Microarray-based Detection of Multidrug Resistance in Human Tumor Cells by Expression Profiling of ATP-binding Cassette Transporter Genes

Multidrug resistance (MDR) in tumor treatment is characterized by resistance to a broad spectrum of structurally unrelated cytotoxic drugs and is of prognostic relevance for treatment outcome (1). Drug resistance may develop after prolonged exposure to a drug and is mediated by different cellular mechanisms, including the expression of ATP-binding cassette (ABC) transporters (2). The ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (MXR and BCRP) genes are the best known genes associated with MDR, and at least seven other ABC transporters are also capable of transporting drugs (2). The presence of other ABC transporters such as ABCC7 and ABCA4 are linked to other diseases such as cystic fibrosis or Stargardt’s disease (3). However, several ABC transporters have still unknown or undefined functions. In recent years, MDR has been diagnosed in tumors by various types of assays. There is actually no technique for obtaining a full picture of the different MDR parameters but mostly determination of individual genes or proteins of the ABC family. High-density DNA microarray analysis have been done to analyze differentially expressed genes in tumor cells after drug treatment (4). Although the use of high-density DNA microarray is very useful for the screening of new markers, the quantitation of gene expression is rather poor. Low-density DNA microarrays are more suitable for routine applications because of their simplicity, good reproducibility, easy data management, and low costs. The aim of the present study was to test a novel low-density microarray for the simultaneous expression analysis of 38 ABC transporter genes in multidrug-resistant tumor cells. To investigate the usefulness of such a new tool, we investigated three multidrug-resistant sublines that are known to express three different ABC transporter genes, e.g., ABCB1(MDR1), ABCC1 (MRP1), and ABCG2 (MXR/BCRP; refs. 5, 6, 7). The parental sensitive cell lines are used as reference and their corresponding drug-resistant subline as test.

Human T-lymphoblastoid leukemic ABCB1 (MDR1) expressing CCRF/ADR5000 cells selected with doxorubicin and parental ABCB1 (MDR1)-negative CCRF-CEM cells were obtained from Dr. Axel Sauerbrey (Department for Pediatrics, University of Jena, Jena, Germany). These cells were cultured as described previously (5).

Promyelocytic ABCC1 (MRP1)-overexpressing HL60/AR leukemia cells and parental ABCC1 (MRP1)-negative HL60 drug-sensitive cells were obtained from Dr. Sauerbrey and seeded in RPMI 1640 supplemented with 10% fetal calf serum and 100 mmol/L daunorubicin for HL60/AR. Parental HL-60 cells were maintained under the same conditions without daunorubicin exposure (6).

Human breast carcinoma parental MCF7 cell line and the multidrug-resistant MCF7/CH1000 subline were kindly provided by Dr. Douglas D. Ross (University of Maryland Greenebaum Cancer Center, Department of Medicine, University of Maryland School of Medicine, and the Baltimore Veterans Affairs Medical Center; Baltimore, MD; ref. 7).

The panel of 60 human tumor cell lines of the Developmental Therapeutics Programme of the National Cancer Institute (NCI; Bethesda, MD) consisted of leukemia, melanoma, non–small cell lung cancer, colon cancer, renal cancer, ovarian cancer cell lines, cell lines of tumors of the central nervous system, prostate carcinoma, and breast cancer. Their origin and processing have been described previously (8).

PolyA⁺ RNA was isolated with the FastTrack 2.0 mRNA isolation kit (Invitrogen, Merelbeke, Belgium) with the manufacturer’s protocol for isolating mRNA starting from 4 × 10⁷ cells. RNA integrity was verified by capillary electrophoresis on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

Labeled cDNA were prepared with 1 μg of mRNA. Three synthetic polyA⁺-tailed RNA samples were spiked at three different amounts (10, 1, and 0.1 ng per reaction) into the purified mRNA as internal standard to assist in quantification and estimation of experimental variation introduced during labeling and analysis. The detailed procedure was already reported previously (9).

The genes on the low-density microarray (termed DualChip human ABC) are presented in Table 1. It contained two arrays per slide with a range of 41 transporter genes composed of 38 ABC transporters, 1 cationic transporter, and 2 ATP-sensitive potassium channels. To evaluate the reliability of the experimental data, several positive and negative hybridization and detection controls are included on the microarray. Three internal standard controls and eight housekeeping genes are arrayed on the slides for the normalization. DualChip human ABC is composed of single-strand DNA probes attached to the glass support by a covalent link. Each DNA probe is present in triplicates (Fig. 1 A). The length of the DNA probes has been optimized, and the design of the probes has been done as described previously. Recent update and the absence of several clones, 11 ABC transporters are absent on the array. The homology between the different genes of this superfamily is very high. It is 35 to 40% in average and for certain genes >60 to 70%. For this reason, in five cases, the capture-probe was complementary of two closely related genes (ABCA2/3, ABCB1/4, ABCC6/8/9, and Kir 6.1/6.2). The specificity of the capture-probe was checked by testing the binding of PCR-amplified ABC transporter clones.

The DualChip human ABC hybridization and the statistical analysis was carried out according to de Longueville et al. (9, 10). Briefly, the detection was done by using a Cy3-conjugated anti-biotin IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) on biotinylated cDNA.

The statistical analysis consisted in the calculation of the statistically significant change in the ratio of the drug-resistant compared with their parental drug-sensitive cells.

The cDNA were synthesized from 0.5 μg of mRNA according the RNA-labeling protocol described in ref. 9 with the following minor modifications: (a) a DNase treatment of mRNA was done before cDNA synthesis; (b) the deoxynucleoside triphosphate mixture contained dGTP, dATP, dTTP, and dCTP each at 500 μmol/L but no biotinylated dCTP; and (c) the second addition of reverse transcriptase was omitted.

Gene-specific primers corresponded to the gene sequences present on the DualChip human ABC (Eppendorf Array Technologies, Namur, Belgium). Forward and reverse primers for real-time PCR amplification were designed with the Primer Express Software (PE Applied Biosystems, Foster City, CA).

Real-time reverse transcription-PCR (RT-PCR) was done on 16 genes, namely ABCA2, ABCA3, ABCA4, ABCA7, ABCB1/4, ABCB2, ABCC1, ABCC3, ABCC4, ABCC5, ABCC6, ABCC8, ABCC9, ABCG1, ABCG2, and α-tubulin (housekeeping gene). mRNA of sensitive and multidrug-resistant cell lines were used in the real-time RT-PCR (n = 2), and each reaction was done in triplicate.

The detailed procedure for PCR reaction mixtures is reported elsewhere (10).

Fluorescence emission was detected for each PCR cycle, and the threshold cycle (CP) values were determined. The CP value was defined as the actual PCR cycle when the fluorescence signal increased above the background threshold. Average CP values from duplicate PCR reactions were normalized to average CP values for housekeeping genes from the same cDNA preparations. The relative expression ratio of a target gene in resistant cells is calculated based on E (efficience) and the CP deviation of a resistant sample versus a sensitive sample and expressed in comparison to a reference gene (housekeeping gene): ratio = (E_target)^{δCP target (resistant-sensitive)}/(E_reference)^{δCP reference (resistant-sensitive)}. The detailed procedure is reported elsewhere (11). Values were reported as average of triplicate analysis.

The sulforhodamine B assay for the determination of drug sensitivity in these cell lines has been reported previously (12). The inhibition concentration 50% (IC₅₀) values for drugs are included in the Standard Agents Database of the Developmental Therapeutics Programme of the NCI.⁶ The mRNA expression values of 60 cell lines of 31 ABC transporter genes (represented by 68 different clones with individual GenBank accession numbers) were selected from the NCI’s database. The other 18 members of the ABC transporter gene family were not represented in the database. The mRNA expression has been determined by microarray analysis as reported previously (13, 14). COMPARE analysis were done to produce rank-ordered lists of genes expressed in the 60 NCI cell lines. The methodology has been previously described in detail (15). Briefly, every standard drug of the NCI’s database is ranked for similarity of its IC₅₀ values to the mRNA expression for a given ABC transporter. To derive COMPARE rankings, a scale index of correlations coefficients (R values) is created. In the standard COMPARE approach, greater mRNA expression in cell lines correlate with greater IC₅₀ values, e.g., drug resistance.

Fig. 1,B shows the data of one representative experiment from the drug-resistant CCRF/ADR5000 subline compared with the parental sensitive-drug cell line CCRF-CEM. The reliability and the reproducibility between assays were assessed by repeating the experiments three times for each cell line (n = 3). The variability observed in triplicate experiments from the drug-resistant CCRF-ADR5000 subline compared with the parental sensitive drug CCRF-CEM cell line was 11.8% (calculated as the mean of variation coefficient for every quantitative significant gene) ranging from 6.3 to 25.9%. In two others experiments, the drug-resistant HL60/AR subline compared with the parental sensitive drug HL60 cell line and the multidrug-resistant MCF7/CH1000 subline compared with the parental sensitive drug MCF7 line were, respectively, 16.1% ranging from 9.2 to 31.9 and 16.4% ranging from 6.2 to 26.7%. The fluorescence intensity ratios for genes, which were statistically significant for each subline, are presented in Fig. 2.

The CCRF/ADR5000 drug-resistant subline is known to be ABCB1 (MDR1) positive (5). Indeed, we found that the ratio of ABCB1gene expression in this resistant subline was >100-fold higher compared with the drug-sensitive parental line by the microarray technique. The expression of the genes in the parental line was very low, making the ratio calculation to be qualitative. The multidrug-resistant HL60/AR subline overexpresses the ABCC1 (MRP1) gene (6). This gene was indeed found to be overexpressed by a factor of 10.46 compared with the parental line with the DualChip human ABC.

The multidrug-resistant subline MCF7/CH1000 has been reported to overexpress the ABCG2 (MXR/BCRP) gene (7), which was indeed observed with an overexpression of 10 times.

Besides the expected overexpression of the specific ABC transporter genes, the three cell lines also showed several other overexpressed genes. In the CCRF/ADR5000 subline, six other ABC genes were overexpressed in addition to ABCB1. The expression pattern for the promyelocytic resistant cell line HL60/AR showed seven overexpressed ABC transporter genes. Several ABC transporter genes involved in drug resistance were found to be overexpressed: ABCA2, which is known to be involved in drug resistance (16), ABCC1, and ABCC4. In the MCF7/CH1000 drug-resistant cell line, three important ABC transporter genes included in drug resistance are overexpressed: ABCC3, ABCC5, and ABCG2, although ABCA7 was down-regulated. We have also observed an overexpression of ABCB6, ABCF3, and ABCG1.

The expression of ABC transporter genes with expression ratios > 2 and one gene with an expression ratio near one selected at random in the multidrug-resistant cell lines were corroborated by quantitative real-time RT-PCR as independent test method. Three genes were quantified on the parental drug-sensitive CCRF-CEM cell line and on its drug-resistant CCRF/ADR5000 subline: ABCA7, ABCB1/4, and ABCF2 (Fig. 3 A). On the microarray, the overexpression of the ABCB1/4 was found to be qualitative and estimated >100. The real-time RT-PCR gave a ratio of 799. The overexpression was found for ABCA7 to be 2.15 on the microarray and 2.7 by real-time RT-PCR. For the ABCF2, the overexpression was 1.28 on the microarray and 1.64 in real-time RT-PCR.

The data obtained for the parental promyelocytic HL60 cell line and its drug-resistant subline HL60/AR are summarized in Fig. 3 B. The overexpression of the 10 genes tested in multidrug-resistant HL60/AR cells was corroborated by real-time RT-PCR. ABCA4, ABCA7, ABCB2, ABCC1, and ABCC4 were also found overexpressed in real-time RT-PCR with ratios slightly higher than on the microarray. We have dissected ABCC6/8/9 and ABCA2/3 capture-probes. ABCC8 and ABCC9 were undetected by real-time RT-PCR in the drug-resistant cells, whereas the ABCC6 was highly overexpressed. The chips gives only a qualitative result with an overexpression > 3 for the sum of the three genes. We obtained a ratio for ABCA2 of 12.56, although ABCA3 was down-regulated with a ratio of 0.022 by real-time RT-PCR. If we sum up the two results, we obtain a ratio for ABCA2+A3 in real-time RT-PCR of 0.59. The ratio of the capture-probe ABCA2/3 was 1.8 on the array.

Fig. 3 C summarizes the data obtained for the parental MCF7 cell line and its multidrug-resistant MCF7/CH1000 subline. Four overexpressed genes were confirmed with real-time RT-PCR with very close ratios obtained in the two methods and even an identical ratio of 2 for the ABCC5 gene.

Finally, we correlated the IC₅₀ values for compounds included into the NCI’s Standard Agent Database with the baseline mRNA expression level of 31 human ABC transporters (represented by 68 different clones with individual GenBank accession numbers) of the 60 NCI cell lines with the COMPARE algorithm. Drugs whose IC₅₀ values correlated with microarray-based mRNA expression of ABC transporter genes with COMPARE correlation coefficients of r > 0.4 are listed in Table 2. This approach was applied to explore which of the members of the ABC transporter gene family could be involved in drug transport processes and resistance. The results indicate that 17 of 31 ABC transporters investigated might act as drug transporters (ABCA1, ABCA2, ABCA3, ABCA5, ABCA12, ABCB1, ABCB4, ABCB6, ABCB7, ABCB11, ABCC1, ABCC3, ABCC4, ABCC6, ABCF2, ABCF3, and ABCG2) because compounds of the Standard Agents Database whose IC₅₀ values correlated with the mRNA expression of ABC transporters could be identified. Vice versa, no candidate substrates were assigned to 14 other ABC transporters (ABCA4, ABCA6, ABCA8, ABCB8, ABCB10, ABCC2, ABCC5, ABCC7, ABCC8, ABCD2, ABCD3, ABCE1, ABCF1, and ABCG1; Table 2).

Many studies have been done in tumors based on various methods to detect the expression of single members of the ABC transporter gene family, e.g., the MDR-conferring ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (BCRP) genes (17). The family of ABC transporters is still increasing, and the role of many members of the MDR is still unknown or not fully understood. To explore the MDR phenotype in its entirety, it may be helpful to analyze the expression of the ABC transporters simultaneously. DNA microarray technology is one of the technologies that allows gene expression profiling and that has been applied in tumor cells associated with the treatment responsiveness (18).

In the present investigation, we described the use of a low-density DNA microarray for the analysis of 38 ABC transporter genes and three other transporters. IMPT1 (alias SLC22A1L solute carrier family 22) was added as an organic cation transporter for chloroquine and quinidine-related compounds. Kir 6.1 and 6.2 are also present because they form channels in association with the sulfonylurea receptor SUR (such ABCC8 and ABCC9). DualChip human ABC has been elaborated as a first attempt to determine the cell resistance pattern. Other transporter genes could be added later on because the gene family will grow.

The DualChip human ABC was developed to give quantitative results. The variability from one experiment to the other showed that the results were rather well reproducible with mean coefficient of variation between 11.8 and 16.4% from one experiment to the other. We observed that the high variability generally occurs for low-expressed genes. This explains partly the spread of variability from one gene to the other.

The results presented in this article clearly showed an increased expression of several times of the expected ABC transporter genes for the three resistant cell lines analyzed. In addition, we also observed significant overexpression of several other genes (Fig. 2). The values for these overexpressions were >60%, except for the ABCF2 in the CEM cells. Some of these genes are known to confer drug resistance such as ABCA2, ABCB1, ABCC1, ABCC3, ABCC4, ABCC5, and ABCG2. Such overexpression could be seen as typical of the adaptation of the cells after prolonged incubation in the presence of drugs.

Three of these genes have been extensively studied. ABCB1 (MDR1) was the first human ABC transporter cloned and characterized through its ability to confer a MDR phenotype to cancer cells (19). Analysis of multidrug-resistant cells not expressing ABCB1 led to the discovery of the ABCC1 (MRP1) protein, which plays a role in protecting cells from chemical toxicity and oxidative stress and in mediation of inflammatory responses involving cysteinyl leukotrienes (20). Finally, ABCG2 (MXR/BCRP) was identified as a drug transporter in multidrug-resistant cells, which do not express ABCB1 and ABCC1 (21).

As a strategy to explore which ABC transporters might function as drug transporters, we performed COMPARE analysis with compounds included in the NCI’s Standard Agent database⁶ and 31 ABC transporters whose mRNA expression in 60 NCI cell lines has been determined by microarrays (13, 14). The COMPARE computation provided a list of drugs that could be considered as substrates for ABC transporters. Although such correlation analysis does not provide clear evidence for a compound as being a true ABC transporter substrate, this strategy can be used to generate testable hypothesis. In many cases, the COMPARE analysis point to previously validated substrates, i.e., doxorubicin, vinblastine, paclitaxel, dactinomycin, and so forth for the ABCB1 (MDR1) gene. We only considered drugs whose COMPARE correlation coefficient was r > 0.4, and the list of candidate substrates is considerably longer if correlations of r < 0.3 were also taken into account. Surprisingly, well-known substrates of some ABC transporters did not show up by COMPARE analysis with r > 0.4, i.e., doxorubicin, etoposide, vincristine, or methotrexcate as substrates for ABCC1 (MRP1) or doxorubicin, mitoxantrone, or topotecan as substrates for ABCG2 (BCRP). This raises the possibility that other still unknown compounds might be better substrates for these ABC transporters. This is in accord with recently published results on the ABC transporter expression detected by real-time RT-PCR in the same NCI cell line panel as used here (22). Our aim was, however, not to provide a complete list of possible substrates for ABC transporters but to obtain information that ABC transporters could be considered as candidate drug transporters. In this respect, the COMPARE analysis point to 17 of 31 ABC transporters analyzed. The results of the COMPARE analysis reinforce the use of the DualChip human ABC as a tool to detect ABC transporter-associated drug resistance.

As recently reviewed, 11 ABC transporters (ABCA2, ABCB1, ABCB4, ABCB11, ABCC1–6, and ABCG2) have been associated with drug resistance and drug transport as of yet (2). Importantly, the role of ABC transporters, apart from the well-known ABCB1 (MDR1), ABCC1 (MRP1), and ABCG2 (BCRP) for clinical drug resistance, treatment outcome, and patient survival is increasingly recognized. A recent study of Steinbach et al. (23) provides first evidence on the association between the clinical response to chemotherapy and the expression of ABCC2, ABCC3, ABCC4, and ABCC5 in childhood acute myeloid leukemia. This study strongly suggest that ABCC3 is involved in drug resistance in this disease and represents an interesting marker for risk-adapted therapy and a possible target for the development of specific drugs to overcome MDR. Moreover, patients who expressed high levels of ABCC2 and ABCC3 had a particularly poor prognosis (23). The expression of ABCC3 is also associated with a poor outcome in childhood acute lymphoblastic leukemia (24). MRP3 expression correlates with unfavorable clinical outcome in ovarian carcinoma (25). The same applies for MRP2 expression in non–small cell lung cancer (26). The MRP3 and MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs were significantly higher than those in tissues from nonexposed patients (27, 28). In lung cancer cell lines, Young et al. (29) could demonstrate a good correlation of mRNA and protein levels of ABCC2 and ABCC3 in response to cytostatic drugs. Tada et al. (30) showed that the expression of MDR1, MRP1, MRP2, and MRP3 in recurrent and residual bladder tumors after chemotherapeutic treatment was higher than that in untreated primary tumors.

The role of other ABC transporters for drug resistance remains to be shown, i.e., ABCA7, whose substrates and function are still unknown. We have detected ABCA2 in our panel of cell lines (16). Interestingly, antisense treatment against ABCA2 of drug-resistant ovarian carcinoma cells increased their drug sensitivity, suggesting that ABCA2 may also function as a drug efflux pump. The present investigation also points to ABC transporter genes that were found overexpressed and have not thus far been recognized as associated with MDR (ABCA4, ABCA7, ABCB2, ABCB3, ABCB6, ABCB8, ABCB9, ABCC6, ABCF2, ABCF3, and ABCG1). Developing a low-density microarray enabled us to explore the potential implication of many ABC transporter genes currently unknown to be associated with MDR. Future studies have to address the substrates and function for drug resistance of these novel ABC transporter genes.

In conclusion, we have shown that gene expression profiles of ABC transporter genes in multidrug-resistant cell lines can be obtained by quantitative analysis with low-density microarrays. The overexpression of all of the genes observed with the arrays were confirmed with real-time PCR analysis. The determination of the expression profiles of ABC transporter genes in multidrug-resistant cells lines may open new avenues for the diagnosis of MDR in the clinic and for monitoring expression profiles in clinical biopsies and their correlation to clinical treatment.

We thank the Institut of Pathology and Genetic of Loverval (Loverval, Belgium) for its support and the members of Eppendorf Array Technologies Company of Namur (Namur, Belgium) for their collaboration. We also thank Drs. Axel Sauerbrey and Douglas D. Ross (University of Maryland Greenebaum Cancer Center, Department of Medicine, University of Maryland School of Medicine, and the Baltimore Veterans Affairs Medical Center; Baltimore, MD), respectively, for the generous provision of CCRF-CEM, HL60, and MCF7 drug-sensitive and drug-resistant cell lines.