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ABCC2) Is Associated with Decreased Formation of Platinum-DNA Adducts and Decreased G2-Arrest in Melanoma Cells Resistant to Cisplatin
ORIGINAL ARTICLE
Overexpression of cMOAT (MRP2/ABCC2) Is Associated with Decreased Formation of Platinum-DNA Adducts and Decreased G2 -Arrest in Melanoma Cells Resistant to Cisplatin Bernd Liedert,n Verena Materna, Dirk Schadendorf,w Jˇrgen Thomale,n and Hermann Lage 1
Institute of Pathology, ChariteŁ Campus Mitte, Humboldt University Berlin, Berlin, Germany; n1Institute of Cell Biology, University of Essen, Germany, wSkin Cancer Unit, German Cancer Research Center (DKFZ), and University Heidelberg, Clinic of Dermatology Mannheim, Mannheim, Germany
Resistance to various anti-neoplastic agents is a common observation in clinical management of melanoma. The biologic mechanisms conferring these di¡erent drug-resistant phenotypes, including resistance against the commonly used anti-cancer drug cisplatin, are unclear. In order to elucidate the role of the membrane adenosine triphosphate binding cassette-transporter cMOAT (canalicular multispeci¢c anion transporter) (MRP2/ABCC2) in cisplatin resistance of melanoma, the expression of this protein was analyzed in the platinum drug-resistant cell line MeWo CIS 1. Cisplatinresistant melanoma cells showed a distinct overexpression of cMOAT on mRNA and protein level. This observation was accompanied by a reduced formation of platinum-induced intrastrand cross-links in the nuclear DNA measured by an immunocytologic assay.
This decrease in DNA platination was accompanied by an accelerated re-entry into the cell cycle after the typical cisplatin-induced G2 arrest, and a resistance to undergo apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity for Pt-d(GpG) adducts was not elevated in platinum drug-resistant melanoma cells. The decrease in platinum-DNA adduct formation in cisplatin-resistant melanoma cells was rather a re£ection of the protecting activity of the transporter cMOAT. In conclusion, the functional inhibition of cMOAT might be a promising strategy in the reversal of resistance to platinum-based anti-cancer drugs in human melanoma. Key words: ABCC2/cisplatin/cMOAT/drug resistance/melanoma/MeWo/ MRP2. J Invest Dermatol 121:172 ^176, 2003
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have not been identi¢ed (Helmbach et al, 2001). Despite the unsatisfactory response rates, anti-cancer drug-based chemotherapy will continue to be the ¢rst line treatment of disseminated melanoma, although there is an urgent need for improvement. One strategy to achieve this, is to identify the molecular mechanisms conferring drug resistance in melanoma, and, based on this knowledge, to develop new treatment modalities to improve response rates in melanoma patients. In order to investigate drug resistance in human melanoma, we have previously established and analyzed a panel of various melanoma-derived cell variants exhibiting di¡erent drug resistance pro¢les (Kern et al, 1997; Lage et al, 1999, 2000, 2001; Nessling et al, 1999; Grottke et al, 2000; Rˇnger et al, 2000; Sinha et al, 2000; Christmann et al, 2001; Helmbach et al, 2002). One of these cell models, MeWo CIS 1, exhibiting a cisplatin-resistant phenotype, was established by continuous exposure of the melanoma cell line MeWo to cisplatin. Although only about 1% of the intracellular cisplatin reacts with nuclear DNA, this is presumably the critical event in cisplatin-mediated cytotoxicity (Gonzalez et al, 2001). The two major adducts are Pt-d(GpG) and Pt-d(ApG) intrastrand cross-links representing about 90% of the DNA platination. The other lesions include Pt-(dG) monoadducts, Pt-d(GpNpG) intrastrand crosslinks, and Pt(dG)2 interstrand cross-links (Fichtinger-Schepman et al, 1985). The formation of these platinium-DNA adducts per se may not be su⁄cient to cause cell death, whereby the exact cascade of the downstream events leading to cell death is not yet clear. It is generally accepted, however, that the formation of
elanoma showed rapidly increasing incidence and mortality rates, and is characterized by a high risk for metastasis. A successful curative treatment is possible only by an opportune, adequate surgical resection. The bene¢t of both adjuvant and palliative systemic chemotherapy is not unequivocally established. Therapeutic regimens based on dacarbazine are the most commonly applied strategies for the treatment of disseminated melanoma; however, only a minority of patients obtain long-lasting response rates. Various response rates have been reported for polychemotherapy treatment protocols, frequently containing cisplatin [cis-dichlorodiamine-platinum (II)]. Although cisplatin is one of the most active and widely applied antineoplastic agents, partial response in melanoma is observed only for about 10% of the patients. Chemotherapeutic strategies are extremely ine¡ective and unsatisfactory for treating melanoma due to the pronounced drug-resistant characteristics of this malignancy. Data from in vitro studies suggest the presence of intrinsic biologic mechanisms conferring this high drug resistance (Schadendorf et al, 1994), whereby the precise cellular mechanisms Manuscript received November 14, 2002; revised February 11, 2003; accepted for publication February 27, 2003 Address correspondence and reprint requests to: PD Dr H. Lage, Institute of Pathology, ChariteŁ Campus Mitte, Humboldt University Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany. Email: hermann.lage @charite.de 1 Both institutes contributed equally to this study.
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platinum-DNA adducts and subsequent triggering of cellular signal transduction pathways leading to apoptosis may be the primary cytotoxic mechanism of cisplatin (Fisher, 1994). In vitro studies demonstrated that cellular mechanisms causing resistance to cisplatin can be associated with detoxi¢cation by methallothioneins (Kelley et al, 1988), increased glutathione (gglutamylcysteinyl glycine) level as well as increased activity of glutathione-S-transferases (Zhang et al, 1998), increased DNA repair (Masuda et al, 1988), decreased activity of the DNA mismatch repair system (Lage and Dietel, 1999), reduced apoptotic response (Perego et al, 1996), or enhanced expression of the ABC (adenosine triphosphate binding cassette)-transporter cMOAT (canalicular multispeci¢c anion transporter) (Taniguchi et al, 1996), also designated as MRP2 or ABCC2. All these studies, however, were performed using carcinoma cells derived from various nonectodermal-derived cells, but not melanoma-derived cells.
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RESULTS AND DISCUSSION
Human melanoma is well known for its high resistance to many anti-neoplastic agents used for therapy. In this study, the cellular mechanisms conferring resistance to the commonly used anticancer drug cisplatin were analyzed in the cisplatin-resistant melanoma cell line MeWo CIS 1. Previous studies have demonstrated that this cisplatin-resistant phenotype was accompanied by: (1) a de¢cient DNA mismatch repair pathway (Lage et al, 1999); (2) a modulation of DNA repair activities (Rˇnger et al, 2000); and (3) an apoptosis de¢ciency (Helmbach et al, 2002). The fundamental biologic principle causing the cisplatin-resistant phenotype in MeWo CIS 1, however, remained unclear. Earlier studies demonstrated that a cisplatin-resistant phenotype could be associated with the overexpression of the ABCtransporter cMOAT. In these studies carcinoma-derived cells with acquired cisplatin resistance (Taniguchi et al, 1996; Kool et al, 1997) or kidney cells transfected with a cMOAT encoding cDNA (Cui
MATERIALS AND METHODS Cell culture Establishment and cell culture conditions of the cisplatinresistant subline MeWo CIS 1, derived from the drug-sensitive human melanoma cell line MeWo (Fogh et al, 1978), was described earlier (Kern et al, 1997; Lage et al, 2001). Cell proliferation assay Chemoresistance was tested proliferation assay as described previously (Lage et al, 2001).
using a
Northern blot Expression of the cMOAT encoding mRNA was determined by northern blot analysis applying standard procedures as described previously (Lage et al, 2001). A 453 bp cMOATspeci¢c hybridization probe was generated by reverse transcription^polymerase chain reaction (reverse transcription^PCR) using cMOAT-speci¢c oligonucleotide primers designed after alignment with published sequences (GenBank accession number NM 000392): cMOAT-forward, 50 -GGA ACA ATT GTA GAG AAA GGA TC-30; and cMOAT-reverse, 50 -CAC AAA CGC AAG GAT GAT GAA GAA-30. As control for equal RNA loading the membranes were rehybridized with a glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA probe, prepared as described in the next subsection (‘Quantitative reverse transcription^PCR’). Quantitative reverse transcription^PCR For quantitative reverse transcription^PCR, a real-time reverse transcription^PCR was carried out using a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). Oligodeoxynucleotide primers used for ampli¢cation were cMOAT-forward and cMOAT-reverse (see above) and as control GAPDH-forward, 50 -CAC CGT CAA GGC TGA GAA C-30; and GAPDH-reverse, 50 -ACC ACT GAC ACG TTG GCA G-30 yielding an expected ampli¢cation product of 556 bp speci¢c for the house keeping gene encoding GAPDH (GenBank accession number NM 002046). Western blot Western blot experiments were performed as described previously (Lage and Dietel, 2002). The antibodies used were a mouse monoclonal antibody (MoAb) directed against cMOAT (clone M2I-4; Sanbio, Uden, the Netherlands), and as control for equivalent protein loading, a mouse MoAb directed against actin (Chemicon, Temecula, California; no. MAB 1501R). Protein^antibody complexes were visualized by chemoluminescence (ECL system, Amersham Aylesbury, UK). Cell cycle analyses by £ow cytometry For cell-cycle analysis in exponentially growing cell cultures, melanoma cells were exposed to 5 mg cisplatin per mL. After 24 h, 48 h, or 72 h cells were ¢xed in ice-cold 75% ethanol and stored at ^201C. After washing cells were resuspended in PBS containing 200 mg DNase-free RNase A per mL, 50 units per mL RNAse T1 and 20 mg propidium iodide per mL and incubated for 30 min at 251C. Analysis was performed on a FACScan £ow cytometer (Calibur 750; Becton Dickinson, Mountain View, California) using Cellquest software. Detection of platinum-DNA adducts Cisplatin-induced Pt-d(GpG) intrastrand cross-links in the nuclear DNA of single cells were visualized and measured by an immunocytologic assay using adduct-speci¢c MoAb as described previously (Buschfort-Papewalis et al, 2002).
Figure 1. (A) Northern blot analysis of cMOAT mRNA expression in human melanoma cells. RNA was hybridized with a 32P-labeled cMOAT-speci¢c cDNA. As control, the blots were probed with a cDNA probe of GAPDH. (B) Relative cMOAT mRNA expression level after normalization against GAPDH expression level determined by quantitative real time reverse transcription^PCR using a LightCycler instrument. Cisplatin-resistant MeWo CIS 1 cells showed a 3.7-fold enhanced expression of the cMOAT mRNA. (C) Western blot analysis using a mouse MoAb M2I- 4 directed against cMOAT. As control, a mouse MoAb directed against actin was used.
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et al, 1999; Kawabe et al, 1999) were used. The conclusiveness of these studies, however, was hampered by the lack of functional assays. Thus, here the involvement of cMOAT in platinum resistance of human melanoma was investigated. Expression analyses revealed that cMOAT is overexpressed in the cisplatin-resistant melanoma cell MeWo CIS 1 on the mRNA level (Fig 1A,B) as well as on the protein level (Fig 1C). No expression of alternative drug resistance-associated ABC-transporters, such as MDR1/P-glycoprotein, MRP1, or BCRP was found in MeWo CIS 1 (data not shown); neither on mRNA level as measured by reverse transcription^PCR and northern blot, nor on protein level as determined by western blot. For examination whether the cross-resistant phenotype of MeWo CIS 1 is in accordance with the typical cMOAT-speci¢c cross-resistance pattern (Cui et al, 1999), the cytotoxicities of di¡erent anti-cancer drugs were determined (Table I). As expected for a cMOAT-mediated cross-resistance, the cisplatin-resistant cell variant exhibited a high cross-resistance to carboplatin, whereas no or only a weak cross-resistance could be observed against the remaining drugs. Flow cytometry-based cell cycle analysis revealed a typical cisplatin-triggered G2 arrest (Chu, 1994) in drug-sensitive melanoma cells as well as in cisplatin-resistant melanoma cells (Fig 2). After treatment with a low cisplatin concentration (5 mg per mL) both cell variants were able to re-enter the cell cycle, but with a distinct delay in MeWo cells (Fig 2A,B). Exposure to a higher dose of cisplatin (20 mg per mL) resulted in a complete G2 arrest in MeWo cells even after 72 h with clear signs of apoptosis (Fig 2C), whereas the resistant MeWo CIS 1 cells were able to re-enter the cell cycle (Fig 2D). The changes in cell cycle distribution of
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MeWo CIS 1 cells after high dosage cisplatin (20 mg per mL) were comparable with the e¡ect observed in the parental MeWo cells exposed a 4 -fold lower dose of the drug, which is in complete concordance with the relative resistance factor of 3.7 (Table I). To elucidate whether this decreased inclination to undergo cisplatin-induced cell cycle arrest and cell death is caused by a decreased platinum-DNA adduct formation, by more e⁄cient adduct repair, or by augmented tolerance to DNA damage, the kinetics of formation and elimination of nuclear Pt-d(GpG) intrastrand cross-links, the major product of cisplatin-induced DNA adducts were measured. In the ¢rst hours after cisplatin Table I. Cross resistance pattern of cisplatin-resistant melanoma cells Cell Lines
Drugs
MeWo IC501
MeWo CIS 1 IC501 - RR2 p-value3
cisplatin carboplatin daunoblastin etoposide vincristine
5.5 55.3 0.0121 0.1957 0.000793
20.3^3.7^o 0.001 158.4^2.9^o 0.001 0.0162^1.3^o 0.01 0.3139^1.6^o 0.05 0.000778^1.0^n.s.4
1
IC50, inhibitory concentration (mM). RR, relative resistance (x-fold). p-value, calculated by Student’s T-test. 4 n.s., no signi¢cant di¡erence. 2 3
Figure 2. Cell cycle analyses of drug-sensitive (MeWo) and cisplatin-resistant (MeWo CIS 1) melanoma cells after propidium iodide staining using £ow cytometry. Analyses were performed at 0 h, 24 h, 48 h, and 72 h after cisplatin treatment. (A) Exposure of MeWo to 5 mg cisplatin per mL: t ¼ 0 h, 31.4% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 80.0% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 67.2% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 56.1% in S þ G2 phase. (B) Exposure of MeWo CIS 1^5 mg cisplatin per mL: t ¼ 0 h, 30.9% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 58.3% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 40.5% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 29.6% in S þ G2 phase. (C) Exposure of MeWo to 20 mg cisplatin per mL: t ¼ 0 h, 31,4% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 87,4% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 92.2% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 98,7% in S þ G2 phase. (D) Exposure of MeWo CIS 1^20 mg cisplatin per mL: t ¼ 0 h, 30,9% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 79.2% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 66,6% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 63.7% in S þ G2 phase.
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exposure, in both melanoma variants the detected nuclear amounts of Pt-d(GpG) adducts corresponded to the relationship of the ongoing formation of platinum-DNA intrastrand crosslinks and the initiating adduct elimination by activated cellular DNA repair (Fig 3). After a time period of 8 h, the peak of nuclear platinum-DNA adduct content was reached and then amounts of adducts started to drop, ¢rst caused predominantly by prevailing DNA repair and later supported by ‘‘adduct dilution’’due to ongoing cell proliferation. At each time point after cisplatin exposure, the nuclear levels of platinum-DNA adducts were lower in drug-resistant cells compared with the cisplatin-sensitive cell line (Fig 3). MeWo CIS 1 cells did not show a faster or more e⁄cient removal of Ptd(GpG) cross-links in comparison with the parental MeWo cell line, indicating that in both cell variants similar DNA repair activities were triggered by these lesions. The fact that adduct levels were already 2-fold lower in MeWoCIS 1 cells as early as 2 h after cisplatin exposure (t0) clearly suggests a decreased adduct formation in these cells resulting in more convenient conditions for proliferation, reduced cell cycle e¡ects, and less induction of apoptosis. This situation is re£ected by the early re-entry of MeWo CIS 1 cells into the cell cycle following G2 arrest and is accompanied by a drug-resistant phenotype as measured in the cell proliferation assay (Table I). Both measures, peak cross-link formation and integrated ‘‘adducts under curve’’ values strongly suggest a signi¢cantly reduced DNA platination by cisplatin in MeWo CIS 1. To induce an adduct burden comparable with that in the sensitive parental line MeWo, cisplatin-resistant cells had to be exposed to a four times higher dose of cisplatin (20 mg per mL) (Fig 3). Altogether, the data imply, that the biologic mechanism leading to the decreased drug sensitivity of MeWo CIS 1 cells acts very early after cisplatin exposure. This instantly acting resistance mechanism might be represented by the ABC-transporter cMOAT that can extrude platinum-based compounds from cells
cMOAT IN MELANOMA CELLS
175
directly after in£ux of the drug. Thus, cMOAT was identi¢ed as the most decisive cellular mechanism to prevent the formation of cisplatin-induced DNA lesions, and therewith to circumvent the activation of signal transduction pathways leading to cell cycle arrest and apoptosis in MeWo CIS 1 cells. DNA-repair analyses from a previous study (Rˇnger et al, 2000) and the data from this study imply that neither an increase in the activity of the nucleotide excision repair pathway nor in the base excision repair pathway is involved in the platinum-resistant phenotype of MeWo CIS 1. The decreased mismatch repair pathway in MeWo CIS 1 (Lage et al, 1999) has most likely no in£uence on the elimination of platinum-DNA adducts. The mismatch repair system rather participates in the recognition of these adducts, and as a result, trigger pathways that end in programmed cell death (Lage and Dietel, 1999). Lower levels of mismatch repair components therefore may lead to a reduction of apoptotic signals and therewith to an elevated survival of drug-treated tumor cells. A decreased activation of distinct apoptotic pathways has been reported for the cisplatin-resistant melanoma cells. After treatment with cisplatin (15 mg per mL) these cells showed a reduction in caspase 9-like activity, in cytochrome c release, and in poly (adenosine diphosphate ribose) polymerase cleavage (Helmbach et al, 2002). In the light of the ¢ndings in this study, however, these di¡erences are most likely due to the reduced DNA-adduct formation in MeWo CIS 1 cells. In summary, the data from this study taken together with previously published results indicate that the cisplatin-resistant phenotype of MeWo CIS 1 cells is mediated predominantly by the decreased formation of platinum-DNA adducts due to the overexpression of cMOAT. A 4 -fold higher dose of cisplatin is necessary in MeWo CIS 1 compared with MeWo cells to achieve: (1) the same cytotoxic e¡ects; (2) a similar degree of cell cycle disturbance; and (3) the same burden of DNA-platination. The data obtained in this study may be important for clinical management of melanoma as they demonstrate that the crucial e¡ect for the mediation of the cytotoxic e¡ects of platinum-based anti-neoplastic agents is the formation of platinum-DNA adducts. Thus, the prevention of a decreased platinum-DNA adduct formation in cisplatin-resistant melanoma cells is the most promising strategy to overcome cisplatin resistance. All consecutive cellular events are merely a direct consequence of the platinumDNA adduct formation. Accordingly, the inhibition of the biologic activity of cMOAT should be the main goal in overcoming resistance to platinum-containing anti-cancer drugs in human melanoma. Moreover, cMOAT appears as an excellent candidate factor for the diagnosis of clinical platinum resistance in melanoma. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) to H.L. (LA 1039/1-3) and to J.T. (FOR 236/2).We like to thank Beate Karow and Birgit Schaefer for skillful technical assistance.
REFERENCES
Figure 3. Time kinetics of cisplatin-induced Pt-d(GpG) adduct formation in the drug-sensitive melanoma line MeWo and its cisplatinresistant subline MeWo CIS 1 as measured by an immunocytologic assay. Cisplatin was added at t ¼ ^ 2 h. The cells were exposed to 5 or 20 mg per mL cisplatin for 2 h and nuclear Pt-d(GpG) adduct content was determined after di¡erent time periods by £uorescence microscopy using the rat anti-Pt-d(GpG) adduct MoAb ‘‘18G10’’ and a £uorescein isothiocyanate-based double sandwich staining procedure. Signals were ampli¢ed and recorded by a dual mode CCD camera, and fed into a four parameter image analysis program. Displayed values represent the mean, the vertical error bars SD.
Buschfort-Papewalis C, Moritz T, Liedert B, Thomale J: Down-regulation of DNA repair in human CD34( þ ) progenitor cells corresponds to increased drug sensitivity and apoptotic response. Blood 100:845^853, 2002 Christmann M, Pick M, Lage H, Schadendorf D, Kaina B: Acquired resistance of melanoma cells to the antineoplastic agent fotemustine is caused by reactivation of the DNA repair gene MGMT. Int J Cancer 92:123^129, 2001 Chu G: Cellular responses to cisplatin. The roles of DNA-binding proteins and DNA repair. J Biol Chem 269:787^790, 1994 Cui Y, K˛nig J, Buchholz JK, Spring H, Leier I, Keppler D: Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 55:929^937, 1999 Fichtinger-Schepman AM, van der Veer JL, den Hartog JH, Lohman PH, Reedijk J: Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA. Formation, identi¢cation, and quantitation.. Biochemistry 24:707^713, 1985 Fisher DE: Apoptosis in cancer therapy: Crossing the threshold. Cell 78:539^542, 1994
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Lage H, Christmann M, Kern MA, Dietel M, Pick M, Kaina B, Schadendorf D: Expression of DNA repair proteins hMSH2, hMSH6, hMLH1, O6 -methylguanine-DNA metyltransfease and N-methylpurine-DNA glycosylase in melanoma cells with acquired drug resistance. Int J Cancer 80:744^750, 1999 Lage H, Helmbach H, Dietel M, Schadendorf D: Modulation of DNA topoisomerase II activity and expression in melanoma cells with acquired drug resistance. Br J Cancer 82:488^491, 2000 Lage H, Helmbach H, Grottke C, Dietel M, Schadendorf D: DFNA5 (ICERE-1) contributes to acquired etoposide resistance in melanoma cells. FEBS Lett 494:54^59, 2001 Masuda H, Ozols RF, Lai GM, Fojo A, Rothenberg M, Hamilton TC: Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum(II) in human ovarian cancer cell lines. Cancer Res 48:5713^5716, 1988 Nessling M, Kern MA, Schadendorf D, Lichter P: Association of genomic imbalances with resistance to therapeutic drugs in human melanoma cell lines. Cytogenet Cell Genet 87:286^290, 1999 Perego P, Giarola M, Righetti SC, et al: Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Res 56:556^562, 1996 Rˇnger TM, Emmert S, Schadendorf D, Diem C, Epe B, Hellfritsch D: Alterations of DNA repair in melanoma cell lines resistant to cisplatin, fotemustine, or etoposide. J Invest Dermatol 114:34^39, 2000 Schadendorf D, Worm M, Algermissen B, Kohlmus CM, Czarnetzki BM: Chemosensitivity testing of human malignant melanoma. A retrospective analysis of clinical response and in vitro drug sensitivity. Cancer 73:103^108, 1994 Sinha P, Kohl S, Fischer J, et al: Identi¢cation of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis. Electrophoresis 21:3048^3057, 2000 Taniguchi K, Wada M, Kohno K, et al: A human canalicular multispeci¢c organic anion transporter (cMOAT) gene is overexpressed in cisplatin-resistant human cancer cell lines with decreased drug accumulation. Cancer Res 56:4124^4129, 1996 Zhang K, Mack P, Wong KP: Glutathione-related mechanisms in cellular resistance to anticancer drugs. Int J Oncol 12:871^882, 1998
Overexpression of cMOAT (MRP2/ABCC2) Is Associated with Decreased Formation of Platinum-DNA Adducts and Decreased G2 -Arrest in Melanoma Cells Resistant to Cisplatin Bernd Liedert,n Verena Materna, Dirk Schadendorf,w Jˇrgen Thomale,n and Hermann Lage 1
Institute of Pathology, ChariteŁ Campus Mitte, Humboldt University Berlin, Berlin, Germany; n1Institute of Cell Biology, University of Essen, Germany, wSkin Cancer Unit, German Cancer Research Center (DKFZ), and University Heidelberg, Clinic of Dermatology Mannheim, Mannheim, Germany
Resistance to various anti-neoplastic agents is a common observation in clinical management of melanoma. The biologic mechanisms conferring these di¡erent drug-resistant phenotypes, including resistance against the commonly used anti-cancer drug cisplatin, are unclear. In order to elucidate the role of the membrane adenosine triphosphate binding cassette-transporter cMOAT (canalicular multispeci¢c anion transporter) (MRP2/ABCC2) in cisplatin resistance of melanoma, the expression of this protein was analyzed in the platinum drug-resistant cell line MeWo CIS 1. Cisplatinresistant melanoma cells showed a distinct overexpression of cMOAT on mRNA and protein level. This observation was accompanied by a reduced formation of platinum-induced intrastrand cross-links in the nuclear DNA measured by an immunocytologic assay.
This decrease in DNA platination was accompanied by an accelerated re-entry into the cell cycle after the typical cisplatin-induced G2 arrest, and a resistance to undergo apoptosis. Kinetics of formation and elimination of platinum-DNA adducts suggest that the DNA repair capacity for Pt-d(GpG) adducts was not elevated in platinum drug-resistant melanoma cells. The decrease in platinum-DNA adduct formation in cisplatin-resistant melanoma cells was rather a re£ection of the protecting activity of the transporter cMOAT. In conclusion, the functional inhibition of cMOAT might be a promising strategy in the reversal of resistance to platinum-based anti-cancer drugs in human melanoma. Key words: ABCC2/cisplatin/cMOAT/drug resistance/melanoma/MeWo/ MRP2. J Invest Dermatol 121:172 ^176, 2003
M
have not been identi¢ed (Helmbach et al, 2001). Despite the unsatisfactory response rates, anti-cancer drug-based chemotherapy will continue to be the ¢rst line treatment of disseminated melanoma, although there is an urgent need for improvement. One strategy to achieve this, is to identify the molecular mechanisms conferring drug resistance in melanoma, and, based on this knowledge, to develop new treatment modalities to improve response rates in melanoma patients. In order to investigate drug resistance in human melanoma, we have previously established and analyzed a panel of various melanoma-derived cell variants exhibiting di¡erent drug resistance pro¢les (Kern et al, 1997; Lage et al, 1999, 2000, 2001; Nessling et al, 1999; Grottke et al, 2000; Rˇnger et al, 2000; Sinha et al, 2000; Christmann et al, 2001; Helmbach et al, 2002). One of these cell models, MeWo CIS 1, exhibiting a cisplatin-resistant phenotype, was established by continuous exposure of the melanoma cell line MeWo to cisplatin. Although only about 1% of the intracellular cisplatin reacts with nuclear DNA, this is presumably the critical event in cisplatin-mediated cytotoxicity (Gonzalez et al, 2001). The two major adducts are Pt-d(GpG) and Pt-d(ApG) intrastrand cross-links representing about 90% of the DNA platination. The other lesions include Pt-(dG) monoadducts, Pt-d(GpNpG) intrastrand crosslinks, and Pt(dG)2 interstrand cross-links (Fichtinger-Schepman et al, 1985). The formation of these platinium-DNA adducts per se may not be su⁄cient to cause cell death, whereby the exact cascade of the downstream events leading to cell death is not yet clear. It is generally accepted, however, that the formation of
elanoma showed rapidly increasing incidence and mortality rates, and is characterized by a high risk for metastasis. A successful curative treatment is possible only by an opportune, adequate surgical resection. The bene¢t of both adjuvant and palliative systemic chemotherapy is not unequivocally established. Therapeutic regimens based on dacarbazine are the most commonly applied strategies for the treatment of disseminated melanoma; however, only a minority of patients obtain long-lasting response rates. Various response rates have been reported for polychemotherapy treatment protocols, frequently containing cisplatin [cis-dichlorodiamine-platinum (II)]. Although cisplatin is one of the most active and widely applied antineoplastic agents, partial response in melanoma is observed only for about 10% of the patients. Chemotherapeutic strategies are extremely ine¡ective and unsatisfactory for treating melanoma due to the pronounced drug-resistant characteristics of this malignancy. Data from in vitro studies suggest the presence of intrinsic biologic mechanisms conferring this high drug resistance (Schadendorf et al, 1994), whereby the precise cellular mechanisms Manuscript received November 14, 2002; revised February 11, 2003; accepted for publication February 27, 2003 Address correspondence and reprint requests to: PD Dr H. Lage, Institute of Pathology, ChariteŁ Campus Mitte, Humboldt University Berlin, Schumannstr. 20/21, D-10117 Berlin, Germany. Email: hermann.lage @charite.de 1 Both institutes contributed equally to this study.
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platinum-DNA adducts and subsequent triggering of cellular signal transduction pathways leading to apoptosis may be the primary cytotoxic mechanism of cisplatin (Fisher, 1994). In vitro studies demonstrated that cellular mechanisms causing resistance to cisplatin can be associated with detoxi¢cation by methallothioneins (Kelley et al, 1988), increased glutathione (gglutamylcysteinyl glycine) level as well as increased activity of glutathione-S-transferases (Zhang et al, 1998), increased DNA repair (Masuda et al, 1988), decreased activity of the DNA mismatch repair system (Lage and Dietel, 1999), reduced apoptotic response (Perego et al, 1996), or enhanced expression of the ABC (adenosine triphosphate binding cassette)-transporter cMOAT (canalicular multispeci¢c anion transporter) (Taniguchi et al, 1996), also designated as MRP2 or ABCC2. All these studies, however, were performed using carcinoma cells derived from various nonectodermal-derived cells, but not melanoma-derived cells.
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RESULTS AND DISCUSSION
Human melanoma is well known for its high resistance to many anti-neoplastic agents used for therapy. In this study, the cellular mechanisms conferring resistance to the commonly used anticancer drug cisplatin were analyzed in the cisplatin-resistant melanoma cell line MeWo CIS 1. Previous studies have demonstrated that this cisplatin-resistant phenotype was accompanied by: (1) a de¢cient DNA mismatch repair pathway (Lage et al, 1999); (2) a modulation of DNA repair activities (Rˇnger et al, 2000); and (3) an apoptosis de¢ciency (Helmbach et al, 2002). The fundamental biologic principle causing the cisplatin-resistant phenotype in MeWo CIS 1, however, remained unclear. Earlier studies demonstrated that a cisplatin-resistant phenotype could be associated with the overexpression of the ABCtransporter cMOAT. In these studies carcinoma-derived cells with acquired cisplatin resistance (Taniguchi et al, 1996; Kool et al, 1997) or kidney cells transfected with a cMOAT encoding cDNA (Cui
MATERIALS AND METHODS Cell culture Establishment and cell culture conditions of the cisplatinresistant subline MeWo CIS 1, derived from the drug-sensitive human melanoma cell line MeWo (Fogh et al, 1978), was described earlier (Kern et al, 1997; Lage et al, 2001). Cell proliferation assay Chemoresistance was tested proliferation assay as described previously (Lage et al, 2001).
using a
Northern blot Expression of the cMOAT encoding mRNA was determined by northern blot analysis applying standard procedures as described previously (Lage et al, 2001). A 453 bp cMOATspeci¢c hybridization probe was generated by reverse transcription^polymerase chain reaction (reverse transcription^PCR) using cMOAT-speci¢c oligonucleotide primers designed after alignment with published sequences (GenBank accession number NM 000392): cMOAT-forward, 50 -GGA ACA ATT GTA GAG AAA GGA TC-30; and cMOAT-reverse, 50 -CAC AAA CGC AAG GAT GAT GAA GAA-30. As control for equal RNA loading the membranes were rehybridized with a glyceraldehyde-3 phosphate dehydrogenase (GAPDH) cDNA probe, prepared as described in the next subsection (‘Quantitative reverse transcription^PCR’). Quantitative reverse transcription^PCR For quantitative reverse transcription^PCR, a real-time reverse transcription^PCR was carried out using a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). Oligodeoxynucleotide primers used for ampli¢cation were cMOAT-forward and cMOAT-reverse (see above) and as control GAPDH-forward, 50 -CAC CGT CAA GGC TGA GAA C-30; and GAPDH-reverse, 50 -ACC ACT GAC ACG TTG GCA G-30 yielding an expected ampli¢cation product of 556 bp speci¢c for the house keeping gene encoding GAPDH (GenBank accession number NM 002046). Western blot Western blot experiments were performed as described previously (Lage and Dietel, 2002). The antibodies used were a mouse monoclonal antibody (MoAb) directed against cMOAT (clone M2I-4; Sanbio, Uden, the Netherlands), and as control for equivalent protein loading, a mouse MoAb directed against actin (Chemicon, Temecula, California; no. MAB 1501R). Protein^antibody complexes were visualized by chemoluminescence (ECL system, Amersham Aylesbury, UK). Cell cycle analyses by £ow cytometry For cell-cycle analysis in exponentially growing cell cultures, melanoma cells were exposed to 5 mg cisplatin per mL. After 24 h, 48 h, or 72 h cells were ¢xed in ice-cold 75% ethanol and stored at ^201C. After washing cells were resuspended in PBS containing 200 mg DNase-free RNase A per mL, 50 units per mL RNAse T1 and 20 mg propidium iodide per mL and incubated for 30 min at 251C. Analysis was performed on a FACScan £ow cytometer (Calibur 750; Becton Dickinson, Mountain View, California) using Cellquest software. Detection of platinum-DNA adducts Cisplatin-induced Pt-d(GpG) intrastrand cross-links in the nuclear DNA of single cells were visualized and measured by an immunocytologic assay using adduct-speci¢c MoAb as described previously (Buschfort-Papewalis et al, 2002).
Figure 1. (A) Northern blot analysis of cMOAT mRNA expression in human melanoma cells. RNA was hybridized with a 32P-labeled cMOAT-speci¢c cDNA. As control, the blots were probed with a cDNA probe of GAPDH. (B) Relative cMOAT mRNA expression level after normalization against GAPDH expression level determined by quantitative real time reverse transcription^PCR using a LightCycler instrument. Cisplatin-resistant MeWo CIS 1 cells showed a 3.7-fold enhanced expression of the cMOAT mRNA. (C) Western blot analysis using a mouse MoAb M2I- 4 directed against cMOAT. As control, a mouse MoAb directed against actin was used.
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et al, 1999; Kawabe et al, 1999) were used. The conclusiveness of these studies, however, was hampered by the lack of functional assays. Thus, here the involvement of cMOAT in platinum resistance of human melanoma was investigated. Expression analyses revealed that cMOAT is overexpressed in the cisplatin-resistant melanoma cell MeWo CIS 1 on the mRNA level (Fig 1A,B) as well as on the protein level (Fig 1C). No expression of alternative drug resistance-associated ABC-transporters, such as MDR1/P-glycoprotein, MRP1, or BCRP was found in MeWo CIS 1 (data not shown); neither on mRNA level as measured by reverse transcription^PCR and northern blot, nor on protein level as determined by western blot. For examination whether the cross-resistant phenotype of MeWo CIS 1 is in accordance with the typical cMOAT-speci¢c cross-resistance pattern (Cui et al, 1999), the cytotoxicities of di¡erent anti-cancer drugs were determined (Table I). As expected for a cMOAT-mediated cross-resistance, the cisplatin-resistant cell variant exhibited a high cross-resistance to carboplatin, whereas no or only a weak cross-resistance could be observed against the remaining drugs. Flow cytometry-based cell cycle analysis revealed a typical cisplatin-triggered G2 arrest (Chu, 1994) in drug-sensitive melanoma cells as well as in cisplatin-resistant melanoma cells (Fig 2). After treatment with a low cisplatin concentration (5 mg per mL) both cell variants were able to re-enter the cell cycle, but with a distinct delay in MeWo cells (Fig 2A,B). Exposure to a higher dose of cisplatin (20 mg per mL) resulted in a complete G2 arrest in MeWo cells even after 72 h with clear signs of apoptosis (Fig 2C), whereas the resistant MeWo CIS 1 cells were able to re-enter the cell cycle (Fig 2D). The changes in cell cycle distribution of
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MeWo CIS 1 cells after high dosage cisplatin (20 mg per mL) were comparable with the e¡ect observed in the parental MeWo cells exposed a 4 -fold lower dose of the drug, which is in complete concordance with the relative resistance factor of 3.7 (Table I). To elucidate whether this decreased inclination to undergo cisplatin-induced cell cycle arrest and cell death is caused by a decreased platinum-DNA adduct formation, by more e⁄cient adduct repair, or by augmented tolerance to DNA damage, the kinetics of formation and elimination of nuclear Pt-d(GpG) intrastrand cross-links, the major product of cisplatin-induced DNA adducts were measured. In the ¢rst hours after cisplatin Table I. Cross resistance pattern of cisplatin-resistant melanoma cells Cell Lines
Drugs
MeWo IC501
MeWo CIS 1 IC501 - RR2 p-value3
cisplatin carboplatin daunoblastin etoposide vincristine
5.5 55.3 0.0121 0.1957 0.000793
20.3^3.7^o 0.001 158.4^2.9^o 0.001 0.0162^1.3^o 0.01 0.3139^1.6^o 0.05 0.000778^1.0^n.s.4
1
IC50, inhibitory concentration (mM). RR, relative resistance (x-fold). p-value, calculated by Student’s T-test. 4 n.s., no signi¢cant di¡erence. 2 3
Figure 2. Cell cycle analyses of drug-sensitive (MeWo) and cisplatin-resistant (MeWo CIS 1) melanoma cells after propidium iodide staining using £ow cytometry. Analyses were performed at 0 h, 24 h, 48 h, and 72 h after cisplatin treatment. (A) Exposure of MeWo to 5 mg cisplatin per mL: t ¼ 0 h, 31.4% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 80.0% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 67.2% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 56.1% in S þ G2 phase. (B) Exposure of MeWo CIS 1^5 mg cisplatin per mL: t ¼ 0 h, 30.9% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 58.3% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 40.5% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 29.6% in S þ G2 phase. (C) Exposure of MeWo to 20 mg cisplatin per mL: t ¼ 0 h, 31,4% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 87,4% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 92.2% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 98,7% in S þ G2 phase. (D) Exposure of MeWo CIS 1^20 mg cisplatin per mL: t ¼ 0 h, 30,9% in S þ G2 phase; t ¼ 24 h after cisplatin treatment, 79.2% in S þ G2 phase; t ¼ 48 h after cisplatin treatment, 66,6% in S þ G2 phase; t ¼ 72 h after cisplatin treatment, 63.7% in S þ G2 phase.
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exposure, in both melanoma variants the detected nuclear amounts of Pt-d(GpG) adducts corresponded to the relationship of the ongoing formation of platinum-DNA intrastrand crosslinks and the initiating adduct elimination by activated cellular DNA repair (Fig 3). After a time period of 8 h, the peak of nuclear platinum-DNA adduct content was reached and then amounts of adducts started to drop, ¢rst caused predominantly by prevailing DNA repair and later supported by ‘‘adduct dilution’’due to ongoing cell proliferation. At each time point after cisplatin exposure, the nuclear levels of platinum-DNA adducts were lower in drug-resistant cells compared with the cisplatin-sensitive cell line (Fig 3). MeWo CIS 1 cells did not show a faster or more e⁄cient removal of Ptd(GpG) cross-links in comparison with the parental MeWo cell line, indicating that in both cell variants similar DNA repair activities were triggered by these lesions. The fact that adduct levels were already 2-fold lower in MeWoCIS 1 cells as early as 2 h after cisplatin exposure (t0) clearly suggests a decreased adduct formation in these cells resulting in more convenient conditions for proliferation, reduced cell cycle e¡ects, and less induction of apoptosis. This situation is re£ected by the early re-entry of MeWo CIS 1 cells into the cell cycle following G2 arrest and is accompanied by a drug-resistant phenotype as measured in the cell proliferation assay (Table I). Both measures, peak cross-link formation and integrated ‘‘adducts under curve’’ values strongly suggest a signi¢cantly reduced DNA platination by cisplatin in MeWo CIS 1. To induce an adduct burden comparable with that in the sensitive parental line MeWo, cisplatin-resistant cells had to be exposed to a four times higher dose of cisplatin (20 mg per mL) (Fig 3). Altogether, the data imply, that the biologic mechanism leading to the decreased drug sensitivity of MeWo CIS 1 cells acts very early after cisplatin exposure. This instantly acting resistance mechanism might be represented by the ABC-transporter cMOAT that can extrude platinum-based compounds from cells
cMOAT IN MELANOMA CELLS
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directly after in£ux of the drug. Thus, cMOAT was identi¢ed as the most decisive cellular mechanism to prevent the formation of cisplatin-induced DNA lesions, and therewith to circumvent the activation of signal transduction pathways leading to cell cycle arrest and apoptosis in MeWo CIS 1 cells. DNA-repair analyses from a previous study (Rˇnger et al, 2000) and the data from this study imply that neither an increase in the activity of the nucleotide excision repair pathway nor in the base excision repair pathway is involved in the platinum-resistant phenotype of MeWo CIS 1. The decreased mismatch repair pathway in MeWo CIS 1 (Lage et al, 1999) has most likely no in£uence on the elimination of platinum-DNA adducts. The mismatch repair system rather participates in the recognition of these adducts, and as a result, trigger pathways that end in programmed cell death (Lage and Dietel, 1999). Lower levels of mismatch repair components therefore may lead to a reduction of apoptotic signals and therewith to an elevated survival of drug-treated tumor cells. A decreased activation of distinct apoptotic pathways has been reported for the cisplatin-resistant melanoma cells. After treatment with cisplatin (15 mg per mL) these cells showed a reduction in caspase 9-like activity, in cytochrome c release, and in poly (adenosine diphosphate ribose) polymerase cleavage (Helmbach et al, 2002). In the light of the ¢ndings in this study, however, these di¡erences are most likely due to the reduced DNA-adduct formation in MeWo CIS 1 cells. In summary, the data from this study taken together with previously published results indicate that the cisplatin-resistant phenotype of MeWo CIS 1 cells is mediated predominantly by the decreased formation of platinum-DNA adducts due to the overexpression of cMOAT. A 4 -fold higher dose of cisplatin is necessary in MeWo CIS 1 compared with MeWo cells to achieve: (1) the same cytotoxic e¡ects; (2) a similar degree of cell cycle disturbance; and (3) the same burden of DNA-platination. The data obtained in this study may be important for clinical management of melanoma as they demonstrate that the crucial e¡ect for the mediation of the cytotoxic e¡ects of platinum-based anti-neoplastic agents is the formation of platinum-DNA adducts. Thus, the prevention of a decreased platinum-DNA adduct formation in cisplatin-resistant melanoma cells is the most promising strategy to overcome cisplatin resistance. All consecutive cellular events are merely a direct consequence of the platinumDNA adduct formation. Accordingly, the inhibition of the biologic activity of cMOAT should be the main goal in overcoming resistance to platinum-containing anti-cancer drugs in human melanoma. Moreover, cMOAT appears as an excellent candidate factor for the diagnosis of clinical platinum resistance in melanoma. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) to H.L. (LA 1039/1-3) and to J.T. (FOR 236/2).We like to thank Beate Karow and Birgit Schaefer for skillful technical assistance.
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Figure 3. Time kinetics of cisplatin-induced Pt-d(GpG) adduct formation in the drug-sensitive melanoma line MeWo and its cisplatinresistant subline MeWo CIS 1 as measured by an immunocytologic assay. Cisplatin was added at t ¼ ^ 2 h. The cells were exposed to 5 or 20 mg per mL cisplatin for 2 h and nuclear Pt-d(GpG) adduct content was determined after di¡erent time periods by £uorescence microscopy using the rat anti-Pt-d(GpG) adduct MoAb ‘‘18G10’’ and a £uorescein isothiocyanate-based double sandwich staining procedure. Signals were ampli¢ed and recorded by a dual mode CCD camera, and fed into a four parameter image analysis program. Displayed values represent the mean, the vertical error bars SD.
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