Bates, Susan E.

Betticher, Daniel C.

Borner, Markus M.

Breuleux, Madlaina

Hochmeister, Manfred

Hotz, Michel A.

Marti, Andreas

Pirnia, Farzaneh

Schneider, Erasmus

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Uncertain Identity ofDoxorubicin-Resistant MCF-7Cell Lines ExpressingMutated p53The doxorubicin (ADR)-selected hu-man breast cancer cell line (MCF-7/ADR-RES) has been a useful model fora multidrug-resistant subline in cancerresearch. However, a recent letter to theJournal has alerted the scientific com-munity about the finding that MCF-7/ADR-RES is not a MCF-7-derived cellline. This report led to a change of no-menclature from MCF-7/ADR-RES toNCI/ADR-RES (1).Another cell line (MCF-7 TH) hasbeen independently selected by doxoru-bicin treatment at the National CancerInstitute (NCI) and demonstrated someof the same, interesting characteristicsof NCI/ADR-RES, such as high levelsof MDR-1 and P-glycoprotein expres-sion and a mutated p53 tumor suppres-sor protein (2). Of interest, both celllines exhibit the same 21-base-pair de-letion beginning at exon 5 spanningcodons 126–133 (3). We used this cellline to analyze the effect of the multi-drug-resistant phenotype and p53 on theefficacy of anticancer drugs with differ-ent mechanisms of action. Various anti-cancer drugs were more effective in theMCF-7 TH subline than in the parentalMCF-7 line, despite a dysfunctional p53and multidrug-resistant phenotype.These agents induced apoptosis inMCF-7 TH cells as demonstrated by thecharacteristic morphologic features. Be-cause the activity of specific caspasesmight affect the propensity of a cell toundergo apoptosis, we examined expres-sion level and cleavage pattern of differ-ent caspases in drug-treated MCF-7 THand parental cells. The MCF-7 TH cellline strongly expressed caspase-3 (Fig.1, A) and cisplatin treatment led to asignificant increase of caspase-3-like ac-tivity as measured by use of the fluoro-genic caspase-3-specific substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (DEVD-afc) (Fig. 1, B). TheMCF-7 cell line has lost caspase-3 be-cause of a 47-base-pair deletion withinJournal of the National Cancer Institute, Vol. 92, No. 18, September 20, 2000 CORRESPONDENCE 1535exon 3 of the CASP-3 gene (4). Thesefindings thus suggest that MCF-7 THalso is not derived from MCF-7. Thishypothesis was confirmed by DNA fin-gerprinting, which demonstrated that pa-rental MCF-7 and MCF-7 TH cannothave the same donor, because they differin all loci except the sex chromosomelocus. The analyses were repeated withseveral different batches of parentalMCF-7 and TH cells. Furthermore,MCF-7 parental cells from differentsources were compared and all showedan identical allelic distribution profile ofthe short tandem repeat loci by DNAfingerprinting. The only cell line show-ing identity with MCF-7 TH by DNAfingerprinting was NCI/ADR-RES.Thus, MCF-7 TH, like NCI/ADR-RES,is not a MCF-7-related cell line.This finding is important becausedoxorubicin-resistant cell lines are apopular tool for new drug development.In a recent article in the Journal, Leoniet al. (5) used NCI/ADR-RES to inves-tigate a novel microtubule-binding inda-none. Despite the fact that these authorsmislabeled the cell line MCF-7/ADR-RES (1), they did not realize that theirobservation of caspase-3 activation bythe drug would not have been possiblein a MCF-7-derived cell line, whichcontains no caspase-3 (4) (Fig. 1, A).The use of a mislabeled cell line canclearly introduce a major source of mis-interpretation and possibly wrong strate-gic decisions in drug development. We,thus, strongly suggest that the identity ofcell lines be assessed regularly by DNAfingerprinting because this is a simpleand reliable method for identity valida-tion.FARZANEH PIRNIAMADLAINA BREULEUXERASMUS SCHNEIDERMANFRED HOCHMEISTERSUSAN E. BATESANDREAS MARTIMICHEL A. HOTZDANIEL C. BETTICHERMARKUS M. BORNERREFERENCES(1) Scudiero DA, Monks A, Sausville EA. Cellline designation change: multidrug-resistantcell line in the NCI anticancer screen [letter].J Natl Cancer Inst 1998;90:862.(2) Wosikowski K, Regis JT, Robey RW, AlvarezM, Buters JT, Gudas JM, et al. Normal p53status and function despite the development ofdrug resistance in human breast cancer cells.Cell Growth Differ 1995;6:1395–403.(3) Ogretmen B, Safa AR. Expression of the mu-tated p53 tumor suppressor protein and its mo-lecular and biochemical characterization inmultidrug resistant MCF- 7/Adr human breastcancer cells. Oncogene 1997;14:499–506.(4) Janicke RU, Sprengart ML, Wati MR, PorterAG. Caspase-3 is required for DNA fragmen-tation and morphological changes associatedwith apoptosis. J Biol Chem 1998;273:9357–60.(5) Leoni LM, Hamel E, Genini D, Shih H, Car-rera CJ, Cottam HB, et al. Indanocine, a mi-crotubule-binding indanone and a selective in-ducer of apoptosis in multidrug-resistant can-cer cells. J Natl Cancer Inst 2000;92:217–24.(6) Krajewska M, Wang HG, Krajewski S, ZapataJM, Shabaik A, Gascoyne R, et al. Immuno-histochemical analysis of in vivo patterns ofexpression of CPP32 (Caspase-3), a cell deathprotease. Cancer Res 1997;57:1605–13.(7) Marti A, Jaggi R, Vallan C, Ritter PM, BaltzerA, Srinivasan A, et al. Physiological apoptosisin hormone-dependent tissues: involvement ofcaspases. Cell Death Differ 1999;6:1190–200.NOTESAffiliations of authors: F. Pirnia, M. Breuleux,D. C. Betticher, M. M. Borner (Institute of Medi-cal Oncology), M. A. Hotz (Department of Ear,Nose and Throat Surgery), Inselspital, Bern, Swit-zerland; M. Hochmeister (Department of ForensicMedicine), A. Marti (Department for Clinical Re-search), University of Bern; E. Schneider, Divi-sion of Molecular Medicine, Wadsworth Center,New York State Department of Health, Albany,NY; S. E. Bates, Medicine Branch, National Can-cer Institute, Bethesda, MD.Correspondence to: Markus M. Borner, M.D.,Institute of Medical Oncology, Inselspital, 3010Bern, Switzerland (e-mail: mborner@insel.ch).Fig. 1. Analysis of caspase-3 protein expressionand activity in MCF-7 and MCF-7 TH cell lines.A) Immunoblot analysis of baseline caspase-3 ex-pression in Jurkat (positive control), MCF-7, andMCF-7 TH cells using a caspase-3 polyclonal an-tibody (6). B) Activation of caspase-3 by cisplatin.MCF-7 TH cells were treated with 10 g/mL cis-platin. At the indicated time points, caspase-3 ac-tivity was measured with the specific fluorogenicsubs t r a t e Asp-Glu -Va l -Asp-7 -amino-4 -trifluoromethyl coumarin (DEVD-afc) (7).1536 CORRESPONDENCE Journal of the National Cancer Institute, Vol. 92, No. 18, September 20, 2000postlabeling technique in which two cy-clobutane pyrimidine dimers, T  Tand T C, and two 6-4 photoproducts,T–T and T–C, were quantified based onexternal standards. Many of the findingschallenge results from earlier, publishedin vitro assays. Repair kinetics of dimersand 6-4 photoproducts encompassed fastand slow components, probably relatingto repair of transcribed and nontran-scribed sequences, respectively (3).Dimers were removed considerablymore slowly than 6-4 photoproducts,with 50% removal times of approxi-mately 15 and 5 hours, respectively;dimers at TT sites were repaired slowerthan those at TC sites (3). A 20-fold in-terindividual variation in repair rateswas observed. Because we have appliedthis test to relatively small numbers ofcase subjects and matched control sub-jects, we need to refrain from makinggeneral conclusions. However, age doesnot cause a decline in repair of UVRdamage in situ, in contrast to resultsfrom the host-mediated assay (4). Asevidence of further contradictions, basalcell carcinoma patients have not dis-played lower rates of DNA repair (5),and melanoma patients appear to repairDNA damage like healthy matchedcontrol subjects (6). The most recent de-velopment is a postlabeling assay for ex-creted urinary photoproducts, informa-tive of the total-body UV damage (7).Valid DNA repair tests will also beexceedingly valuable to analyze thefunctional effects of DNA repair genepolymorphisms, which is currently apopular research area. Here we concurwith Berwick and Vineis who state, inreference to the bewildering literatureon attempts to match metabolic geno-types to risk of any cancers: “It is notclear that conducting these studies with-out concomitant studies of expressionand/or function will be fruitful.” Con-ducting DNA repair tests in the relevanthuman organs will be fruitful and rel-evant.KARI HEMMINKIGUOGANG XUFRANK LE CURIEUXREFERENCES(1) Berwick M, Vineis P. Markers of DNA repairand susceptibility to cancer in humans: an epi-demiologic review. J Natl Cancer Inst 2000;92:874–97.(2) Bykov VJ, Jansen CT, Hemminki K. High lev-els of dipyrimidine dimers are induced in hu-man skin by solar-simulating UV radiation.Cancer Epidemiol Biomarkers Prev 1998;7:199–202.(3) Bykov VJ, Sheehan JM, Hemminki K, YoungAR. In situ repair of cyclobutane pyrimidinedimers and 6-4 photoproducts in human skinexposed to solar simulating radiation. J InvestDermatol 1999;112:326–31.(4) Xu G, Snellman E, Bykov VJ, Jansen CT,Hemminki K. Effect of age on the formationand repair of UV photoproducts in human skinin situ. Mutat Res 2000;459:195–202.(5) Xu G, Snellman E, Jansen CT, Hemminki K.Levels and repair of cyclobutane pyrimidinedimers and 6-4 photoproducts in skin of spo-radic basal cell carcinoma patients. J InvestDermatol 2000;115:95–9.(6) Xu G, Snellman E, Bykov VJ, Jansen CT,Hemminki K. Cutaneous malignant melanomapatients have normal repair kinetics of ultra-violet-induced DNA damage in skin in situ. JInvest Dermatol 2000;114:628–31.(7) Hemminki K, Xu G, Le Curieux F. Ultravioletradiation-induced photoproducts in humanskin DNA as biomarkers of damage and itsrepair. IARC Sci Publ. No. 154. In press 2000.NOTESAffiliation of authors: Department of Biosci-ences at Novum, Karolinska Institute, Huddinge,Sweden.Correspondence to: Kari Hemminki, M.D.,Ph.D., Karolinska Institute, Department of Biosci-ences at Novum, 141 57 Huddinge, Sweden.RESPONSEWe are pleased that Hemminki et al.have taken seriously the challenge in ourrecent review (1) to develop definitivemolecular assays for DNA repair. Theirconclusions echo ours: neither they norwe really know what the studies in lym-phocytes measure. Clearly, their re-search publications in 1999 and 2000will be valuable in developing the tech-niques for studying DNA repair.We find it interesting that the twostudies using kinetic tests on skin biop-sies, which Hemminki et al. believe ful-fill the criterion of biologic relevance,had positive and statistically significantresults (although carried out in only afew subjects). Roth et al. (2) showed thatmelanoma subjects had lower DNA re-pair rates than control subjects (P <.001), and Alcalay et al. (3) showed thatdimers in basal cell carcinoma subjectswere eliminated less rapidly than in con-trol subjects (P < .05).The overall message that we derivefrom our analysis of the literature is thata considerable number of studies consis-tently found an association between bio-logic tests, whose significance is stillobscure, and cancer at several sites.Looking at it this way, we agree withHemminki and colleagues that furtherclarification of the biologic backgroundis crucial. However, at this time, wethink that focusing our attention on thedesign of the studies, reproducibility,sample size, and selection of controlsubjects was necessary to draw validconclusions at the population level. Theissue of biologic relevance of studies ofDNA repair in lymphocytes is criticalfor epidemiologic studies where asso-ciations are derived from numerous sub-jects to define small reproducible alter-ations that may be important. Most ofthe work Hemminki et al. cite has beenconducted in very small groups and hasnot yet been reproduced.In sum, the issues raised by Hem-minki et al. are critical for the appropri-ate assessment of DNA repair. We hopethat other eminent scientists will jointheir efforts to develop appropriate as-says for DNA repair.MARIANNE BERWICKPAOLO VINEISREFERENCES(1) Berwick M, Vineis P. Markers of DNA repairand susceptibility to cancer in humans: an epi-demiologic review. J Natl Cancer Inst 2000;92:874–97.(2) Roth M, Muller H, Boyle JM. Immunochemi-cal determination of an initial step in thyminedimer excision repair in xeroderma pigmento-sum variant fibroblasts and biopsy materialfrom the normal population and patients withbasal cell carcinoma and melanoma. Carcino-genesis 1987;8:1301–7.(3) Alcalay J, Freeman SE, Goldberg LH, WolfJE. Excision repair of pyrimidine dimers in-duced by simulated solar radiation in the skinof patients with basal cell carcinoma. J InvestDermatol 1990;95:506–9.NOTESAffiliations of authors: M. Berwick, Departmentof Epidemiology and Biostatistics, MemorialSloan-Kettering Cancer Center, New York, NY; P.Vineis, Unit of Cancer Epidemiology, Universityof Torino, and Ospedale S. Giovanni Battista,Torino, Italy.Correspondence to: Marianne Berwick, Ph.D.,Department of Epidemiology and Biostatistics,Memorial Sloan-Kettering Cancer Center, 1275York Ave., Box 588, New York, NY 10021 (e-mail: berwickm@mskcc.org).Journal of the National Cancer Institute, Vol. 92, No. 18, September 20, 2000 CORRESPONDENCE 1537

Uncertain Identity of Doxorubicin-Resistant MCF-7 Cell Lines Expressing Mutated p53

Uncertain Identity of Doxorubicin-Resistant MCF-7 Cell Lines Expressing Mutated p53

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