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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3498-3505
NEOPLASIA
From the Department of Adult Oncology, Dana Farber Cancer Institute,
Department of Medicine, Brigham and Women's Hospital, and Department
of Medicine, Harvard Medical School, Boston, MA.
The tyrosine kinase activity of the Bcr/Abl oncogene is required for
transformation of hematopoietic cells. The tyrosine kinase inhibitor
STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits
BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and
viability of cells transformed by any of these ABL oncogenes. Here we
report the generation of 2 BCR/ABL-positive cell lines that have
developed partial resistance to STI571. BCR/ABL-transformed Ba/F3
hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571
over a period of several months to generate resistant lines. Resistant
Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger
RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold
increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210
cells, drug-resistant K562 cells did not undergo detectable
amplification of the BCR/ABL gene, although they displayed a 2-fold to
3-fold increase in p210BCR/ABL protein. The addition of STI571 to both
resistant Ba/F3.p210 and K562 cells resulted in a rapid reduction of
tyrosine phosphorylation of cellular proteins, similar to that observed
for nonresistant cells. However, the inhibition of kinase activity was
transient and partial and was not accompanied by apoptosis. The results
suggest that resistance to STI571 may be multifactorial. Increased
expression of the target protein BCR/ABL was observed in both lines,
and resulted from oncogene amplification in one line. However, altered
drug metabolism, transport, or other related mechanisms may also
contribute to drug resistance.
(Blood. 2000;95:3498-3505)
Chronic myelogenous leukemia (CML) is a clonal
myeloproliferative disorder that is characterized by excessive
production, and premature release, of both mature and immature myeloid
cells. CML is caused by the 210-kd BCR/ABL oncoprotein, a chimeric
fusion of either exon 2 or exon 3 of c-Bcr and exon 2 of c-Abl, which results from the Philadelphia (Ph) chromosome
translocation t (9,22).1-3 One of the hallmarks of BCR/ABL
transformation is the greatly elevated ABL tyrosine kinase activity,
which is possibly related to oligomerization of BCR/ABL owing to the
presence of a coiled-coil motif in BCR.4-7
Since transformation by BCR/ABL is absolutely dependent on tyrosine
kinase activity,6 it has been evident for some time that a
drug that inhibited ABL tyrosine kinase activity could be of
therapeutic value. Synthesis of a class of tyrosine kinase-specific inhibitors called tyrphostins8 led to the discovery of an
inhibitor of ABL tyrosine kinase toxic to the human CML cell line
K562.9,10 More recently, STI571, a more potent and highly
selective ABL tyrosine kinase inhibitor of the 2-phenyl-amino
pyrimidine class, was developed.11,12 This compound,
synthesized using the structure of the ATP binding site of protein
kinases, selectively inhibits the anchorage-independent growth and
tumorigenicity of v-Abl-transformed cells,11 inhibits the
ability of BCR/ABL-positive cells to proliferate and develop tumors,
and suppresses the formation of BCR/ABL colony formation in cells
derived from peripheral blood or bone marrow from CML
patients.12,13 STI571 induces apoptosis in a variety of
BCR/ABL-positive cell lines and Ph+ cells.14-16 In
addition to the p210 form of BCR/ABL (B2A2), STI571 also inhibits the
p190 form of BCR/ABL (B1A2).16,17 STI571 is also effective
in inhibiting the kinase activity of platelet-derived growth factor
receptor (PDGFR)11 and TEL-PDGFR, an activated
PDGFR kinase.17 Preliminary results from a phase I
dose-escalating clinical trial suggest that STI571 has significant
activity in stable and advanced phases of CML.18
A recent study demonstrated the need for a continuous block of BCR/ABL
tyrosine kinase activity by STI571 to effectively cure BCR/ABL-positive tumor-bearing nude mice treated with the
inhibitor.19 Since chronic or repeated administration of
STI571 to CML patients may be necessary for successful treatment, we
were interested in identifying potential mechanisms of resistance to
this compound. Two cell lines were selected for study. K562 is a human
Ph+ cell line derived from a patient with CML. Ba/F3.p210 was derived
in our laboratory by transfecting a BCR/ABL complementary DNA (cDNA) into the nontransformed murine hematopoietic cell line Ba/F3. In this
study, we have compared the mechanisms of resistance to STI571 in these
2 cell lines and find both similarities and distinct differences.
Cells and cell culture
Antibodies
Proteasome/protease inhibitors Calpain Inhibitor I (MG101; ALLN; N-Ac-Leu-Leu-norleucinal) was purchased from Calbiochem (La Jolla, CA), resuspended as a 200-mmol/L stock in dimethyl sulfoxide (DMSO), and stored at 20°C. AEBSF, hydrochloric acid (4-[2-Aminoethyl]
benzenesulfonylfluoride, HCl), was purchased from Calbiochem,
resuspended as a 100-mmol/L stock in water, and stored at
20°C. Proteasome Inhibitor I (PSI; z-Ile-Glu
[OtBu]-Ala-Leu-CHO) was purchased from Calbiochem, resuspended as a
50-mmol/L stock in DMSO, and stored at 20°C.
Clasto-Lactacystin  -Lactone was purchased from Calbiochem,
resuspended as a 10-mmol/L stock in DMSO, and stored at
20°C. MG132 (z-Leu-Leu-Leu-H [aldehyde]) was
purchased from Peptide Institute (Osaka, Japan),
resuspended as a 10-mmol/L stock in DMSO, and stored at
20°C.
Immunoblotting Cells were lysed in lysis buffer containing 0.02 mol/L Tris, pH 8.0, 0.15 mol/L NaCl, 10% glycerol, 1% NP-40 (wt/vol), 0.1 mol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L 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), and values were normalized accordingly. Protein lysates were dissolved in Laemmli's sample buffer by boiling for 5 minutes. Whole cell lysates 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 either for 1 hour at 25°C or overnight at 4°C with 5% nonfat dry milk in 1 × TBS (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl), and then probed for 2 hours at 25°C or overnight at 4°C with antibody in 1 × TBST buffer (10 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.05% Tween20). Following 3 washes with 1 × TBST, membranes were incubated for 1 hour at 25°C with antimouse immunoglobulin (horseradish peroxidase linked whole antibody from sheep) or antirabbit immunoglobulin (horseradish peroxidase linked whole antibody from donkey) (Amersham Life Science, Arlington Heights, IL). The membrane was washed 5 times 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 mol/L Tris, pH 6.8, and 0.7% 2-mercaptoethanol) for 30 minutes at 50°C prior to probing with additional antibodies.Northern blotting Total RNAs were extracted from 50 to 100 × 106 cells with the use of Trizol Reagent (Gibco BRL Life Technologies, Gaithersburg, MD), and then messenger RNA (mRNA) was extracted with the use of the Messagemaker Reagent Assembly Kit (Gibco BRL Life Technologies). Approximately 2 µg of mRNA per lane were electrophoresed overnight at 20 V on an ethidium-bromide-stained formaldehyde gel, denatured at room temperature for 30 minutes in 50 mmol/L NaOH, neutralized in 50 mmol/L Tris (pH 7.0) for 30 minutes, and transferred to a Genescreen Plus nylon membrane (NEN Life Science Products) overnight via capillary action. Messenger RNA was fixed to the membrane by UV crosslinking with a Stratalinker (Stratagene, La Jolla, CA), and prehybridized overnight in 50% deionized formamide, 4 × SSC, 0.02 mol/L Tris, 1 × Denhardt's Reagent (50 × Denhardt's Reagent contains 1% Ficoll, Type 400, 1% polyvinylpyrrolidone, and 1% bovine serum albumin [BSA], Fraction V), 0.5% SDS, and 10% dextran sulfate. 32P-labeled cDNA probe (1 to 2 × 106 cpm/mL), labeled by a Random Primed DNA Labeling Kit (Boehringer Mannheim GmbH, Germany) and purified by ProbeQuant G-50 Micro Columns (Amersham Pharmacia Biotech, Piscataway, NJ), was added to the prehybridization solution along with 10 µg/mL denatured salmon sperm DNA (Gibco BRL Life Technologies). Following overnight hybridization at 42°C, the membrane was washed for 20 minutes at 25°C with low-stringency wash buffer (2 × SSC, 0.1% SDS) and washed for 15 minutes at 25°C with high-stringency wash buffer (0.2 × SSC, 0.1% SDS). Filters were exposed to film for varying lengths of time.Southern blotting Genomic DNA (100 to 150 kilobases [kb]) was extracted from approximately 50 × 106 cells with the use of extraction buffer (10 mmol/L Tris-Cl, pH 8.0, 0.1 mol/L EDTA, pH 8.0, 20 µg/mL pancreatic RNAase, and 0.5% SDS) and treated with 100 µg/mL of proteinase K for 3 hours at 50°C. The solution was then extracted 3 times with phenol:chloroform:isoamyl alcohol (25:24:1), and genomic DNA was precipitated with 0.3 mol/L sodium acetate, pH 5.2, and 2 volumes of 100% ethanol. Pure genomic DNA was spooled out and washed twice with 70% ethanol. DNA was finally resuspended in 1 × TE buffer (10 mmol/L Tris-Cl, pH 8.0, 1 mmol/L EDTA, pH 8.0). DNA was digested with EcoRI, HindIII, StuI, and XhoI, and restriction fragments were separated by electrophoresis overnight at 20 V on a 0.8% ethidium bromide-stained agarose gel. Following electrophoresis, DNA was treated for 15 minutes at 25°C with 0.2 N HCl (for partial depurination), 30 minutes at 25°C with 0.4 N NaOH-0.6 mol/L NaCl (for hydrolysis of phosphodiester backbone), and 30 minutes at 25°C with 1.5 mol/L NaCl-0.5 mol/L Tris-Cl, pH 7.4 (for neutralization). Capillary transfer of DNA to a nylon filter, hybridization, and washing steps were carried out as described for Northern blotting.Probes Probes used in Northern and Southern analysis were generated by polymerase chain reaction (PCR) amplification of a BCR/ABL cDNA cloned in a pBluescript vector. PCR primers used for amplification of c-Bcr were 5'GGCGGCGCTCAGGTCCAACTTC3' (upper primer) and 5'CCAGCTCCTCATCGGGCACCAG3' (lower primer), which yield a product spanning the coding region of the c-Bcr gene from base pair (bp) 453 to bp 2332. PCR primers used for amplification of c-Abl were 5'TCGGAAGAGGGCAGGGGAGAAC3' (upper primer) and 5'GCCGGACCACTGCCTGCTGACG3' (lower primer), which yield a product spanning the C-terminus of the c-Abl gene coding region from bp 2289 to bp 3305. A human glyceraldehyde phosphodehydrogenase cDNA probe was generated by reverse transcriptase (RT)-PCR, subcloned into a TA vector (Invitrogen, Carlsbad, CA), and gel-purified. Primers used for amplification of a 332-bp portion of the 3' end of the gene were 5'TTCAAGGGGTCTACATGGCAACTG3' (upper primer) and 5'GGG- CATCCTGGGCTACACTG3' (lower primer).Quantitative PCR for Bcr/Abl Total RNA was isolated from DMSO-treated and STI571-resistant Ba/F3.p210 cells with the use of Trizol Reagent (Gibco BRL Life Technologies).Reverse transcription of 1 µg of total RNA isolated from DMSO-treated and STI571-resistant Ba/F3.p210 cells (see discussion of Northern blotting under "Results") was achieved by mixing the RNA with 2 µL of 10 × PCR buffer, 0.4 µL of deoxynucleotides (Perkin Elmer, Norwalk, CT), 0.65 µL of Random Hexamers (50 A260 Units) (Pharmacia, Piscataway, NJ), 0.25 µL RNAase Inhibitor (10 U/µL) (Gibco BRL Life Technologies), and 0.5 µL of Superscript II Reverse Transcriptase (200 U/µL) (Gibco BRL Life Technologies) in a 20-µL volume. This was followed by incubation of the mixture at 42°C for 60 minutes, and then for 5 minutes at 95°C.
FACS analysis Approximately 1 × 105 cells were incubated with Mdr-1 antibody (diluted 1:100) for 1 hour at 4°C. Cell/antibody mixtures were diluted with 1% BSA in 1 × PBS (PBSA) and centrifuged for 10 minutes at 2000 rpm. Cell pellets were then resuspended in fluorescein isothiocyanate (FITC)-conjugated antirabbit immunoglobulin (Ig)-G (H+L) (0.5 µg/mL) (Zymed, San Francisco, CA) and incubated for 45 minutes at 4°C. Samples were diluted with PBSA and centrifuged for 10 minutes at 2000 rpm. Cell pellets were resuspended in 3% formaldehyde in PBSA and analyzed by FACS analysis. As controls, cells were incubated at 4°C in the absence of Mdr-1 and FITC-conjugated antirabbit IgG antibodies, and cells were incubated at 4°C in the presence of only FITC-conjugated antirabbit IgG antibody. FACS analysis was performed as described above using MRP1 antibody (diluted 1:100) and antigoat IgG (whole molecule) FITC conjugate (Sigma, St Louis, MO).
STI571 inhibits tyrosine phosphorylation in Ba/F3.p210 and K562 cells The Abl tyrosine kinase inhibitor STI571 has previously been shown to inhibit the kinase activity of BCR/ABL.11,12 The concentration of STI571 that inhibits tyrosine phosphorylation by at least 50% in the BCR/ABL-transformed hematopoietic cell line, Ba/F3.p210, was determined by measuring tyrosine phosphorylation of cellular substrates of BCR/ABL by immunoblotting. Figure 1A shows that at 2 hours, levels of tyrosine phosphorylation began to decrease at 1 µmol/L STI571 and were barely detectable at 10 µmol/L STI571. Figure 1B shows that after 24 hours, 1 µmol/L STI571 decreased cellular tyrosine phosphorylation by more than 50%, as compared with DMSO-treated and untreated Ba/F3.p210 cells. STI571 did not appear to affect expression levels of BCR/ABL and c-Abl after 2 hours, although treatment for 24 hours with, respectively, 1 and 10 µmol/L STI571 resulted in a small reduction in the level of BCR/ABL. As with Ba/F3.p210 cells, tyrosine phosphorylation in K562 cells was potently inhibited by culturing cells in the presence of STI571 for 2 hours (Figure 1C).
Development of STI571-resistant Ba/F3.p210 cells Ba/F3.p210 cells were cultured in the presence of gradually increasing concentrations (0.01 µmol/L to 0.2 µmol/L) of STI571 over a period of 56 days. A subpopulation of these cells was placed in the presence of 1 µmol/L STI571, and the concentration of STI571 increased to 2.5 µmol/L in 4 steps over a period of 31 days. Protein, RNA, and DNA analyses were performed on cells resistant to 2 µmol/L STI571. Interestingly, although the cells achieved complete resistance to 2.5 µmol/L STI571, they were rapidly killed by 3 µmol/L STI571, even following several months of culturing in the presence of 2.5 µmol/L STI571 (data not shown). Whereas nonresistant Ba/F3.p210 cells rapidly die within 2 days of culture in the presence of 1 µmol/L STI571, drug-resistant Ba/F3.p210 cells grow well in 1 µmol/L STI571 (Figure 2A).
BCR/ABL protein expression and cellular tyrosine phosphorylation in STI571-resistant Ba/F3.p210 cells Protein lysates were prepared from STI571-resistant cells with the use of lysis buffer with a standard cocktail of protease inhibitors, including aprotinin, phenyl methyl sulfonyl fluoride, and leupeptin. BCR/ABL was then detected by immunoblotting with a monoclonal antibody directed against the SH2 domain of c-Abl (3F12). There was an increase in BCR/ABL protein in STI571-resistant Ba/F3.p210 cells, but multiple proteolytic cleavage products were observed (data not shown). STI571-resistant cells were then treated with a variety of proteasome and protease inhibitors while being continuously cultured in the presence of STI571. MG-132 (20 µmol/L, 4 hours), a reversible proteasome inhibitor that diminishes 26S complex-mediated degradation of ubiquitin-conjugated proteins, and the 20S proteasome inhibitors, Proteasome Inhibitor I (30 µmol/L to 100 µmol/L, 4 hours) and clasto-Lactacystin-lactone (30 µmol/L, 4 hours) did not reduce proteolytic cleavage.
Calpain Inhibitor I, an inhibitor of calpain I, calpain II, cathepsin B, cathepsin L, and neutral cysteine proteases (20 µmol/L and 50 µmol/L, 6 hours) had a
modest effect. However, AEBSF, a specific, cell-permeable, irreversible
inhibitor of serine proteases, such as trypsin, chymotrypsin, plasmin,
thrombin, and kallikrein, used at a concentration of 1 mmol/L for 20 minutes effectively reduced accumulation of
truncated BCR/ABL-related products and led to the reappearance of
apparently full-length p210 BCR/ABL. Therefore, all subsequent
experiments included AEBSF as indicated to reduce proteolytic cleavage
of the amplified BCR/ABL proteins occurring primarily during cell lysis.
Expression of BCR/ABL RNA in STI571-resistant and nonresistant
Ba/F3.p210 cells
Analysis of BCR/ABL gene in STI571-resistant and
nonresistant Ba/F3.p210 cells
Development of STI571-resistant K562 cells
BCR/ABL protein expression and cellular tyrosine
phosphorylation in STI571-resistant K562 cells
Expression of BCR/ABL RNA in STI571-resistant and nonresistant
K562 cells
Analysis of BCR/ABL gene in STI571-resistant and nonresistant K562 cells The BCR/ABL gene was investigated for amplification/alteration in STI571-resistant K562 cells. Southern analysis was performed on HindIII- and EcoRI-digested genomic DNA isolated from DMSO-treated nonresistant K562 cells and STI571-resistant K562 cells as described above. No differences were observed between nonresistant and STI571-resistant K562 cells in either DNA-banding pattern or signal intensity (data not shown).Mdr-1 and MRP1 protein expression in STI571-resistant K562 cells Flow cytometry was performed to determine if drug-resistant K562 cells had altered p-glycoprotein (Pgp) expression, as compared with nonresistant cells. No significant difference in levels of Mdr-1 or MRP1 was observed between the 2 cell populations (data not shown).
STI571 inhibits c-Abl tyrosine kinase activity by acting as a competitor for ATP.11 As a result of this inhibition, the proliferation of cells transformed by v-Abl, p190Bcr/Abl, p210Bcr/Abl, and TEL-ABL is suppressed.11-13,17 STI571 also inhibits colony formation and tumorigenicity of BCR/ABL- and Ph+ cells.11-13 STI571 induces apoptosis in BCR/ABL-positive cells14-16 without causing cell differentiation14; STI571-induced programmed cell death is accompanied by caspase activation.15
We are grateful to Dr Jerry Ritz and Antoinette Chillemi from the Dana Farber Cancer Institute for technical assistance with quantitative PCR.
Submitted August 5, 1999; accepted January 18, 2000.
Reprints: James D. Griffin, Dana Farber Cancer Institute, Department of Adult Oncology, 44 Binney St, Boston, MA 02115; e-mail: james_griffin{at}dfci.harvard.edu.
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.
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Top
Abstract
Introduction
1 of Bcr/Abl
plasmid DNA were used as reference standards of fluorescent signals for
copy number as described previously.
