however, in vitro kinase assays may not be accurate at predicting in vivo substrates. This has been observed repeatedly when comparing c-ABL and BCR/ABL, where most of the known substrates for c-ABL are nuclear proteins and most of the known substrates for BCR/ABL are cytoplasmic proteins. Similarly, subcellular localization of ARG and ABL is distinct: whereas ABL is located in the nucleus and cytoplasm, ARG is located only in the cytoplasm.33 Although not a goal of the current studies, it is clear from examining the phosphotyrosine immunoblots shown in Figures 2 and 4 that the overall patterns of cellular phosphorylation induced by TEL/ARG, TEL/ABL, and BCR/ABL in Ba/F3 cells are not the same. Although ARG is widely expressed,7 its only known function is in the central nervous system. ARG is abundant in the brain and is concentrated in synapses.8 ARG knockout mice develop normally but have neuronal dysfunction manifested by multiple behavioral disorders.8 Interestingly, ABL/ARG double knockout mice have severe defects in neurulation, including failure to close the neural tube.8 Because these defects are more significant than in either single knockout mouse, it is likely that ABL and ARG cooperate during development of the nervous system, perhaps in regulating cytoskeletal structure or function. ARG is not known to be required for normal hematopoiesis and, unlike c-ABL, has not been linked to the regulation of DNA repair processes. Fusion of the TEL and ARG genes has been
reported in 2 patients with leukemia, with a specific chromosomal
translocation, t(1;12)(q25;p13).9,10 In both cases, the
fusions included the Pointed (PNT) domain of TEL (ETV6) and the SH3,
SH2, and kinase domains of ARG. The reciprocal ARG/TEL transcript was
detected in one patient but not in the other. One patient had acute
myeloblastic leukemia (AML)-M39 and also had the t(15;17)
translocation characteristic of acute promyelocytic leukemia. The
second patient had AML-M4.10 Interestingly, cells from
both patients displayed eosinophilic differentiation, suggesting that
activation of ARG might modulate the differentiation potential of
leukemic cells. Based on previous studies with TEL/PDGF Overall, the results presented here extend the spectrum of
kinases known to be inhibited by STI571 to a fourth kinase, c-ARG. STI571 appears in early trials to represent a major advance in the
therapy of CML and other ABL-oncogene related leukemias. Patients in
chronic phase are reported to have high rates of hematologic response
and significant rates of cytogenetic response. Patients in more
advanced stages also have high rates of at least short-term responses.
The remarkable lack of serious side effects suggests either that the
PDGF
Submitted August 21, 2000; accepted December 21, 2000.
K.O. is supported by Grants in Aid (no. 11671011) from the Ministry of Education, Science, and Culture of Japan.
K.O. and E.W. contributed equally to the manuscript.
J.D.G. has declared a financial interest in Novartis Pharmaceuticals.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: James D. Griffin, Department of Adult Oncology, Dana Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; e-mail: james_griffin{at}dfci.harvard.edu.
1.
Buchdunger E, Zimmermann J, Mett H, et al.
Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative.
Cancer Res.
1996;56:100-104 2. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566[CrossRef][Medline] [Order article via Infotrieve]. 3. Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest. 2000;105:3-7[Medline] [Order article via Infotrieve]. 4. Druker BJ, Talpaz M, Resta D, et al. Clinical efficacy and safety of an Abl-specific tyrosine kinase inhibitor as targeted therapy for chronic myelogenous leukemia [abstract]. Blood. 1999;94:1639.
5.
Kruh GD, King CR, Kraus MH, et al.
A novel human gene closely related to the abl proto-oncogene.
Science.
1986;234:1545-1548
6.
Kruh GD, Perego R, Miki T, Aaronson SA.
The complete coding sequence of arg defines the Abelson subfamily of cytoplasmic tyrosine kinases.
Proc Natl Acad Sci U S A.
1990;87:5802-5806 7. Perego R, Ron D, Kruh GD. Arg encodes a widely expressed 145-kDa protein-tyrosine kinase. Oncogene. 1991;6:1899-1902[Medline] [Order article via Infotrieve]. 8. Koleske AJ, Gifford AM, Scott ML, et al. Essential roles for the Abl and Arg tyrosine kinases in neurulation. Neuron. 1998;21:1259-1272[CrossRef][Medline] [Order article via Infotrieve].
9.
Iijima Y, Ito T, Oikawa T, et al.
A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12) (q25;p13) translocation.
Blood.
2000;95:2126-2131
10.
Cazzaniga G, Tosi S, Aloisi A, et al.
The tyrosine kinase abl-related gene ARG is fused to ETV6 in an AML-M4Eo patient with a t(1;12)(q25;p13): molecular cloning of both reciprocal transcripts.
Blood.
1999;94:4370-4373 11. Golub TR, Barker GF, Stegmaier K, Gilliland DG. Involvement of the TEL gene in hematologic malignancy by diverse molecular genetic mechanisms [review]. Curr Top Microbiol Immunol. 1996;211:279-288[Medline] [Order article via Infotrieve]. 12. Golub TG, Barker G, Lovett M, Gilliland DG. Fusion of platelet-derived growth factor beta to a novel ets-like gene in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 1994;77:307-316[CrossRef][Medline] [Order article via Infotrieve].
13.
Greenberger JS, Sakakeeny MA, Humphries RK, Eaves CJ, Eckner RJ.
Demonstration of permanent factor-dependent multipotential (erythroid/neutrophil/basophil) hematopoietic progentior cell lines.
Proc Natl Acad Sci U S A.
1983;80:2931-2935 14. Sattler M, Salgia R, Okuda K, et al. The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway. Oncogene. 1996;12:839-846[Medline] [Order article via Infotrieve]. 15. Okuda K, Golub TR, Gilliland DG, Griffin JD. p210BCR/ABL, p190BCR/ABL, and TEL/ABL activate similar signal transduction pathways in hematopoietic cell lines. Oncogene. 1996;13:1147-1152[Medline] [Order article via Infotrieve]. 16. Schwaller J, Frantsve J, Aster J, et al. Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes. EMBO J. 1998;17:5321-5333[CrossRef][Medline] [Order article via Infotrieve].
17.
Carroll M, Tomasson MH, Barker GF, Golub TR, Gilliland DG.
The TEL/platelet-derived growth factor beta receptor (PDGF beta R) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGF beta R kinase-dependent signaling pathways.
Proc Natl Acad Sci U S A.
1996;93:14845-14850
18.
Carroll M, Ohno-Jones S, Tamura S, et al.
CGP57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins.
Blood.
1997;90:4947-4952 19. Bravo P, Agustin BD, Bellas C, et al. Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation. Am J Hematol. 2000;63:226-229[CrossRef][Medline] [Order article via Infotrieve]. 20. Naeem R, Singer S, Fletcher JA. Translocation t(8;13)(p11;q11-12) in stem cell leukemia/lymphoma of T-cell and myeloid lineages. Genes Chromosomes Cancer. 1995;12:148-151[Medline] [Order article via Infotrieve]. 21. Kempski H, Macdonald D, Michalski AJ, et al. Localization of the 8;13 translocation breakpoint associated with myeloproliferative disease to a 1.5 Mbp region of chromosome 13. Genes Chromosomes Cancer. 1995;12:283-287[Medline] [Order article via Infotrieve].
22.
Peeters P, Raynaud SD, Cools J, et al.
Fusion of TEL, the ETS-variant gene 6 (ETV6), to the receptor-associated kinase JAK2 as a result of t(9;12) in a lymphoid and t(9;15;12) in a myeloid leukemia.
Blood.
1997;90:2535-2540 23. Levitzki A. Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol Ther. 1999;82:231-239[CrossRef][Medline] [Order article via Infotrieve].
24.
Carter SK.
Clinical strategy for the development of angiogenesis inhibitors.
Oncologist.
2000;5:51-54 25. Huettner CS, Zhang P, Van Etten RA, Tenen DG. Reversibility of acute B-cell leukaemia induced by BCR-ABL1. Nat Genet. 2000;24:57-60[CrossRef][Medline] [Order article via Infotrieve].
26.
Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ.
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia.
Blood.
1994;83:2038-2044 27. Beran M, Cao X, Estrov Z, et al. Selective inhibition of cell proliferation and BCR-ABL phosphorylation in acute lymphoblastic leukemia cells expressing Mr 190,000 BCR-ABL protein by a tyrosine kinase inhibitor (CGP-57148). Clin Cancer Res. 1998;4:1661-1672[Abstract].
28.
Deininger MW, Goldman JM, Lydon N, Melo JV.
The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells.
Blood.
1997;90:3691-3698 29. Dan S, Naito M, Tsuruo T. Selective induction of apoptosis in Philadelphia chromosome-positive chronic myelogenous leukemia cells by an inhibitor of BCR-ABL tyrosine kinase, CGP57148. Cell Death Differ. 1998;5:710-715[CrossRef][Medline] [Order article via Infotrieve].
30.
Schindler T, Bornmann W, Pellicena P, et al.
Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase.
Science.
2000;289:1938-1942
31.
Baskaran R, Chiang GG, Mysliwiec T, Kruh GD, Wang JY.
Tyrosine phosphorylation of RNA polymerase II carboxyl-terminal domain by the Abl-related gene product.
J Biol Chem.
1997;272:18905-18909 32. Voss J, Posern G, Hannemann JR, et al. The leukaemic oncoproteins Bcr-Abl and Tel-Abl (ETV6/Abl) have altered substrate preferences and activate similar intracellular signalling pathways. Oncogene. 2000;19:1684-1690[CrossRef][Medline] [Order article via Infotrieve]. 33. Wang B, Kruh GD. Subcellular localization of the Arg protein tyrosine kinase. Oncogene. 1996;13:193-197[Medline] [Order article via Infotrieve]. 34. Golub TR, Barker GF, Lovett M, Gilliland DG. Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation. Cell. 1994;77:307-316. 35. Golub TR, Goga A, Barker GF, et al. Oligomerization of the ABL tyrosine kinase by the Ets protein TEL in human leukemia. Mol Cell Biol. 1996;16:4107-4116[Abstract].
36.
Papadopoulos P, Ridge SA, Boucher CA, Stocking C, Wiedemann LM.
The novel activation of ABL by fusion to an ets-related gene, TEL.
Cancer Res.
1995;55:34-38
37.
Eguchi M, Eguchi-Ishimae M, Tojo A, et al.
Fusion of ETV6 to neurotrophin-3 receptor TRKC in acute myeloid leukemia with t(12;15)(p13;q25).
Blood.
1999;93:1355-1363
38.
McWhirter JR, Galasso DL, Wang JY.
A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins.
Mol Cell Biol.
1993;13:7587-7595 39. Hannemann JR, Healy LE, Ridge SA, Wiedemann LM. The second ETV6 allele is not necessarily deleted in acute leukemias with a ETV6/ABL fusion. Genes Chromosomes Cancer. 1998;21:256-259[CrossRef][Medline] [Order article via Infotrieve]. 40. Andreasson P, Johansson B, Carlsson M, et al. BCR/ABL-negative chronic myeloid leukemia with ETV6/ABL fusion. Genes Chromosomes Cancer. 1997;20:299-304[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
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Top
Abstract
Introduction
) and 72 hours (
), respectively, and cultured in the absence or
presence of 15% WEHI-conditioned medium, used as a source of IL-3
(left panel). Growth curves for TEL/ABL-Ba/F3 cells treated as
described for TEL/ARG-Ba/F3 cells (right panel). Experiments were
performed in duplicate for each cell line, and viability counts were
determined by trypan blue exclusion and shown as percentage control.
(B) Annexin-PI analysis of TEL/ARG-Ba/F3 cells (left panel) and
TEL/ABL-Ba/F3 cells (right panel), treated as described in panel A.
PDGF