MCF-7/ADR cells

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Nov 30, 2010 - irregular pseudopodia. With increasing concentrations of. ADR, the DNA fingerprints of MCF-7/ADR0 cells were always identical to the parental ...
Med Oncol (2011) 28:S135–S141 DOI 10.1007/s12032-010-9747-1

ORIGINAL PAPER

MCF-7/ADR cells (re-designated NCI/ADR-RES) are not derived from MCF-7 breast cancer cells: a loss for breast cancer multidrug-resistant research Weifeng Ke • Pei Yu • Jianfeng Wang • Ruitao Wang • Chongyong Guo • Ling Zhou Changchun Li • Ke Li



Received: 31 October 2010 / Accepted: 10 November 2010 / Published online: 30 November 2010 ! Springer Science+Business Media, LLC 2010

Abstract MCF-7/ADR cells have been widely used as a multidrug-resistant breast cancer cell model in cancer research. The origin of MCF-7/ADR has been a matter of debate since MCF-7/ADR cells were re-designated NCI/ ADR-RES in 1998. Many recent studies still describe MCF-7/ADR cells as originating from the breast cancer cell line MCF-7. Thus, the real origin of MCF-7/ADR cells remains more unclear. In this study, a new adriamycin (ADR)-resistant cell line MCF-7/ADR0 was reproduced using the same procedure employed during the initial establishment of MCF-7/ADR. Since the MCF-7/ADR0 cell line was definitely derived from parental MCF-7 cells, we were able to directly compare these cell lines together with MCF-7/ADR using immunocytochemical, morphological, and consecutive DNA fingerprinting analyses to determine the true origin of MCF-7/ADR. Both ADR-resistant cell lines displayed some similar phenotypic characteristics, such as high levels of P-glycoprotein (P-gp) expression, increased vacuolation, abundant filamentous material, and irregular pseudopodia. With increasing concentrations of ADR, the DNA fingerprints of MCF-7/ADR0 cells were always identical to the parental MCF-7 cells. However, the DNA fingerprints of MCF-7/ADR cells did not relate to MCF-7 or MCF-7/ADR0 . MCF-7/ADR and the breast cancer cell line MCF-7 are not of the same origin. Longtime culture in the presence of ADR does not cause significant changes in DNA fingerprint patterns.

W. Ke ! P. Yu ! J. Wang ! R. Wang ! C. Guo ! L. Zhou ! C. Li ! K. Li (&) Department of General Surgery, First People’s Hospital Affiliated to Shanghai Jiaotong University, No. 100 Haining Road, Hongkou District, Shanghai 200080, China e-mail: [email protected]

Keywords MCF-7/ADR ! NCI/ADR-RES ! Breast cancer ! Misidentification ! DNA fingerprinting Abbreviations ADR Adriamycin P-gp P-glycoprotein MDR Multidrug-resistant NCI National cancer institute SNP Single-nucleotide polymorphism STR Short tandem repeat PBS Phosphate-buffered saline

Introduction In 1986, the adriamycin (ADR)-resistant cell line MCF-7/ ADR was established by Batist et al. [1] by incubating the breast cancer cell line MCF-7 cells with increasing concentrations of the anthracycline antibiotic ADR. This cell line has been widely used as a stable multidrug-resistant (MDR) experimental model because of its high expression level of P-glycoprotein (P-gp) [2–4]. The two cell lines have been frequently used as a pair of drug-resistant and drug-sensitive cell lines since they were collectively included in the National Cancer Institute’s Developmental Therapeutics Program 60 (NCI 60) cell line panel in 1990. However, reports that MCF-7/ADR cells were not derived from MCF-7 cells on the basis of their DNA fingerprinting data led to a change in nomenclature from MCF-7/ADR to NCI/ADR-RES in 1998 [5]. After this, the true origin and exact identity of MCF-7/ADR have been a matter of debate [5–12]. In 2002, Mehta et al. demonstrated that MCF-7, MCF-7/ADR as well as another ADR-resistant subline of MCF-7 all possessed a unique full-length caspase-3 gene

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and proposed that the original nomenclature of MCF-7/ ADR should be retained [8, 13]. However, DNA fingerprinting was not used to further confirm their observations. Recently, it has been reported that MCF-7/ADR cells were derived from the human ovarian carcinoma cell line OVCAR-8. These assertions were based on DNA fingerprinting analyses of the already-established cell lines and did not reflect the DNA fingerprint changes that occur during the acquisition of ADR resistance [10–12]. Moreover, the mechanism of ADR is likely to initiate DNA damage. Based on short tandem repeat (STR) profiling of hypervariable regions within the DNA, DNA fingerprinting can provide an international reference standard for every cell line [14]. In this study, DNA fingerprints of cell lines were continuously detected during the establishment of an ADR-resistant cell line. Since the MCF-7/ADR0 cell line was known to be derived from the parental MCF-7 cell line in our laboratory, we were able to directly compare the STR profiles of these cell lines together with MCF-7/ADR and found that MCF-7/ ADR cells are not derived from MCF-7.

Materials and methods Cell lines and cell culture The human breast cancer cell line MCF-7 and ADRresistant cancer cell line MCF-7/ADR cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and were originally imported from ATCC, USA. All these cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin. MCF-7/ADR cells’ medium was further supplemented with 10 lL ADR. All the cells were incubated at 37"C in a humidified atmosphere of 95% air/ 5% CO2. The ADR-resistant MCF-7 cell sublines (MCF-7/ADR0 ) were established by culturing parental MCF-7 cells with increasing concentrations of ADR (from 0.01 to 5 lL). When cells could survive at any given concentration of the drug, they were passaged in concentrations that were 1.5- to 2-fold higher. Cells were subsequently obtained, which were able to survive in 5 lL ADR and thereafter maintained in medium containing that concentration of ADR. MCF-7/ADR0 and MCF-7/ADR cells were passaged for 2–4 weeks in ADR-free medium prior to use [1].

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bromide (MTT)] assay. Briefly, rapidly growing cells were harvested, counted, and inoculated in 96-well microtiter plates (4,000 cells/well) in 200 ll of medium containing 10% fetal bovine serum. After 24 h, medium containing varying concentrations of ADR was applied to triplicate culture wells, and cultures were incubated for 3 days at 37"C. MTT (Sigma, Germany) was prepared at 5 mg/ml in phosphate-buffered saline (PBS, pH: 7.2–7.4) and stored at 4"C. On the fourth day, 20 ll of MTT was added to every well. After 4 h incubation at 37"C, the supernatant was removed from each well, and 150 ll of dimethylsulfoxide (DMSO) was added to solubilize the MTT-formazan product. After thorough mixing with a mechanical plate mixer for 10 min, spectrometric absorbance at 490 nm was measured with a microplate reader. Assays were carried out as described previously [15]. The IC50 value was determined by the dose of drug that resulted in 50% cell viability. Immunofluorescence and confocal microscopy Cells were seeded on glass coverslips that were coated with 0.1% gelatin and fixed in cold methanol for 10 min. The cells were washed with PBS and blocked with 1% BSA/ PBS at room temperature. Then, cells were incubated with a mixture of mouse anti-human P-gp monoclonal antibody (1:120, Santa-Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. Cy3-conjugated goat anti-mouse IgG (1:1000, Santa-Cruz Biotechnology) was used as the secondary antibody. Nuclear DNA was stained with 4, 6-diamidino-2phenylindole (DAPI). Fluorescence images were captured with a Zeiss LSM-510 laser confocal microscope using a Zeiss 40 9 1.3 numerical aperture oil immersion lens (Carl Zeiss Inc., Thornwood, NJ, USA). Transmission electron microscopy Transmission electron microscopy was used to observe morphological changes in the cells. Cells were fixed in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4"C for 2 h followed by washing in 0.1 M cacodylate buffer solution for 16–18 h. To achieve the isotonic state, sucrose (50 g/l) was added. Cell post-fixation was performed in 2% osmium tetraoxide with further dehydration in alcohols and embedding in araldite. The ultrathin sections were prepared on LKB-8800 ultratome and treated with the contrast agents, uranyl acetate, and lead citrate. Images were collected using a CM 120 transmission electron microscope with an acceleration voltage of 60 kV.

MTT assay DNA fingerprinting The relative sensitivity of ADR-resistant cell lines and MCF-7 cells to ADR was measured using the colorimetric [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium

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DNA fingerprinting was performed using the commercially available kit, PowerPlex# 1.2 System (Promega, Madison,

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WI, USA). This system allows the co-amplification and the two-color detection of nine loci (eight STR loci and the gender-determining Amelogenin). The following STR loci were tested: CSF1PO, TPOX, TH01, vWA, D16S539, D7S820, D13S317, and D5S818. PCR amplification was performed according to the manufacturer’s recommended protocol. Allele size was determined by electrophoresis of the PCR products in 6% denaturing polyacrylamide gels and compared to ROX 500 size standards (Applied Biosystems, Foster City, CA, USA), using the ABI PRISM# 3100 Genetic Analyzer (Applied Biosystems). The fluorescent signals from the different size alleles were recorded using GENESCAN (version 3.1) software and analyzed using GENOTYPER (version 2.1) software (Applied Biosystems). Allele size evaluation and data analysis were carried out by two independent operators. The experiments were repeated twice.

Results The establishment of ADR-resistant cell lines In this study, ADR-resistant cell sublines MCF-7/ADR0 were established from parental MCF-7 by continuous exposure to increasing drug concentrations (Batist et al. [1]) over a time period of 1 year. As shown in Table 1, these sublines showed gradually increasing resistance levels to ADR with the development of a drug-resistant phenotype. The largest resistance index of MCF-7/ADR0 to ADR was 85, and the cells could passage at a concentration of 5 lL ADR. Thus, MCF-7/ADR0 was considered to be a new ADR-resistant cell line and was employed for this study. Expression and cellular localization of P-gp in cells Mdr1 gene amplification followed by P-gp over-expression is one of the mechanisms involved in the development of

Table 1 Sensitivity of MCF-7/ADR0 and MCF-7 cells to ADR. With increasing concentrations of ADR (from 0.01 to 5.0 lL), IC50 values for MCF-7/ADR0 cells were also gradually increased (from 0.263 to 9.202 lL). The resistance index (RI) represent fold resistant to ADR between IC50 (MCF-7/ADR0 ) and IC50 (MCF-7) Concentration of ADR (lL)a

MCF-7/ADR0 IC50 (lL)

MCF-7 IC50 Resistance (lL) index (RI)

0.01

0.263

0.105

0.1

0.637

0.097

6.6

1.0

3.051

0.116

26.3

5.0

9.202

0.108

85.2

a

drug resistance. The expression and localization of P-gp was studied by immunofluorescence and confocal microscopy in the above three cell lines. P-gp was over-expressed and concentrated in the plasmalemma of the majority of ADR-resistant cells (MCF-7/ADR0 and MCF-7/ADR) but was virtually undetectable in MCF-7 cells (Fig. 1). Therefore, the development of ADR resistance seemed to follow the drug resistance mechanism of P-gp overexpression. These findings were consistent with the previous reports [16]. Morphological features of cells that are sensitive and resistant to ADR It is known that the development of a drug-resistant phenotype could be accompanied by changes in morphological structure. As shown in Fig. 2A, spindle-shaped cells were observed in all cell lines, especially MCF-7/ADR cells. In contrast to MCF-7 and MCF-7/ADR0 cells, which grow in clumps, MCF-7/ADR cells were more dispersed. As shown using ultrastructural analysis (Fig. 2B), ADR-treated cells exhibited increased vacuoles, abundant filamentous material, and irregular pseudopodia [16, 17]. Therefore, MCF-7/ ADR0 cell lines displayed similar resistant phenotypic changes to MCF-7/ADR cells in ultrastructural organization. DNA fingerprinting analysis In order to clarify whether MCF-7/ADR cells originated from the breast cancer cell line MCF-7, genotypic changes in the newly established ADR-resistant cell line MCF-7/ADR0 were continuously detected during the establishment of the cell line. Moreover, DNA fingerprints of the three cell lines were compared under the known premise that MCF-7/ADR0 cells originated from MCF-7 cells. As shown in Fig. 3, the DNA fingerprints of MCF-7/ADR0 did not change remarkably during the course of the development of ADR resistance. Furthermore, the DNA fingerprints of MCF-7/ADR0 and the parental MCF-7 cell line were almost identical, indicating that the cells shared the same origin. However, the DNA fingerprints of MCF-7/ADR cells were very different from the other MCF-7 cell lines in all loci except the sex chromosome locus (XX chromosomes). Thus, we confirmed that MCF-7/ADR cells did not originate from the breast cancer cell line MCF-7.

2.5

0

The concentration of ADR to maintain MCF-7/ADR resistance phenotype

Discussion Since 1998, when Scudiero et al. reported that MCF-7/ ADR cells might be derived from other cell lines rather than the breast cancer cell line MCF-7 (leading to a change

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Fig. 1 The expression and localization of P-gp in MCF-7, MCF-7/ ADR0 , and MCF-7/ADR cells were examined by immunofluorescence confocal microscopy. P-gp was over-expressed and found to be

concentrated in the plasmalemma of the majority of ADR-resistant cells but absent in MCF-7 cells

Fig. 2 Morphological differences in MCF-7, MCF-7/ADR0 , and MCF7/ADR cells. Phase-contrast images display that MCF-7 and the newly established ADR-resistant subline MCF-7/ADR0 cells grew in clumps, whereas MCF-7/ADR cells were more dispersed (panel A). Electron

microscopy ultrastructural analysis show that ADR-treated cells displayed increased vacuoles (solid arrowhead), abundant filamentous material (solid arrow), and irregular pseudopodia (arrow), especially the MCF-7/ADR0 cells compared with parental MCF-7 cells (panel B)

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Fig. 3 DNA fingerprinting analyses of MCF-7, MCF-7/ADR0 , and MCF-7/ADR cell lines at nine STR markers. The DNA fingerprints of MCF-7/ADR0 showed unremarkable changes during the development of ADR resistance and were similar to the DNA fingerprints of the

parental cell line MCF-7 (a). The DNA fingerprint of MCF-7/ADR was significantly different in all loci except the sex chromosome locus (XX chromosome) to the other MCF-7 cell lines (b). The allele sizes are indicated under each allele

in nomenclature from MCF-7/ADR to NCI/ADR-RES), the true origin of MCF-7/ADR cells has been a matter of debate [5–12]. This is not only a simple academic question, since MCF-7/ADR cells has been widely used as a drugresistant breast cancer cell model, and conclusions from many studies were based on comparisons between MCF-7/ ADR and their presumed parental cell line MCF-7. A

search of the medical literature for tumor MDR studies using MCF-7/ADR retrieved over 250 manuscripts in PubMed, about two-thirds papers still being published that describe these cells as being derived from MCF-7, despite an increasing number of articles citing NCI/ADR-RES (Fig. 4). In fact, many investigators that routinely use MCF-7/ADR without apparent awareness of its real origin

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Fig. 4 Number of recent research publications, citing the use of the cell line MCF-7/ADR (or NCI/ADR-RES), that were retrieved from PubMed. The queries used in the PubMed search included ‘‘NCI/ ADR-RES’’ and ‘‘(MCF-7/ADR OR MCF-7/ADM OR MCF-7/AdrR) AND MCF-7’’, respectively

in recent papers further slowing the spread of concerns regarding these cells. Controversy over the origin of MCF-7/ADR cells has persisted, partly due to inconsistent reports [6, 8, 13]. In 2000, Pirnia et al. [6] reported that MCF-7 TH (another cell line that has been independently selected by ADR treatment at the NCI) and not MCF-7 had identical DNA fingerprints with MCF-7/ADR. Although both ADR-resistant cell lines expressed high levels of P-gp and caspase-3 protein, caspase-3 protein was absent in the presumed parental MCF-7 cells because of a 47-base-pair deletion in exon 3 of the caspase-3 gene. However, Mehta et al. demonstrated that the parental MCF-7 cells harbor the same full-length caspase-3 gene as the ADR-resistant cell lines and proposed that the original nomenclature of MCF7/ADR should be retained [8, 13]. Unfortunately, their studies were not accompanied by DNA fingerprinting analysis of the novel ADR-resistant cell lines established by the team. Further studies identified another origin of MCF-7/ADR cells. When spectral karyotyping of the NCI-60 cell lines was evaluated, the ovarian cancer cell line OVCAR-8 cells were reported to share many of the same karyotypic abnormalities as MCF-7/ADR [18]. In support of the karyotypic analysis, single-nucleotide polymorphism (SNP) array analysis showed that MCF-7/ADR and OVCAR-8 cells share 98.7% of over 10,000 individual SNPs, indicating that MCF-7/ADR was highly likely to be derived from the same individual as OVCAR-8 [10, 11]. However, these results were based on the already-established cell lines and maybe omit the DNA fingerprint changes that occur during the acquisition of ADR resistance. The mechanism of action of ADR is intercalation into DNA leading to inhibition of DNA biosynthesis, initiation of DNA damage via free radical formation, interference with DNA strand separation, and the inhibition of topoisomerase II [19, 20]. As these effects could influence DNA

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fingerprints, it is difficult to determine whether long-time induction of ADR or other reasons such as cross-contamination led to the changes in DNA fingerprints. To directly address this concern, STR profiling, an efficient and reliable means of detecting cross-contamination, was used during the establishment of ADR-resistant cell line in this study. In our experiment, the serial STR profiles of MCF-7/ ADR0 cells were always consistent with their parental MCF-7 cells during the establishment of ADR-resistant cell line and so the possibility of cross-contamination is excluded. The MCF-7/ADR0 cell line was known to be derived from MCF-7 cell line. However, the DNA fingerprints of MCF-7/ADR cells were very different from them. Therefore, we could conclude that MCF-7/ADR cells do not originate from MCF-7 cells, and long-time culture in the presence of ADR does not cause significant changes in DNA fingerprint patterns by this improved method. Moreover, we also tested DNA fingerprints of the cell lines MCF-7/ADR and MCF-7 from two different sources (Breast Cancer Institute/Cancer Hospital and the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai). DNA fingerprints of MCF-7 or MCF-7/ ADR cells from the two sources were also identical (data not shown). Here, we successfully derived MCF-7/ADR0 cell line from MCF-7 cells using the same procedure employed for the initial establishment of MCF-7/ADR cell line. Both ADR-resistant cell lines exhibited some similar phenotypic characteristics, such as abundant filamentous material and irregular pseudopodia. These phenotypic changes most likely increase the invasive ability of tumor cells. Our own MCF-7/ADR0 cells are developed by prolonged continuous selection for ADR resistance and express high levels of P-gp. Thus, MCF-7/ADR0 cells can also serve as a valuable cell model in cancer research. Many important conclusions based on the comparison between MCF-7/ADR and MCF-7 should be reexamined using MCF-7/ADR0 cells. The misidentification of MCF-7/ADR is by no means an isolated occurrence [21, 22]. In fact, the phenomenon of cell line cross-contamination is already a serious problem in cell culture systems [23]. If cell lines can be identified by DNA fingerprinting prior to experiments and the regulations concerning the use of cell lines can be strictly followed [24], such as the use of only one cell line at any given time and separation of culture materials for each cell line, cross-contamination of cell lines will be readily detectable and reduced to a minimum. Acknowledgments We thank Dr. Li-quan Hong (Second People’s Hospital of Hangzhou, The Affiliated Hospital of Hangzhou Normal University, Zhejiang) for his helpful advice. We also thank Dr. Quan Wang (First People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai) for his technical support.

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