121-128 8/6/07 10:39 Page 121 INTERNATIONAL JOURNAL OF ONCOLOGY 31: 121-128, 2007 121 Cytogenetic characterisation and proteomic profiling of the Imatinib-resistant cell line KCL22-R JULIA ROSENHAHN1*, ANJA WEISE3*, SUSANNE MICHEL4, KATRIN HENNIG2, ISABELL HARTMANN2, JANA SCHIEFNER2, KATRIN SCHUBERT1, THOMAS LIEHR3, FERDINAND VON EGGELING1 and IVAN F. LONCAREVIC2 1Core Unit Chip Application, Institute of Human Genetics and Anthropology, 2Tumor Genetics, Institute of Human Genetics and Anthropology, 3Molecular Cytogenetics, Institute of Human Genetics and Anthropology, 4Institute of Human Genetics and Anthropology, Medical Faculty at the Friedrich Schiller University Jena, Germany Received February 5, 2007; Accepted March 26, 2007 Abstract. Approximately 30% of chronic myeloid leukemia in chronic myeloid leukemia (CML). It acts by inhibiting the patients show initially no response to Imatinib, a potent tyrosine kinase BCR-ABL, a characteristic feature of CML inhibitor of BCR-ABL. This intrinsic resistance may be due that results predominantly from a reciprocal chromosomal to BCR-ABL-independent cell growth. Here we analysed the translocation t(9;22)(q34;q11) (1,2). The BCR-ABL gene cytogenetic anomalies and the proteomic profiling in KCL22-S encodes for a constitutively active tyrosine kinase which is and KCL22-R, two Imatinib-sensitive and -resistant derivative involved in a multitude of signalling and survival pathways cell lines of KCL22. A tetrasomy 8 and a non-reciprocal trans- that result in enhanced proliferation, decreased apoptosis and location +der(6)t(6;13)(p11.1;q12) were found only in KCL22- reduced adhesion of tumor cells (3-5). R as new evolved anomalies. Chromosome der(6)t(6;13) Although Imatinib is a very potent drug for targeting showed four variants differing in the chromatin content of patients in the chronic phase of the disease, the response 13q14-13qter including the retinoblastoma gene. Due to rates decrease during disease progression (6). This type of these sub-clones, approximately 65-79% of the Imatinib- primary Imatinib resistance strictly differs from the drug- treated KCL22-R cells showed a disomy and 21-35% a induced acquired resistance. The latter is caused either by monosomy for 13q14. Imatinib removal reduced the main mutations in the ATP-binding site of BCR-ABL or by its clone to approximately 20% in the benefit of the monosomic overexpression (7-9). Patients showing acquired resistance sub-clones. This was accompanied by an increased apoptosis respond initially to Imatinib but relapse within 12 months. rate and was revertible by Imatinib re-treatment. This effect Primary resistance to Imatinib, on the other hand, is observed may be connected with genes located in 13q14-qter. Proteomic in 20-30% of newly diagnosed patients (5) and in 22-50% of profiling of the cell lines performed with ProteinChip tech- patients in accelerated or blastic phase (10). It is characterised nology (SELDI) revealed several differentially expressed by a proliferation of tumor cells independent of BCR-ABL proteins (n=45). In summary, we demonstrate here the complex activity. However, what substitutes BCR-ABL function in changes on the cytogenetic and proteomic level which could those Imatinib-resistant cells is not well understood. be caused by Imatinib and the resistance resulting from it. Surprisingly, the role of chromosomal changes has not been investigated in the context of primary Imatinib resistance to Introduction date albeit chromosomal changes and clonal evolution are clearly associated with disease progression in CML (11). Imatinib (STI571, Gleevec®) is a relatively new type of anti- Approximately 71% of patients with CML in blast crisis cancer medication that can effectively block the proliferation show chromosomal changes in addition to t(9;22). The most frequent secondary aberrations are gains of the Philadelphia _________________________________________ chromosome, chromosomes 1, 2, 6, 7, 8 16, 17, 19 or 21 as well as a loss of chromosomes 3, 5, 7, 17 or 19 (12). Chromosomal changes have also been shown to develop from Imatinib Correspondence to: Dr Ferdinand von Eggeling, Core Unit Chip therapy. These aberrations affect neither additional chromo- Application, Institute of Human Genetics and Anthropology, FSU Jena, Germany somes nor do they result in a new Imatinib-resistant clone E-mail: [email protected] (13). So far no relationship between additional chromosomal changes and Imatinib resistance could be conclusively deduced. *Contributed equally ProteinChip technology (SELDI; surface-enhanced laser desorption/ionization) is a proteomic high-throughput technique Key words: chronic myeloid leukemia, BCR-ABL, Imatinib, initial that uses chromatographic surfaces able to retain proteins resistance, karyotype evolution, SELDI depending on their physico-chemical properties followed by direct analysis via time of flight mass spectrometry (TOF-MS) 121-128 8/6/07 10:39 Page 122 122 ROSENHAHN et al: PROFILING OF AN IMATINIB-RESISTANT CELL LINE (14). A multitude of studies using the ProteinChip technology Multiplex-FISH (M-FISH) was performed to confirm were carried out for protein profiling of cells and body fluids GTG-results as described by Weise and co-workers (25). Ten (15-22). metaphase images per cell line were captured on a fluorescence In the present study we used cytogenetic analysis to microscope (Axioplan 2, Zeiss, Germany) with a PCO VC45 characterise the Imatinib-sensitive and -resistant CML cell CCD camera (PCO, Kehl, Germany) and suitable filter lines KCL22-S and -R to discriminate for numerical and combinations (DAPI/FITC/Spectrum Orange/Texas Red/ structural chromosome aberrations and proteomic profiling to Cyanine 5/Cyanine 5.5) using ISIS3-software (MetaSystems, find differentially expressed proteins. Accordingly, we have Altlussheim, Germany). 24-Color-FISH using all 24 human identified characteristic chromosomal aberrations in the whole chromosome painting probes was applied to identify resistant cells and 45 differentially expressed proteins. These interchromosomal rearrangements (25). results provide in vitro evidence that Imatinib has an influence To quantify relevant M-FISH data, interphase nuclei were on specific chromosome aberrations and thereby causes several hybridised with the centromere probe cep 6 (Spectrum Green), alterations in the proteome which may lead conclusively to cep8 (Spectrum Green or Spectrum Aqua) and a retinoblast- resistance. oma (Rb) gene (13q14.3) locus specific probe LSI 13S319 (Spectrum Orange) (Abbott) as previously described (24). Materials and methods Signals of 200-250 interphase nuclei were analysed. The ‘cut-off’ for trisomy/tetrasomy 8 and monosomy 13q14 was Cell lines and cell proliferation/apoptosis analysis. The 4% and 8%, respectively according to lab standards based Imatinib-resistant cell line KCL22-R and its sensitive counter- on the analysis of 200-250 cells. BCR-ABL breakpoint was part KCL22-S were established by Mahon and co-workers (23) confirmed using LSI Dual Color Dual Fusion probe (Abbott) to analyse primary Imatinib resistance in vitro. The parental and by PCR using oligonucleotides as described (26). cell line KCL22 is initially resistant to Imatinib (23) and High resolution multi-color banding (MCB) was performed to consists of sensitive and resistant subpopulations, which can analyse the breakpoints in chromosome der(6)t(6;13)(q11.1;q12). be isolated from the original cell line using methylcellulose. Overlapping microdissected labelled probe sets were hybridised The KCL22-R cells employed in this study neither bear on metaphase spreads as described previously (27). mutations in the ATP-binding site of BCR-ABL nor show BCR-ABL overexpression (23). Therefore, they represent ProteinChip Array analysis. Cells were lysed with 9.5 M urea, an adequate in vitro system to analyse Imatinib resistance 2% CHAPS, 1% DTT for 15 min at RT and centrifuged at which is based on a BCR-ABL-independent growth capacity. 12000 x g for 15 min. Protein concentration of supernatants Cells were cultured in RPMI (Pan Biotech GmbH) with was measured by the Bradford method. Equal amounts of 10% FCS (Biochrom) in a 5% CO2 atmosphere at 37˚C and protein (30-50 mg/ml) in a total volume of 50-100 μl lysate were passaged twice weekly. The Imatinib-resistant cell line were applied to the fractionation using QHyperD columns KCL22-R1+ was grown in the presence of 1 μM Imatinib. (Ciphergen) according to the manufacturer's guidelines. Whole The tyrosine kinase inhibitor Imatinib, kindly provided by cell lysates and fractionated samples were analyzed on IMAC Andrea Uecker (Molecular Cell Biology department, FSU Cu(II) protein arrays (Ciphergen Biosystems Inc, Fremont, CA) Jena), was prepared in sterile DMSO as a 10 mM stock solution as described elsewhere (28). Mass analysis was performed in and diluted to a working concentration of 100 μM with sterile a ProteinChip Reader (series 4000, Ciphergen Biosystems Inc) PBS. We established a non-treated resistant clone from according to an automated data collection protocol. After KCL22-R1+ by growing cells for three to six months without normalization based on total ion current and calibration of Imatinib (KCL22-R1-). These cells were used to analyse data, peaks with a signal-to-noise ratio of 10 were combined as changes associated with Imatinib therapy. clusters, using the CiphergenExpress 3.0 program (Ciphergen For proliferation assays, cells were seeded at a concentration Biosystems). The p values were