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Stem Cells 2003;21:315-321 www.StemCells.com
© 2003 AlphaMed Press

Analysis of Gene Expression Profiles in an Imatinib-Resistant Cell Line, KCL22/SR

Ken Ohminea, Tadashi Nagaia, Takahisa Tarumotoa, Takuji Miyoshia, Kazuo Muroib, Hiroyuki Manoc, Norio Komatsua, Fumimaro Takakua, Keiya Ozawaa

a Divisions of Hematology,
b Cell Transplantation and Transfusion, and
c Functional Genomics, Jichi Medical School, Tochigi, Japan

Key Words. Imatinib mesylate • Drug resistance • KCL22/SR • Ras • MAPK

Tadashi Nagai, M.D., Ph.D., Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan. Telephone: 81-285-58-7353; Fax: 81-285-44-5258; e-mail t-nagai{at}jichi.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The BCR/ABL tyrosine kinase inhibitor, imatinib, has shown substantial effects in blast crises of chronic myelogenous leukemia. However, most patients relapse after an initial clinical response, indicating that drug resistance is a major problem for patients being treated with imatinib. In this study, we generated a new imatinib-resistant BCR/ABL-positive cell line, KCL22/SR. The 50% inhibitory concentration of imatinib was 11-fold higher in KCL22/SR than in the imatinib-sensitive parental cell line, KCL22. However, KCL22/SR showed no mutations in the BCR/ABL gene and no increase in the levels of BCR/ABL protein and P-glycoprotein. Furthermore, the level of phosphorylated BCR/ABL protein was suppressed by imatinib treatment, suggesting that mechanisms independent of BCR/ABL signaling are involved in the imatinib resistance in KCL22/SR cells. DNA microarray analyses demonstrated that the signal transduction-related molecules, RAS p21 protein activator and RhoA, which could affect Ras signaling, and a surface tumor antigen, L6, were upregulated, while c-Myb and activin A receptor were downregulated in KCL22/SR cells. Furthermore, imatinib treatment significantly suppressed the level of phosphorylated p44/42 in KCL22 cells but not in KCL22/SR cells, even when BCR/ABL was inhibited by imatinib. These results suggest that various mechanisms, including disturbance of Ras-mitogen-activated protein kinase signaling, are involved in imatinib resistance.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Imatinib (imatinib mesylate; formally STI571), a specific ABL tyrosine kinase inhibitor, has been reported to have a significant clinical effect on chronic myelogenous leukemia (CML) in blast crisis as well as in the chronic phase [1–2]. However, many patients in blast crisis who are being treated with imatinib relapse at a relatively early time [2], suggesting that leukemia cells tend to acquire resistance to imatinib easily in blast crisis. Thus, drug resistance is a major problem even for CML patients in blast crisis who are being treated with imatinib.

Recently, there have been several studies on the mechanisms of imatinib resistance. These studies have shown that amplification of the BCR/ABL gene, increased expression of BCR/ABL protein, and upregulation of P-glycoprotein (P-gp) occurred in some imatinib-resistant BCR/ABL-positive cell lines [3–5]. P-gp belongs to the ATP-binding cassette (ABC) family and has been shown to expel drugs outside cells. In addition, BCR/ABL gene amplification or point mutations in the ATP-binding pocket of the gene have been observed in patients who had responded to imatinib treatment but finally relapsed [6–7]. This point mutation causes the replacement of a threonine residue with an isoleucine residue, resulting in inhibition of binding of imatinib to the ATP-binding pocket. On the other hand, Passerini et al., who transplanted KU812 human CML cells into nude mice, found that the association between imatinib and .1 acid glycoprotein resulted in inactivation of imatinib [8]. These previous studies strongly suggest that various mechanisms are involved in the acquirement of resistance to imatinib.

In this work, we established a new imatinib-resistant cell line, KCL22/SR, and examined the differences in the gene expression profiles of imatinib-sensitive and imatinib-resistant cells by DNA microarray analyses. We found that BCR/ABL signaling-independent, continuous activation of Ras signaling occurred in KCL22/SR cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines
KCL22 is a Philadelphia chromosome-positive cell line established from peripheral blood of a patient with CML in blast crisis [9]. The cells were grown in RPMI1640 medium supplemented with 10% fetal bovine serum and split every 4 days. To generate imatinib-resistant clones, KCL22 cells were treated with step-wise increasing concentrations of imatinib (0.1–1.0 µM) and colonized on a medium containing methylcellulose; then individual colonies were selected. The clone KCL22/SR had the highest 50% inhibitory concentration (IC50) value of imatinib and was used for further examinations.

Imatinib was kindly provided by Novartis Pharmaceuticals (Basel, Switzerland; http://www.novartis.com). Cells were incubated with various concentrations of imatinib for 3 days, and then numbers of viable cells were counted by trypan blue staining. The fold resistance was calculated by dividing the IC50 of KCL22/SR cells by that of KCL22 cells.

Sequence Analysis of BCR/ABL Gene
Total RNA from KCL22 and KCL22/SR cells was isolated by the acid guanidium thiocyanate-phenol-chloroform method [10]. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using cDNA that had been prepared from total RNA by SuperScript II reverse transcriptase (Invitrogen Corp.; Carlsbad, CA; http://www.invitrogen.com). The primers used for this experiment were 5'-GCGCAA CAAGCCCACTGTCTATGG-3' (forward) and 5'-GCCAG GCTCTCGGGTGCAGTCC-3' (reverse). PCR products were cloned into the pT-Adv vector (Clontech; Palo Alto, CA; http://www.clontech.com). The sequences of both strands of 10 amplified cDNA clones were determined with the forward primer 5'-CACCATGAAGCACAAGCTGG-3' and the reverse primer 5'-CAGCTACCTTCACCAAGTGG-3' by an ABI prism 377 automated sequencer (Applied Biosystems; Foster City, CA; http://www.appliedbiosystems.com).

Western Blot Analysis
Nuclear extracts were prepared from 1 x 107 cells according to the method described previously [11]. Ten µg of nuclear extracts was separated electrophoretically using 10% polyacrylamide gel. Immunoblotting and detection by enhanced chemiluminescence were performed as described previously [11]. Anti-BCR rabbit polyclonal antibody and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (G3PDH) monoclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA; http://www.scbt.com) and Chemicon International (Temecula, CA; http://www.chemicon.com), respectively. Anti-p44/42 (extracellular signal-regulated kinase 1 and 2 [ERK1/2]) mitogen-activated protein (MAP) kinase and anti-phospho p44/42 (ERK1/2) MAP kinase rabbit polyconal antibodies were purchased from Cell Signaling Technology (Beverly, MA; http://www.cellsignal.com). Densitometric analysis was performed to determine the levels of ERK1 protein.

Flow Cytometry Analysis
Cells were incubated with a phycoerythrin (PE)-labeled anti-P-gp antibody (Immunotech; Marseille, France; http://www.immunotech.fr) at room temperature for 30 minutes and then washed with phosphate-buffered saline. PE-labeled mouse IgG1 (Becton Dickinson Immunocytometry Systems; Mountain View, CA; http://www.bd.com) was used as a control. The expression of P-gp was determined by flow cytometry.

DNA Microarray Analysis
Total RNA from KCL22 and KCL22/SR cells was prepared using the acid guanidium thiocyanate-phenol-chloroform method [10]. DNA microarray analyses were performed as described previously [12]. Briefly, biotin-labeled cRNA was synthesized and subjected to hybridization with HO2 and HO3 microarrays (Mergen; San Leandro, CA; http://www.mergen.com) and GeneChip HU95Avs2 microarrays (Affymetrix; Sana Clara, CA; http://www.affymetrix.com) representing a total of 2,304 and 12,625 known human genes, respectively. Hybridization signals were analyzed using a GMS418 Array Scanner (Affymetrix) and GeneSpring 3.2.2. software (Silicon Genetics; Redwood, CA; http://www.sigenetics.com).

RT-PCR and Real-Time PCR Analysis
cDNA was generated from total RNA extracted from KCL22 and KCL22/SR cells by SuperScript II reverse transcriptase. The primers used for RT-PCR and real-time PCR were as follows: Ras p21 protein activator (RASAP1): 5'-CCAACTAACCAGTGGTATCACGG-3' (forward) and 5'-GCAGGGAAGTCTGGCAGTTATC-3' (reverse); RhoA: 5'-TAACGATGTCCAACCCGTCTG-3' (forward) and 5'-CTGACACACCAGGCGCTAATT-3' (reverse); L6: 5'-GGAGTGCTTGGAGGCATATGTGGC-3' (forward) and 5'-GTGGCTCTGTCCTGGGTTGGTTCT-3' (reverse); c-Myb: 5'-CCTGGATTCCAAGGCCCTGGTGCCCTGAGC-3' (forward) and 5'-CCACACCCCTGGTGAGTACCAGA CGCTGCC-3' (reverse); and activin A receptor: 5'-GTG GATCAGCAGACCCCCACCATCCC-3' (forward) and 5'-GAGCTAGGCCTGAGAGGACCGGGTCT-3' (reverse). PCR products were electrophoresed on a 1.2% agarose-formaldehyde gel (RT-PCR) or analyzed using an ABI PRISM 7700 system (Applied Biosystems; Foster City, CA; http://home.appliedbiosystems.com) (real-time PCR). cDNA corresponding to the ß-actin gene was used for the internal control of these real-time analyses.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of an Imatinib-Resistant BCR/ABL-Positive Cell Line
We established a new imatinib-resistant cell line, KCL22/SR, from the human bcr/abl-positive cell line KCL22 by treatment with step-wise increasing concentrations of imatinib (0.1–1.0 µM). The IC50 value of imatinib to KCL22/SR was about 11.6-fold higher than that to KCL22, indicating that KCL22/SR has acquired significant resistance to imatinib.

Recent studies have suggested that several mechanisms, including amplification of the BCR/ABL gene, increased expression of BCR/ABL protein, point mutation in the ATP-binding pocket of the BCR/ABL gene, and overexpression of P-gp, are involved in the resistance to imatinib. However, there was no amplification of or point mutation in the BCR/ABL gene in KCL22/SR cells (data not shown). Immunoblot analysis using an anti-BCR antibody showed that there was no difference between the BCR/ABL protein levels in KCL22 and KCL22/SR cells (Fig.1AGo). Furthermore, the expression levels of P-gp in these two cell lines were almost the same (Fig. 1BGo). We therefore concluded that other unknown mechanisms are involved in the acquirement of resistance to imatinib in KCL22/SR cells.



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  		Figure 1. Levels of BCR/ABL and P-gp in KCL22 and KCL22/SR cells. A) The expression of BCR/ABL protein was determined by Western blot analysis using an anti-BCR antibody. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was demonstrated as an internal control. B) Expression of P-gp in KCL22 and KCL22/SR cells was determined by flow cytometry as described in Materials and Methods.

 
Upregulation of Signal Transduction-Related Molecules in KCL22/SR Cells
The level of autophosphorylation of BCR/ABL protein in KCL22/SR cells was immediately suppressed by imatinib treatment, similar to that in KCL22 cells (Fig. 2Go). This finding suggests that imatinib could inhibit BCR/ABL signaling even in KCL22/SR cells and that other mechanisms that were independent of BCR/ABL signaling were involved in the imatinib resistance. To understand the mechanisms involved in the imatinib resistance in KCL22/SR cells, we then examined the differences in gene expression profiles of KCL22 and KCL22/SR cells. DNA microarray analysis is an appropriate method for this purpose because KCL22 and KCL22/SR cells have common genetic backgrounds. Biotin-labeled cRNA was synthesized from KCL22 and KCL22/SR cells and was subjected to hybridization with an oligonucleotide chip, representing a total of 2,304 known human genes. While only three genes exhibited lower expression levels in KCL22/SR cells, 46 genes were initially demonstrated to be preferentially expressed in KCL22/SR cells. However, only two genes were confirmed to be expressed at higher levels in KCL22/SR cells by RT-PCR and real-time PCR methods. These were RASAP1 and RhoA, which are both signal transduction-related molecules (Table 1Go).



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  		Figure 2. Autophosphorylation of BCR/ABL protein in KCL22 and KCL22/SR cells. KCL22/SR cells were cultured without imatinib for 3 days prior to treatment. Cells were treated with 1 or 5 µM imatinib for 6 hours. Immunoblot analysis using anti-phosphotyrosine antibody was performed as described in Materials and Methods. BCR/ABL autophosphorylation levels normalized on the basis of GAPDH are shown in the lower panel.

 

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  		Table 1. Differently expressed genes identified by DNA microarray analysis using an oligonucleotide chip
 
We next tried another DNA microarray analysis using microarrays (Affymetrix) to identify additional genes whose expressions were up- or downregulated in KCL22/SR cells. By this analysis, four genes that were differently expressed in KCL22 and KCL22/SR cells were identified (Table 2Go). The expression level of complement cytolysis inhibitor (CLI), which may be involved in tumor cell resistance to complement-mediated cytotoxicity [13], was shown to be higher in KCL22/SR cells by microarray analysis. However, this could not be confirmed by real-time PCR because of the failure of RT-PCR to detect an amplified product. On the other hand, real-time PCR clearly demonstrated that the expression of L6 was upregulated and that the expression of c-Myb and activin A receptor was downregulated in KCL22/SR cells (Table 2Go).


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  		Table 2. Differently expressed genes identified by DNA microarray analysis using Affymetrix arrays
 
Ras-MAP Kinase Signaling in KCL22/SR Cells Is not Suppressed by Imatinib Treatment
BCR/ABL activates Ras-MAP kinase signaling, which is involved in cellular proliferation and differentiation. Since a Ras mediator, RASAP1, and RhoA, which has cross-talk with Ras signaling, were expressed at high levels in KCL22/SR cells, we speculated that disturbance of Ras-MAP kinase signaling might have occurred in KCL22/SR cells even when BCR/ABL signaling was inhibited by imatinib. To verify this hypothesis, we examined the level of ERK1/2, which belongs to the family of MAP kinases and plays an important role in many gene expressions, by Western blot analysis using anti-p44/42 MAP kinase and anti-phospho-p44/42 MAP kinase (active form) antibodies. The results showed that the level of phosphorylated ERK1/2 was significantly suppressed by imatinib treatment, with no remarkable change in the total amount of ERK1/2 in KCL22 cells (Fig. 3Go). In contrast, neither the level of phosphorylated ERK1/2 nor that of total ERK1/2 in KCL22/SR cells were changed (Fig. 3Go). These results indicated that continuous activation of Ras signaling occurred in KCL22/SR cells and that this activation was not under the control of BCR/ABL signaling.



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  		Figure 3. Changes in ERK1/2 phosphorylation caused by imatinib in KCL22 and KCL22/SR cells. KCL22/SR cells were cultured without imatinib for 3 days prior to treatment. Cells were treated with 1 or 5 µM imatinib for 6 hours. Immunoblot analysis using anti-phospho ERK1/2 (upper panel) and anti-ERK1/2 (lower panel) antibodies was performed as described in Materials and Methods. ERK1 protein levels normalized on the basis of GAPDH are shown in the figure.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently, various new anticancer agents that target specific oncogenic molecules have been developed. Imatinib is one of the most promising reagents among them [14]. However, one of the major problems in imatinib treatment, especially for patients in blast crisis, is acquirement of resistance. Many previous works have shown that several ABC family proteins such as P-gp, multidrug-resistant protein and lung cancer-resistant protein play important roles in multidrug resistance in leukemic cells [15–18]. It is still not clear, however, whether these proteins are involved in resistance to imatinib. Although overexpression and mutations of BCR/ABL have been suggested to be major causes of resistance to imatinib [6], neither overexpression nor mutations of BCR/ABL have been found in some imatinib-resistant cell lines and patients. We therefore tried to determine other mechanisms involved in resistance to imatinib.

For this purpose, we established a new imatinib-resistant cell line, KCL22/SR, in this work. KCL22/SR showed very strong resistance to imatinib, whereas no mutation in the BCR/ABL gene and no increase in BCR/ABL protein and P-gp levels were observed (Fig. 1A–BGo). To the best of our knowledge, two resistant cell lines in which these changes were not observed were previously reported [5], but the mechanisms involved in the resistance of those cells have not been elucidated. The level of autophosphorylation of BCR/ABL protein in KCL22/SR cells was decreased 6 hours after the addition of imatinib, as was also found in KCL22 cells (Fig. 2Go). These results strongly suggest that some mechanisms that are not under the control of BCR/ABL play important roles in the resistance to imatinib in KCL22/SR cells.

To identify imatinib resistance-related molecules, we performed DNA microarray analyses. First, using an oligonucleotide chip, we identified two genes that are expressed at higher levels in KCL22/SR cells than in KCL22 cells (Table 1Go). These were RASAP1 and RhoA, both of which play important roles in signal transduction pathways. RASAP1, which is one of the GTPase-activating proteins and can enhance the intrinsic GTPase activity of Ras proteins, is an effector of Ras protein action [19]. RhoA belongs to the Rho family of small G proteins, which are involved in remodeling of the actin cytoskeleton [20] and cellular proliferation [21]. Rho proteins have also been shown to have cross-talk with Ras signaling [22] and to participate in Ras-mediated induction of carcinogenesis [23, 24]. The upregulation of these molecules strongly suggests that intracellular signal transductions were disturbed in KCL22/SR cells. In fact, while the level of phosphorylated p44/42 was suppressed by imatinib treatment in accordance with the decrease in tyrosine autophosphorylation of BCR/ABL protein in KCL22 cells, it remained high in KCL22/SR cells even when BCR/ABL autophosphorylation was inhibited by imatinib treatment (Fig. 3Go). Since there is no point mutation in the Ras genes in KCL22/SR cells (data not shown), it is possible that the continuous activation of Ras-MAP kinase signaling is caused by unusual expressions of molecules such as RASAP1 and RhoA, and that such disturbance of signal transduction pathways contributes to the resistance to imatinib in these cells.

DNA microarray analysis using Affymetrix microarrays demonstrated that the expressions of L6 and CLI were upregulated and that the expressions of c-Myb and activin A receptor were downregulated in KCL22/SR cells compared with the expressions in KCL22 cells (Table 2Go). L6, whose expression was upregulated 22-fold in KCL22/SR cells, is known to be a surface antigen and to be expressed at high levels in some tumors [25], though its function has not been clarified. CLI may be involved in tumor cell resistance to complement-mediated cytotoxicity [13], but its expression level could not be confirmed by real-time PCR because RT-PCR yields no products. On the other hand, the expression levels of c-Myb and activin A receptor were significantly decreased in KCL22/SR cells.

C-Myb is a transcription factor that is important for the proliferation of early hematopoietic progenitors [26]. A previous study showed that MAP kinase could suppress the transactivating activity of c-Myb through phosphorylation at serine 528 of the carboxy-terminal negative regulatory domain [27]. This finding together with the fact that c-Myb expression is downregulated in KCL22/SR cells suggests that c-Myb function may be suppressed in these cells. Activin A receptor, also called erythroid differentiation factor, is a cell-surface receptor for activin A, which belongs to the transforming growth factor-ß superfamily [28]. Although it remains to be clarified how dysregulation of these molecules contributes to the acquirement of resistance, these results strongly suggest that various mechanisms are involved in the acquirement of resistance to imatinib. Determination of which mechanisms are involved in each case should enable the establishment of effective methods for overcoming the problem of resistance in patients.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to Dr. T. Kondo, Dr. S. Nakano, and Dr. K. Mitsugi for their helpful discussions. We also thank Ms. M. Nakamura for her technical assistance and Ms. E. Yamakawa for her help in preparation of the manuscript.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 30, 2002; accepted for publication February 4, 2003.



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