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Abnormality of C-Kit Oncoprotein in Certain Patients with Chronic

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Abnormality of C-Kit Oncoprotein in Certain Patients with Chronic Leukemia (2002) 16, 170–177 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Abnormality of c-kit oncoprotein in certain patients with chronic myelogenous leukemia – potential clinical significance K Inokuchi, H Yamaguchi, M Tarusawa, M Futaki, H Hanawa, S Tanosaki and K Dan Division of Hematology, Department of Internal Medicine, Nippon Medical School, Tokyo, Japan Chronic myelogenous leukemia (CML) is characterized by the patients, there are two bcr/abl mRNAs for P210BCR/ABL, one Philadelphia (Ph) chromosome and bcr/abl gene rearrangement with and one without exon b3 (b3-a2 type and b2-a2 type).4 which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate In a smaller number of CML patients, there are two other types the biological properties of KIT in CML leukemogenesis, we of bcr/abl mRNAs based on the breakpoint positions of the performed analysis of alterations of the c-kit gene and func- bcr gene, ie m-bcr and ␮-bcr for P190BCR/ABL and P230BCR/ABL, tional analysis of altered KIT proteins. Gene alterations in the respectively.5,6 Extensive studies have been performed on the c-kit juxtamembrane domain of 80 CML cases were analyzed subtypes of the bcr/abl gene and their relation to the prognosis by reverse transcriptase and polymerase chain reaction-single and clinical features.7,8 The established findings regarding the strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT→AAG, Asn→Lys), and influence of the subtype of the bcr/abl gene (b3-a2 or b2-a2) six cases had the same base abnormality at codon 541 on the clinical characteristics have been similar for each sub- (ATG→CTG, Met→Leu) in the juxtamembrane domain. Because type, except for a higher platelet count in patients with the the change from Met to Leu at codon 541 was a conservative b3-a2 type.4,9 P190BCR/ABL in CML maybe associated with one which was also observed in the normal population and nor- monocytosis, and P230BCR/ABL maybe associated with the mal tissues of CML patients, it probably represents a polymor- chronic neutrophilic leukemia variant and marked throm- phic variation. Although samples of hair roots and leukemic 6,10 cells from the chronic phase of one CML patient showed no bocytosis. Other molecular factors which might control the abnormality, an abnormality at codon 541 (ATG→CTG, clinical features and hematological characteristics remain Met→Leu) was found only at blastic crisis (BC) of this case. In unclear. the case with the abnormality at codon 564, the mutation was Recently, the stem cell factor (SCF) c-kit signal system detected only in a sample of leukemic cells collected at BC. To (KIT/SCF) has been shown to playa crucial role in hematopo- examine the biological consequence and biological signifi- iesis.11 The c-kit receptor tyrosine kinase (KIT) is expressed on cance of these abnormalities, murine KITL540 and KITK563 expression vectors were introduced into interleukin-3 (IL-3)- progenitor stem cells as well as mast cells. SCF synergizes in dependent murine Ba/F3 cells to study their state of tyrosine vitro with other cytokines to increase the number and size of phosphorylation and their growth rate. Ba/F3 cells expressing colonies of hematopoietic progenitors.12 Thus, we speculated KITWT,KITL540 and KITK563 showed dose-dependent tyrosine that the KIT/SCF system possibly controls the hematological phosphorylation after treatment with increasing concentrations characteristics of CML. of recombinant mouse stem cell factor (rmSCF). The cells The present studywas designed to investigate the mutations expressing KITL540 and KITK563 were found to have greater tyro- sine phosphorylation than cells expressing KITWT at 0.1 and 1.0 of the c-kit gene and the relationship between the in vitro ng/ml of rmSCF. The Ba/F3 cells expressing KITK563 proliferated function of mutant c-kit and the biological features of CML in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The patients. Ba/F3 cells expressing KITL540 showed a relatively higher pro- liferative response to 0.1 ng/ml of rmSCF than the response of WT cells expressing KIT . These mutations and in vitro functional Materials and methods analyses raise the possibility that the KIT abnormalities influ- ence the white blood cell counts (P Ͻ 0.05) and survival (P Ͻ 0.04) of CML patients. Patients Leukemia (2002) 16, 170–177. DOI: 10.1038/sj/leu/2402341 Keywords: c-kit; CML; Ba/F3; WBC We studied 116 bone marrow (BM) or peripheral blood (PB) samples obtained from 80 patients with CML in various clini- cal phases: 65 in chronic phase (CP), seven in accelerated Introduction phase (AP), and 44 in blastic crisis (BC). The diagnosis of CML was made on the basis of clinical features, hematological data Philadelphia (Ph) chromosome is the cytogenetic hallmark of and Ph chromosome. In 34 patients, both cytogenetic and chronic myelogenous leukemia (CML) and is found in up to molecular analyses were performed in more than two phases, 95% of CML patients.1 The demonstration of bcr/abl mRNA ie CP and BC (30 patients), CP and AP (two patients), and CP, is accepted as a reliable diagnostic marker for CML, and in AP and BC (two patients). Sixty-eight normal BM or PB some cases this evidence is more reliable than the Ph chromo- samples were obtained to studymutation and polymorphism some.2 The clinical signs and hematological findings probably of the c-kit gene. These samples were obtained with the depend partlyon the presence of P210 BCR/ABL, which plays a patients’ and normal volunteers’ informed consent. central role in the pathogenesis of the chronic phase of CML.3 According to the breakpoint site of the bcr gene in most CML Cells Ba/F3,13 a murine IL-3-dependent pro-B lymphoid cell line, Correspondence: K Inokuchi, Division of Hematology, Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo- was cultured in RPMI 1640 medium supplemented with 10% ku, Tokyo 113, Japan; Fax: 81-3-5814–6934. fetal calf serum (FCS) and 10% WEHI-3 cell conditioned Received 19 December 2000; accepted 3 October 2001 medium. Abnormality of KIT in CML K Inokuchi et al 171 Extraction of RNA and DNA a 10% polyacrylamide gel containing 90 mM Tris-borate, pH 8.3, 4 mM EDTA, and 10% glycerol. Electrophoresis was per- The total RNA of BM or PB leukocytes was extracted with an formed at 40 W for 3 h at 18° with cooling using a water RNAzol kit (Biotex Laboratories, Houston, TX, USA), which jacket. The gel was dried on a filter paper and exposed to was based on a technique described previously.14 The total X-rayfilm at −80° for 1–24 h with an intensifying screen. cellular DNA was extracted from BM and PB cells byprotease K digestion, phenol–chloroform extraction and ethanol precipitation. Sequence analysis of the c-kit gene After the RT-PCR products were separated on 2% agarose gels RT-PCR of c-kit mRNA and stained with ethidium bromide, the amplified fragment was excised from the gel, electroeluted, purified with phenol RT-PCR was performed as described elsewhere.15 The sense and precipitated with ethanol. The fragments were subcloned primers were: kit560–1, 5Ј-CTGTTCACTCCTTTGCTGAT-3Ј into the EcoRV site of the pGEM-5Zf(+/−) cloning vector.16 The (residues 1582–1601); kit560–2, 5Ј-TTCGTAATCGT transfected cells were plated on to Luria-Beriani (LB)-ampicil- AGCTGGCAT-3Ј (residues 1605–1624); kit816–1, 5Ј- lin agar plates containing 5-bromo-4-chloro-3-indolyl-␤-D- ATCATGGAGGATGACGAGTTG-3Ј (residues 2287–2306); galactoside (X-Gal), isotransferred to fresh LB-ampicillin agar and kit816–2, 5Ј-CTAGACTTAGAAGACTTGCT-3Ј (residues plates containing X-Gal and isopropylthio-␤-D-galactoside, 2310–2329). The antisense primers were: kit560–3, 5Ј- and cultured overnight for secondaryselection. White colon- CATGTGATTACCAAGGTAA-3Ј (residues 1955–1974); ies were transferred into 150 ml of LB medium containing 50 kit560–4, 5Ј-GCTCCAAGTAGATTCACAAT-3Ј (residues mg/ml ampicillin and cultured at 37°C for 4 h. The cultures 1978–1997); kit816–3, 5Ј-ATTTCAGCAGGTGCGTGTTC-3Ј were sedimented bycentrifugation, resuspended in 20 ml of (residues 2698–2717); and kit816–4, 5Ј-TTTTTA water and heated at 98°C for 10 min. After centrifugation, the GGGGATCTGCATCC-3Ј (residues 2742–2761). Complemen- supernatants were amplified byPCR using the T7 or SP6 taryDNA was synthesizedfrom 500 ng of total cellular RNA primer. Three to five clones of the three independent PCR pro- extracted from cells using 100 ng of primer kit560–4 for analy- ducts were sequenced using a Model 377 ABI sequencer with sis of the sequence in the juxtamembrane domain or kit816– dye terminators (Perkin Elmer, Warrington, UK). All sequences 4 for analysis of the phosphotransferase domain. Briefly, RT were confirmed in both orientations. A mutation was defined reaction mixture contained 32 U of avian myeloblastosis virus as when three or more clones showed the same abnormality (AMV) RT (Takara Biochemicals, Shiga, Japan) in 25 ␮lofa of the base sequence. solution containing 200 ␮mol/l each of all four dNTPs, 80 U of RNase inhibitor, 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, 10 mmol/l dithiothreitol and 3 mmol/l MgCl2. The reac- Site-directed mutagenesis and transfection tion was allowed to proceed for 60 min at 37°C, and the reac- tion mixture was used as the PCR substrate. A 35-cycle PCR To examine for functional abnormalityof c-kit mutations in reaction was performed in a DNA Thermal Cycler with slight activation of c-kit tyrosine kinase activity, site-directed modification of our original protocol.4 Briefly, 25 ␮l of the RT mutagenesis was performed using murine c-kit cDNA as reaction solution was mixed with a mixture containing 250 described byFuritsu et al.17 The genes encoding murine wild- ␮mol/l of each of all four dNTPs, 100 ng of 5Ј-primer ST1, 10 type c-kit and c-kit c-DNA with a mutation at codon 814 mmol/l Tris-HCl (pH 8.3) , 50 mmol/l KCl, 3 U of Taq DNA (GAC→GTC) in the XbaI site of the expression vector, pEF- polymerase (Takara Biochemicals) and 100 ng of primers BOS, were used as wild and mutant controls.18 These two c- kit560–1 and kit560–4 or primers kit816–1 and kit816–4.
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