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Candidate Genes for Cross-Resistance Against DNA-Damaging Drugs1,2

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Candidate Genes for Cross-Resistance Against DNA-Damaging Drugs1,2 [CANCER RESEARCH 62, 6698–6705, November 15, 2002] Candidate Genes for Cross-Resistance against DNA-damaging Drugs1,2 Rainer Wittig, Michelle Nessling, Rainer D. Will, Jan Mollenhauer, Ru¨diger Salowsky, Ewald Mu¨nstermann, Matthias Schick, Heike Helmbach, Brigitte Gschwendt, Bernhard Korn, Petra Kioschis, Peter Lichter, Dirk Schadendorf, and Annemarie Poustka3 Department of Molecular Genome Analysis [R. W., R. D. W., J. M., R. S., E. M., P. K., A. P.], Department of Molecular Genetics, [M. N., P. L.], and Skin Cancer Unit [H. H., B. G., D. S.], Deutsches Krebsforschungszentrum, and Resource Center for Genome Research [M. S., B. K.], D-69120 Heidelberg, Germany ABSTRACT has experienced an increasing attention because of its high and still rising incidence, and the poor prognosis associated with this particular Drug resistance of tumor cells leads to major drawbacks in the treat- kind of skin cancer (11). ment of cancer. To identify candidate genes for drug resistance, we In general, the response of malignant melanoma to chemotherapy is compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired poor, and an improvement of therapeutic protocols is highly desirable. resistance to the DNA-damaging agents cisplatin, etoposide, and fotemus- Several well-characterized drug resistance mechanisms have been tine. Subarray analyses confirmed 57 candidate genes recovered from a studied in regard to their importance for malignant melanoma. Diver- genome-wide scan for differential expression. By specifically addressing gent results have been obtained by these analyses indicating that the cancer genes we retrieved another set of 209 candidates. Exemplary molecular mechanisms underlying drug resistance in melanoma may Northern blot studies indicated qualitative concordance for 110 of 135 be multifaceted and poorly understood (12, 13). (81.4%) data points. Whereas the etoposide-resistant line showed constant The malignant melanoma cell line MeWo has been treated with expression patterns over a period of ϳ2.5 years, the fotemustine- and various cytotoxic compounds, among these the commonly used anti- cisplatin-resistant sublines exhibited considerable variability. Initially cancer drugs cisplatin, fotemustine, and etoposide, to give rise to representing distinct entities, these two sublines finally converged in their sublines with resistance to different concentrations of the respective expression patterns. A total of 110 genes was transiently or permanently deregulated in at least two resistant sublines. Fourteen genes displayed drugs (14). Whereas cisplatin and fotemustine are thought to form differential expression in all three of the sublines. We hypothesize that the DNA adducts, etoposide interferes with topoisomerase II resulting in variations in fotemustine and cisplatin resistance are based on progressive double strand breaks (5, 15–17). Thus, the three drugs have in com- optimization and/or polyclonality. This, in addition to genomic alterations mon the ability to either directly or indirectly lead to DNA damage. investigated by comparative genomic hybridization and evaluation of The resistant cell lines have been characterized in regard to their short-term response genes, can be used as a criterion for the selection of pharmacological properties and genomic alterations (14, 18). Differ- promising candidates. Among these are CYR61, AHCYL1, and MPP1, as ences in gene expression have initially been determined using differ- well as several apoptosis-related genes, in particular STK17A and CRYAB. ential display reverse transcription-PCR and two-dimensional protein As MPP1 and CRYAB are also among the 14 genes differentially expressed gel electrophoresis (19, 20). These efforts have retrieved 15 primary in all three of the drug-resistant sublines, they represent the strongest candidate genes for resistance against the three DNA-damaging drugs, candidates for resistance against DNA-damaging drugs. of which one has been confirmed to date by functional assays (21). The sublines with resistance to DNA-damaging agents have been INTRODUCTION shown to display alterations of pathways involved in the maintenance of DNA integrity (22–25). Drug resistance represents a major problem in cancer therapy. The In specific regard to melanoma therapy cisplatin, but not fotemus- use of in vitro systems has been valuable for the identification and tine or etoposide, presently plays a central role (26). However, the functional verification of several resistance mechanisms. The energy- three MeWo sublines with the highest levels of resistance to cisplatin, dependent drug efflux mediated by members of the ATP binding (MeWoCis1), fotemustine (MeWoFote40), and etoposide (MeWoEto1), cassette transporter protein superfamily (1, 2) is one of the most offer the opportunity to reveal communities in the resistance to important mechanisms for multidrug resistance, and the modulation of DNA-damaging drugs under standardized conditions. Because this is this pathway is in the focus of intense research (3). Other well- of importance for a series of cancer types, we conducted a compre- characterized mechanisms of cellular drug resistance include the de- hensive and detailed analysis of differential gene expression specifi- toxification of drugs via glutathione conjugation (4) and alterations in cally in these three MeWo sublines. the activities or properties of DNA topoisomerases (5). More recently, cellular drug resistance has been associated with alterations in cell death pathways (6, 7), but to date the exact mechanisms remain MATERIALS AND METHODS unclear. Highly parallelized gene expression analyses have been used Cell Culture and Cell Lines. Drug-resistant derivatives of MeWo had to identify additional resistance-associated genes (8–10), because the been selected as described by Kern et al. (14). MeWo cells were grown in presently known mechanisms cannot sufficiently explain the resist- RPMI 1640 supplemented with 10% FCS, the antibiotics penicillin and strep- ance of several tumors. Among these is malignant melanoma, which tomycin (100 units/ml each), and L-glutamine (2 mM). Stable chemoresistant MeWo sublines were grown in supplemented RPMI 1640 with the respective ␮ Received 5/6/02; accepted 9/13/02. drugs: MeWoCis1 with additional cisplatin (1 g/ml), MeWoEto1 with addi- ␮ The costs of publication of this article were defrayed in part by the payment of page tional etoposide (1 g/ml), and MeWoFote40 with additional fotemustine (40 charges. This article must therefore be hereby marked advertisement in accordance with ␮g/ml). In general, cells were grown to 80–90% confluence, and then har- 18 U.S.C. Section 1734 solely to indicate this fact. 1 vested for RNA and DNA isolation. Drug treatment of MeWo cells occurred Supported by the Deutsche Forschungsgemeinschaft Grants Scha 422/7-2 and Scha ϳ 422/7-3 (to D. S.), and the Forschungsfond Mannheim (to D. S.). at 80% confluence for 24 h with the concentrations depicted above. In 2 Supplementary data in this article are available at Cancer Research Online (http:// parallel, MeWo control cells were cultivated for the same time without drugs. cancerres.aacrjournals.org). For the determination of the effects of etoposide treatment on gene expression 3 To whom requests for reprints should be addressed, at Department of Molecular in MeWo , we grew cells with and without etoposide supplementation. For Genome Analysis, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Eto1 Heidelberg, Germany. Phone: 49-6221-424742; Fax: 49-6221-423454; E-mail: the array hybridizations, RNA was prepared from at least three different time [email protected]. points (t1-t3) for each of the cell lines. The cell lines were reconstituted from 6698 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2002 American Association for Cancer Research. DRUG RESISTANCE IN MELANOMA frozen stocks collected during continuous growth over a period of ϳ2.5 years. imaging plates. In the latter case image acquisition was done using a Fuji For the sensitive MeWo reference, the time point t1 represents the parental FLA3000 phosphorimager and AIDA software. cells from which the drug-resistant sublines were established. The time points CGH. Cells were lysed by incubation in 50 mM Tris, 100 mM EDTA, and t2 and t3 represent cells that were continuously cultured for an additional 23 200 mM NaCl (pH 9) with 1% SDS and 0.5 mg/ml proteinase K at 37°C and 42 months, respectively. For all of the drug-resistant sublines, t1 denotes overnight. Genomic DNA was purified by a standard phenol-chloroform the time point of the definition of drug resistance (14). The nomenclature for extraction procedure and subsequent ethanol precipitation. Genomic DNA was M M additional time points of drug-resistant sublines is as follows: MeWoCis1, t2: 12 reconstituted in 10 m Tris and 1 m EDTA (pH 7.5). Probe preparation, hybridization, image acquisition, and analysis of CGH were performed as months and t3: 31 months; MeWoFote40, t2: 11 months and t3: 30 months; and described previously (28). MeWoEto1, t2: 3 months, t3: 22 months, and t4: 46 months after definition of 4 ϩ drug resistance. In CGH analyses, t1 of MeWoCis1 and MeWoFote40 corre- sponds to t1 ϩ 8–9 months. RESULTS cDNA Arrays. The genome-wide cDNA array (RZPD p950 UniGene1) was manufactured as described previously (27). The drug resistance subarray Genome-wide Screen for Differentially Expressed Genes in was composed of
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