American Journal of Pathology, Vol. 147, No. 6, December 1995Copyrnght (0 American Societyfor Investigative Patbology

Short CommunicationMembrane Transport Proteins Associated withDrug Resistance Expressed in HumanMelanoma

Dirk Schadendorf,* Amira Makki,*Christiane Stahr,* Antje van Dyck,*Reinhard Wanner,* George L. Scheffer,tMarcel J. Flens,t Rik Scheper,t andBeate M. Henz*From the Department ofDermatology,* Virchow Clinics,Humboldt University Berlin, Berlin, Germany, andDepartment ofPatbology,t Free University HospitalAmsterdam, Amsterdam, The Netherlands

Melanoma cells often display a multidrug-resis-tantphenotype, but the mechanisms involved arelargely unknown. We have studied here the re-cently identified transport-associated proteins,MRP and LRP, and the well-known drug resis-tance marker P-glycoprotein using a panel of 16human melanoma ceUl lines and 71 benign andmalignant melanocytic tissue samples. By flowcytometry and immunohistochemistry, expres-sion ofP-glycoprotein was not detectable on theprotein level in the 10 ceUl lines analyzed, al-though by reverse transcriptase polymerasechain reaction, MDR-1 gene expression was dem-onstrated in 2 of10 ceU lines. In addition, immu-nohistology revealed P-glycoprotein expressionin only 1 of 71 melanocytic lesions. In contrast,MRP was detected in a subset of melanoma ceUlines by reverse transcriptase polymerase chainreaction and immunohistology (4 of 10). LRP ex-pression was observed in 8 of 10 melanoma ceUlines by immunochemistry and in 10 of 10 byreverse transcriptase polymerase chain reac-tion. Furthermore, MRP was detected immuno-histologically in almost 50% of primary andmetastatic melanoma specimens, although nosignifscant differences were found between me-tastases taken before or after chemotherapy. Ex-

pression ofLRP was detected in a subset ofneviwith nevus ceUls exhibiting up to 25% positiveLRP reactivity. In 13 of 21 primary melanomasand 23 of3 7 metastases, more than 25% oftumorceUs were stained by the LRP-56 monoclonal an-tibody. Particularly in the group of metastaseswith more than 50% ofLRP-positive ceUs, 7 of11of the metastases had been previously exposedto chemotherapeutic drugs. Although the ex-pression of membrane transport proteins mayexplain only the chemoresistance toward li-pophilic, natural compounds and not resistanceagainst alkylating agents, the lack ofP-glycopro-tein expression after chemotherapeutic treat-ment and the significant expression ofMRP andLRP in melanoma ceUs providefirst insights intothe drug-resistantphenotype in melanoma. Addi-tional studies analyzing the role ofMRP andLRPin chemoresistance ofmelanoma are warranted(Am J Pathol 1995, 147:1545-1552)

Chemotherapy is one of the proven strategiesagainst malignant tumors. In patients suffering frommalignant melanoma in its metastatic stage, suchmeasures are, however, highly unsatisfying.1 Thera-peutic protocols produce an overall response rate ofapproximately 20% at its best with a single agentand up to 50% with combination chemotherapy.23However, cure rates and overall survival time are notaffected by these therapeutic approaches.23

Supported in part by a grant from the Deutsche Krebshilfe(W 69/92/Scha 3).Accepted for publication September 1, 1995.

Address reprint requests to Dr. Dirk Schadendorf, Virchow Klini-kum, Department of Dermatology, HU Berlin, Augustenburger Platz1, 13344 Berlin, Germany.

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1546 Schadendorf et alAJP December 1995, Vol. 14 7, No. 6

Freshly isolated melanoma cells from metastasesin patients without any previous exposure to chemo-therapeutic drugs as well as melanoma cells cul-tured in vitro for several passages are known to ex-hibit a high level of intrinsic chemoresistance tovarious cytostatic agents.45 The exact mechanismof chemoresistance exhibited by human melanomacells is currently unknown.

Overexpression of the transmembrane transportprotein P-glycoprotein (Pgp) has been detected inmany multidrug-resistant tumor cell lines and in avariety of tumors from cancer patients and has beenassociated with an increased transport of (lipophilic)natural compounds.6-10 P-glycoprotein is encodedby the human MDR-1 gene, which has been shownto confer resistance to several natural and syntheticchemotherapeutic drugs.8 13 In addition, Cole andco-workers14 have identified and sequenced a sec-ond multidrug-resistance-related protein (MRP) thatis overexpressed in several non-Pgp MDR celllines.15-17 MRP belongs like Pgp to the ABC trans-porter superfamily, suggesting that both proteinsmight cause resistance by extrusion of drugs out ofthe cell.14'18 Transfection experiments and subse-quent pharmacological characterization have dem-onstrated that the MRP gene confers chemoresis-tance to a similiar but not identical spectrum of drugsas Pgp17,19

Recently, a third membrane transport protein hasbeen identified that is expressed in non-Pgp MDRcell lines of different histogenetic origin, termed thelung resistance protein (LRP).20 The gene encodingLRP has been cloned and sequenced, revealing ahigh homology to the major vault protein from thelower eukaryote Dictyostelium discoideum and the ratand which is involved in the transport of a variety ofsubstrates.21To better understand the high chemoresistance of

melanoma cells, the present study was designed toinvestigate the membrane transport-associated pro-teins Pgp, MRP, and LRP for their possible role inconferring multidrug resistance in these cells. Wereport here that the expression of MRP and LRP, butnot Pgp, can be detected on both RNA and proteinlevels in several melanoma cell lines by reversetranscriptase polymerase chain reaction (RT-PCR),immunohistochemistry, and flow cytometry. Further-more, an immunohistological survey of 71 melano-cytic tumors including nevi, primary melanomas, andmelanoma metastases revealed almost no expres-sion of Pgp in human melanoma cells whereas MRPand LRP were expressed in a large subset of mela-nomas and melanoma metastases.

Materials and Methods

Cell Culture

Human melanoma cell lines derived from metastaticlesions of patients with malignant melanoma estab-lished either at the Memorial Sloan Kettering CancerCenter (SK-Mel-13, -23, -29, -37, -147, -173, -177,and -186 and MeWo) or at the University HospitalRudolf Virchow (UKRV-Mel-1, -2, -4, -5, -xl, -x2, and-x3) were cultured in RPMI 1640 medium supple-mented with 10% fetal calf serum, 2 mmol/L L-glu-tamine, and antibiotics, as described previously.22None of the cell lines had been exposed to chemo-therapeutic drugs before. A panel of 16 human mel-anoma cell lines were analyzed by immunohistologyand RT-PCR; a set of 10 cell lines was chosen foreach technique upon availability.

Synthetic OligonucleotidesThe oligonucleotides were designed with the help ofthe PC/Gene computer program (IntelliGenetics,Mountain View, CA). Oligonucleotides for detectionof MDR-1, MRP, and LRP in human melanoma cellswere designed after alignment with published se-quences for MDR- 1,213 MRP,14 and LRP.21 Primersused for amplification of the MDR-1 gene were 5'-TTA CAT TTT CCT TCA CCC AGG C-3' and 5'-GCCTCC TTT GCT GCC CTC ACG A-3', yielding anexpected amplificate of 637 bp; for amplification ofthe MRP gene, 5'-AGA ACC TCA GTG TCG GGCAGC G-3' and 5'-TCG CAT CTC TGT CTC TCC TGGG-3', yielding an expected amplificate of 527 bp;and for amplification of the LRP gene, 5'-CCT CGAGAT CCA TTG TGC TGG-3' and 5'-CAC AGG GTTGGC CAC TGT GCA-3', yielding an expected am-plificate of 300 bp. The identity of the products wasconfirmed by restriction analysis.

Polymerase Chain ReactionPoly A+ mRNA was isolated from cells with the Mi-cro-Fast Track mRNA isolation kit (Invitrogen, SanDiego, CA). Subsequently, the RNA was transcribedinto first strand cDNA with the cDNA cycle kit (In-vitrogen) according to the manufacturer's instruc-tions. A total of 100 ng of cDNA of the preparationswere used as a template for PCR, as describedpreviously.22 Full-length cDNA probes encodingMDR-1 and MRP were used as positive controls.Briefly, PCR was performed with a thermocycler (Hy-baid OmniGene, Teddington, UK). Each sample wasamplified for 35 cycles of denaturation (940C, 45

Chemoresistance and Melanoma 1547AJP December 1995, Vol. 147, No. 6

seconds), annealing (600C, 45 seconds for MDR-1and MRP; 650C, 45 seconds for LRP), synthesis(720C, 1 minute), and a primer extension of 3 sec-onds at 720C after each cycle. The 100-,l reactionmixture contained 50 mmol/L KCI, 0.01 mol/L Tris,pH 7.4, 6.5 mmol/L MgCI2, 10 mg/ml swine gelatin(Sigma Chemical Co., Deisenhofen, Germany), 200,umol/L dNTP, 2 U of Taq polymerase (Gibco/BRL,Berlin, Germany), and oligo primers at a final con-centration of 0.7 to 1 ,mol/L. PCR products wereanalyzed by agarose gel electrophoresis. An aliquotof 1/10 of the PCR reaction was analyzed, and DNAwas visualized by ethidium bromide staining.

Monoclonal AntibodiesTwo mouse monoclonal antibodies, JSB-123 and4E324 (Signet, Dedham, MA), detecting P-glycopro-tein, one mouse (MRPm6) and one rat monoclonalantibody (MRPr1) detecting MRP,25 and one mousemonoclonal antibody (LRP-56) detecting LRP ex-pression20 were used for immunohistochemistry andflow cytometry. Mouse monoclonal TA99 (L. J. Old,New York, NY) detecting the melanosomal antigenTRP-126 was used as internal control.

Tissue HandlingHuman tumor tissue specimens were taken from thetissue bank at the Department of Dermatology, Vir-chow Clinics, Humboldt University of Berlin (Berlin,Germany), and derived from surgical excisions ofmelanocytic lesions done for diagnostic or therapeu-tic reasons. All tissue samples for immunohisto-chemistry were rapidly frozen in liquid nitrogen andkept at -800C. Cryostat sections (4 to 10 ,lm) wereprepared at -250C, placed on poly-L-lysine-coatedslides, dried for 20 minutes, fixed with ice-cold ace-tone, and used immediately or after storage (up to 5days) at -200C.

Patient DocumentationFor immunohistological studies, 13 nevi (3 dysplas-tic, 5 compound, 4 dermal, 1 junctional), 21 primarymalignant melanomas of various tumor thickness(median tumor thickness, 2.1 mm), and 37 mela-noma metastases of 20 patients were evaluated be-fore (n = 19) and during (n = 18) chemotherapy.Patients were treated with 5-(3,3-dimethyl-1-tria-seno-)imidazol-4-carboxamid (DTIC; n = 11), vin-desine (n = 6), 5-FU (n = 3), cis-platin (n = 1), andfotemustine (n = 1). Metastases that had contactwith chemotherapeutic drugs were surgically re-

moved during chemotherapeutic cycles but notlonger than 2 months after the last cycle.

ImmunohistochemisttyBefore processing, sections were fixed for 10 min-utes in acetone at room temperature and subse-quently stained by standard alkaline phosphataseanti-alkaline phosphatase (APAAP) technique, aspreviously described.26

ImmunocytochemisttyCytospins were prepared for 10 human melanomacell lines, 4 of which had been analyzed by RT-PCRregarding MDR-1, MRP, and LRP, at a density of500,000 cells/ml and subsequently stained by theAPAAP technique, as previously described.26 Cyto-spins were evaluated as follows. The percentage ofpositive cells was scored semiquantitatively into fourdifferent categories (-, +, ++, and +++) by twoindependent investigators. Discrepancies in the in-terpretation were resolved by a second parallel read-ing of the slides.

Evaluation of Staining ResultsFor evaluation of immunohistological staining of tis-sue sections, a standard evaluation form was de-signed. Two investigators read all sections indepen-dently as described.26 The percentage of positivetumor cells was then scored according to five differ-ent categories (0 to 5%, 6 to 25%, 26 to 50%, 51 to75%, and 76 to 100%). Discrepancies in the inter-pretation were resolved by a second parallel readingof the slides.

To test the reproducibility of the histological eval-uation procedure, the number of positive cells ex-pressing a distinct resistance-related membraneprotein were evaluated in five specimens and 50slides by the two observers. In addition, each spec-imen was stained with hematoxylin and eoxin and bythe APAAP technique with HMB-45 and TA99 anti-bodies. With these three slides as guidance in iden-tifying melanoma cells, the mean relative differencebetween the two observers was found to be 8.5%.

Statistical AnalysisThe distribution of the scored percentages of posi-tive cells after immunohistological staining in eachgroup was the unit of analysis. SPSS/PC+ software(Chicago, IL) and BMDP Statistical software (LosAngeles, CA) were used for statistical analyses. Sta-

1548 Schadendorf et alAJP December 1995, Vol. 14 7, No. 6

B 123MA 1

MDR-1 * |

Figure 1. Detection of MDR-1, MRP, and LRPgene expression in human melanoma cells byPCR. Analysis of the expression of MDR-1 (A),MRP (B), and LRP (C) on RNA level by RT-PCRin 10 human melanoma cell lines. Arrows in-dicate specific DNA amplfication. Lane 1, SK-Mel-1 77; lane 2, SK-Mel-1.3; lane 3, SK-Mel-1 73;lane M, 1-kb DNA ladder; lane 4, UKRV-Mel-5;lane 5, UKRV-Mel-4; lane 6, K-Mel-29; lane 7,SK-Mel-186; lane M, 1-kb DNA ladder; lane 8,SK-Mel-23; lane 9, SK-Mel-3 7; lane 1 0, MeWo.

tistical comparisons of two groups were made withthe Wilcoxon rank-sum test (two-tailed). To avoiderrors as a result of multiple comparisons, the Pvalues were confirmed by unifactorial analyses ofvariance, including the test of Scheffe. P values ofless than 0.05 were considered to indicate signifi-cance.

Results

Detection of Drug-Resistance-RelatedMembrane Proteins in Melanoma Cell Lines

The expression of drug-resistance-related mem-

brane proteins in melanoma cells was evaluated invitro on the RNA and protein level. On analysis of 10human melanoma cell lines by RT-PCR for mRNAencoding MDR-1, MRP, and LRP, all three mRNAspecies were detectable in subsets of the cell lines(Figure 1, A-C). An MDR-1-specific amplificate wasdetectable in 2 of 10 human melanoma cell lines(SK-Mel-29 and -186) (Figure 1A), MRP was ampli-fied from 4 of 10 cell lines (SK-Mel-23, -29, and -186and UKRV-Mel-5) (Figure 1 B), and LRP was demon-strated in all 10 melanoma cell lines (Figure 1C).On the protein level, Pgp was, however, not de-

tectable in any of the cell lines on cytospin prepara-

tions stained by JSB-1 and 4E3 using the APAAPtechnique (Table 1; Figure 2, A and D). In contrast,MRP protein was found in 4 of 10 melanoma lines(Table 1; Figure 2, B and E), comparable to theresults obtained by RT-PCR (Figure 1 B). The stainingresults of MRPm6 and MRPr1 were similar, with usu-

ally a stronger signal obtained by the rat antibody,with a typical intense membrane staining (Figure

C 1 2 3 M 4 5 6 7 M 8 910

LRP -

2B). LRP was detected in 8 of 10 melanoma cell linesby immunohistochemistry (Table 1). Staining of cy-tospins with LRP-56 showed primarily a cytoplasmicgranular pattern (Figure 2C).The results obtained by immunohistology were

confirmed by flow cytometry with the same primaryantibodies against membrane transport proteins(data not shown). The phenotype of human mela-noma cell lines varied considerably with regard tothe expression of Pgp, MRP, and LRP. Cell lineswithout detectable expression of Pgp and MRP(SK-Mel-37, -147, and -173, MeWo, and UKRV-Mel-2and -x3) (Table 1; Figure 2) were identified as wellas melanoma cell lines exhibiting MRP and LRPco-expression (SK-Mel-23 and UKRV-1, -xl, and-x2).

Table 1. Immunohistological Detection of P-Glycoprotein(JSB-1, 4E3), MRP (MRPm6, MRPrl), and LRP(LRP-56) on Cytospin Preparations of 10Human Melanoma Cell Lines (APAAPTechnique)

Cell line JSB-1 4E3 MRPm6 MRPr1 LRP-56

SK-Mel-23 - - ++ ++ ++SK-Mel-37 - - - - +SK-Mel-147 - - - - ++SK-Mel-173 - - - -

MeWo - - - - ++UKRV-Mel-1 - - ++ +++ +++UKRV-Mel-2 - -

UKRV-Mel-xl - - + + +UKRV-Mel-x2 - - ++ ++ ++UKRV-Mel-x3 - - - - +

-, less than 5% of cells stained; +, 5 to 25% of cells stained;+ +, 26 to 50% of cells stained; + + +, more than 50% ofmelanoma cells stained.

4 MRP

Chemoresistance and Melanoma 1549AJP December 1995, Vol. 14 7, No. 6

Ad.N-wjsii..x1|_ - ~~~.40.Si1 F.e

_XW-tlbkz-_ . w9 :

Figure 2. Immunhistological detection ofmembrane transport proteins on cytospin preparations ofhuman melanoma cell Lines (A to F) andfrozentissue sections ofmelanoma metastases (G and H). Cytospin preparations of UKRV-Mel-x2 (A to C) and SK-Mel-23 (D to F) were stained witb JSB-1detecting Pgp (A and D), MRPm6 detecting MRP(B and E), and LRP-56 detecting LRP (C and F) with theAPAAP technique. Lymph node metastasisstained with high reactivity oftumor cellsforMRPm6 (G) and subcutaneous melanoma metastasis stainedfor LRP-56 (H).

1550 Schadendorf et alAJP December 1995, Vol. 14 7, No. 6

Figure 3. Expression of P-glycoprotein, MRP,and LRP in melanocytic cells during tumorpro-gression. Immunobistological detection ofthe P-glycoprotein (JSB-1; 4E3) (A), MRP (MRPm6;MRPrl) (B), and LRP (LRP-56) (C) in naevi,primary melanoma, and melanoma metastaseswith the APAAP technique. The percentage ofpositive tumor cells isgiven and categorized intosix groups as descrIbed in Materials and Metb-ods. Melanoma metastases without pretreatmentare marked with closed circles and metastasesremoved during or after chemotherapy are iden-tified by black triangles.

00%

75%

50%

25%

5%

0%000000

00000 0000000

00000 0000000

Naevi PrimaryMelanoma

MelanomaMetastases

0 AAAI00000 000AA

00 -0 AAA

00OAA

000

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Melanoma Metastases

OALA

OLA

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Naevi Primary MelanomaMelanoma Metastases

Expression of Drug-Resistance-RelatedMembrane Proteins in Benign andMalignant Melanocytic Lesions in Vivo

Frozen sections of 71 melanocytic tumors includingnevi (n = 13), primary melanoma (n = 21), andmelanoma metastases (n = 37) were examined byinmmohistochemistry for the expression of Pgp(JSB-1, 4E3), MRP (MRPm6, MRPr1) and LRP (LRP-56). P-glycoprotein was observed in only one pri-mary melanoma (Figure 3A) corresponding to thelow detection rate in human melanoma cell lines.MRP as well as LRP were expressed in a subset ofbenign and malignant melanocytic lesions with an

increasing number of cells staining when nevi, pri-mary tumors, and metastases were compared (Fig-ure 3, B and C). Only in 1 of 13 nevi, more than 5%MRP-positive cells were observed, whereas in 9 of21 primary melanomas and 16 of 37 melanoma me-

tastases, more than 5% of the tumor cells stainedwith the MRP antibodies (Figure 3B) (P < 0.01).Further analysis of the metastases stained by MRPantibodies demonstrated no significant difference inMRP expression between metastases taken beforeor during chemotherapy (Figure 3B). Nevi as well as

primary melanomas showed a high intrinsic expres-sion of LRP, with an increasing detection from be-nign to metastatic lesions (P < 0.01). Even more than5% of cells were stained by LRP-56 in 8 of 13 nevi(Figure 3C). In addition, in 13 of 21 primary melano-mas and in 23 of 37 metastases, more than 25% oftumor cells were stained by LRP. Particularly in thegroup of metastases with more than 50% of LRP-positive cells, 7 of 11 of the metastases (63.6%) werepreviously exposed to chemotherapeutic drugs; incontrast, 11 of 26 of the metastases (42.3%) with lessthan 50% of LRP-positive-stained melanoma cellshad no previous contact with chemotherapeuticcompounds (Figure 3C) (P < 0.05).

In conclusion, the phenotype of human melano-mas varied considerably with regard to the expres-sion of Pgp, MRP, and LRP. Some tumors did not

express any membrane transport protein, whereasothers lacked expression of Pgp and MRP but ex-

pressed LRP, in addition to other melanomas exhib-iting MRP and LRP co-expression. No statistical dif-ference between MRP and LRP expression was

observed and the combination of detected markerswas not associated with drugs used previously.

Discussion

Although chemotherapeutic drugs have beenproven to be effective in the cure or palliation ofsome human cancers, both intrinsic and acquireddrug resistance remains the major obstacle in thetreatment of many cancers.7-9 Human malignantmelanoma is particularly known for its high therapeu-tic resistance,23 which is reflected by its markedchemoresistance in vitro.4'5 The mechanisms confer-ring such high drug resistance to melanoma cells are

currently unknown. Mechanisms involved in drug re-

sistance are diverse with transport-associated pro-

teins such as MDR-1, MRP, and LRP conferring re-

sistance to lipophilic compounds, whereassensitivity of melanoma cells to alkylating agents andantimetabolites is not affected by those membranetransporters.9 1112 Notably, LRP overexpression wasrecently found to also predict the clinical response tochemotherapy-containing treatment regimens inovarian cancer patients.2728 The first analyses ofhuman melanoma cell lines in vitro regarding MDR-1gene expression29-32 are in accordance with dataobtained in this study, showing low levels of MDR-1RNA in only a subset of melanoma cells. These invitro data correspond also with the low immunohis-tochemical detection of Pgp expression in only 1 of58 melanoma specimens.

Recently, it has been shown that non-Pgp MDRmay be mediated by other membrane transport-associated proteins like MRP and LRP.14-21 We re-

port here for the first time on the in vitro expression ofMRP and LRP on RNA and protein level in a large

A MDR-1 B MRP C LRP

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O"

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Chemoresistance and Melanoma 1551AJP December 1995, Vol. 14 7, No. 6

proportion of human melanoma cell lines that had notbeen previously exposed to chemotherapeuticdrugs. Moreover, almost two-thirds of primary mela-nomas and melanoma metastases contained signif-icant numbers of immunodetectable tumor cells ex-pressing MRP and LRP molecules. Of particularinterest is the observation that melanoma metasta-ses with more than 50% LRP-positive-staining tumorcells had been exposed to chemotherapeutic agentsin 63.6% of specimens compared with metastaseswith a lower percentage of positively stained tumorcells, suggesting a possible association of LRP ex-pression and previous drug exposure. The elevationobserved may even be an underestimation as vari-ous melanoma metastases studied had been surgi-cally removed up to 2 months after the last chemo-therapy. Whether the expression of those moleculesis responsible for conferring the high intrinsic drugresistance to human melanoma cells needs addi-tional studies.

In conclusion, our data show that Pgp expressionin melanoma cells is a rare event and most likely notresponsible for the known drug resistance of mela-noma. However, the expression of MRP and LRPmolecules that have been found to be associatedwith drug resistance in a substantial number of hu-man melanoma cell lines and in primary as well asmetastatic melanoma lesions opens a new outlookon the chemoresistant phenotype of melanoma cells.Additional studies are warranted to establish the as-sociation between the presence of MRP and LRPand drug resistance in human melanoma.

AcknowledgmentsThe authors are grateful to Dr. L. J. Old for providinghuman melanoma cell lines and to Drs. G. Zaman, A.Schinkel, and P.Borst for providing cDNA probesencoding MDR-1 and MRP.

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