Cancer Diagnosis & Therapy
Induction of MRP5 and SMRP mRNA by adriamycin exposure and its overexpression in human lung cancer cells resistant to adriamycin
Article first published online: 17 AUG 2001
Copyright © 2001 Wiley-Liss, Inc.
International Journal of Cancer
Volume 94, Issue 3, pages 432–437, 1 November 2001
How to Cite
Yoshida, M., Suzuki, T., Komiya, T., Hatashita, E., Nishio, K., Kazuhiko, N. and Fukuoka, M. (2001), Induction of MRP5 and SMRP mRNA by adriamycin exposure and its overexpression in human lung cancer cells resistant to adriamycin. Int. J. Cancer, 94: 432–437. doi: 10.1002/ijc.1490
- Issue published online: 5 OCT 2001
- Article first published online: 17 AUG 2001
- Manuscript Accepted: 11 JUN 2001
- Manuscript Revised: 14 MAY 2001
- Manuscript Received: 24 NOV 2000
- 2nd Term Comprehensive 10-Year Strategy for Cancer Control
Acquired anticancer drug resistance in cancer cells is often a result of an increase in levels of the ATP binding cassette (ABC) transporters that export anticancer drugs from cancer cells, suggesting that anticancer drugs may induce genes that mediate drug resistance in cancer cells. In this study, the induction of anticancer drug transporter gene expression by Adriamycin was examined in human lung cancer cell lines. Increased expression of MDR1, MRP5 and SMRP mRNA was observed 48 hr after the initiation of Adriamycin exposure in human lung cancer PC-14 cells and cisplatin-resistant PC-14/CDDP cells, in a dose-dependent manner as measured by TaqMan real-time RT-PCR. The levels of MRP-1, MRP2 and LRP mRNA were not altered by Adriamycin exposure. The biologic functions of the MRP5 and SMRP genes have not been fully clarified. To elucidate the relationship between Adriamycin resistance and MRP5 and SMRP, mRNA levels of MRP5 and SMRP in Adriamycin-resistant cell lines were compared with the parental cells. Increased expression of MRP5 and SMRP mRNA was observed in all 3 cell lines (SBC-3/ADM, AdR MCF7 and K562/ADM) by Northern blot analysis and RNase protection assay. These results suggest that subacute exposure of lung cancer cells to Adriamycin induced MRP5 and SMRP and that long-term exposure with Adriamycin selected the MRP5- and SMRP-overexpressing lung cancer cells. MRP5 and SMRP is a candidate molecule for acquired Adriamycin resistance in addition to MDR1. © 2001 Wiley-Liss, Inc.
Adriamycin, a DNA topoisomerase II inhibitor, is a standard anticancer agent for solid tumors such as breast carcinoma and stomach carcinoma. Therefore, the acquired resistance to Adriamycin in cancer cells is a major obstacle to achieving treatment success. Increased intracellular detoxification by molecules such as glutathione and superoxide dismutase has been suggested to be involved in Adriamycin resistance.1–3 Adriamycin resistance often may also result from overexpression of P-glycoprotein encoded by the MDR-1 gene.4, 5 However, the increased expression of P-glycoprotein and MDR-1 does not always contribute to clinical resistance, at least in lung cancer patients.
Another ATP binding cassette (ABC) transporter, MRP1, has also been reported to be a possible predictor of resistance to anticancer drugs in lung cancer patients. A glutathione-S-X conjugate pump (GS-X pump), which is distinct from MRP1 and MRP2(cMOAT), has been found to be involved in reducing the accumulation of Adriamycin.6–8 Five new homologues of MRP1 have been identified: MRP3, MRP4, MRP5, MRP6 and MRP7.9–11 The steady-state levels of mRNA for MRP3 and MRP5 are elevated in some drug-selected cancer cell lines, suggesting that overexpression of these MRP1 homologues may contribute to drug resistance. We have cloned a short type of MRP1 homologue, SMRP, which is identical to MRP5, from a human lung cancer cell line resistant to cisplatin (CDDP).12 It was suggested that increased levels of expression of some of the transporters is responsible for the resistance to anticancer drugs.9
Transporter-mediated drug resistance is considered to be related to intrinsic and acquired resistant mechanisms in the clinical situation. Recent progress in cancer chemotherapy is improving the tumor regression rate. The purpose of this study was to elucidate the induction of transporter gene expression by Adriamycin treatment and the expression of these genes in Adriamycin-resistant cells to determine the potency of Adriamycin for acquired and intrinsic resistance in lung cancer.
MATERIAL AND METHODS
Drugs and cells
Adriamycin was obtained from Kyowa Hakko Kogyo Co. (Tokyo, Japan). Adriamycin was dissolved in dH2O and stored at −30°C until use. The PC-7, PC-9 and PC-14 cell lines derived from a human nonsmall cell lung cancer were donated by Prof. Hayata, Tokyo Medical University (Tokyo, Japan). The CDDP-resistant cell lines, PC-7/CDDP, PC-9 and PC-14/CDDP, were obtained from PC-7, PC9 and PC-14, respectively, by stepwise dose escalation of CDDP.13, 14 These sublines showed approximately 10-fold resistance to the growth-inhibitory effect of CDDP as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and this resistant phenotype has been stable for more than 1 year.14 Three Adriamycin-resistant cell lines—SBC-3/ADM,15, 16 K562/ADM17 and AdrR MCF716, 18—and their respective parental cell lines (SBC-3, K562 and MCF7) were obtained from Dr. Kiura (Okayama University, Okayama), Prof. Tsuruo (Tokyo University, Tokyo) and Prof. Cowan (NCI, Bethesda, MD). These 3 Adriamycin-resistant cell lines show a multidrug-resistant phenotype with overexpression of MDR-1 genes. They were maintained in RPMI-1640 medium supplemented with 10% FCS. For the induction experiments, cells were harvested in the late-log phase and seeded into tissue culture dishes at a density of 5 × 105 cells/ml. After a 2-hr preincubation, 5 different concentrations of Adriamycin (0.03–1 μM) were added to the medium and the cells were exposed to the drug under a humidified atmosphere of 5% CO2 and air at 37°C for the 6–48 hr. The concentration of Adriamycin was determined by the IC50 value for a 72-hr incubation in the MTT assay.
Quantitation of PCR products and analysis of mRNA expression
RNA was extracted using a commercial kit (RNeasy Total RNA System, Qiagen, Tokyo). One microgram of total RNA and 2 μl of random hexamer was combined in a total volume of 12 μl, incubated at 70°C for 10 min and allowed to cool to room temperature. After the addition of 2 μl 10× PCR buffer, 2 μl 0.1 M DTT, 1 μl 10 mM dNTP mixture, 2 μl 25 mM MgCl2 and 1 μl SuperScript reverse transcriptase (GIBCO-BRL, Gaithersburg, MD), the mixture was serially incubated at 42°C for 50 min and 70°C for 15 min. One microliter of the RT reaction served as template for the TaqMan real-time PCR technique to quantify mRNA expression.19, 20 Commercial reagents were used (TaqMan PCR Reagent Kit; Perkin-Elmer, Foster City, CA) following the manufacturer's protocol were applied. Briefly, 2.5 μl of cDNA (reverse transcription mixture) and oligonucleotides at a final concentration of 300 nM of primers and 200 nM of TaqMan hybridization probe was analyzed in a 25-μl volume. The oligonucleotides of each target of interest were designed by the Primer Express software (Perkin-Elmer) using uniform selection parameters that allow the application of standard cycle conditions. Within the amplicon defined by a gene-specific PCR primer pair, an oligonucleotide probe labeled with 2 fluorescent dyes was created and designated the TaqMan probe. The emission of the reporter dye (i.e., 6-carboxy-fluorescein, FAM) at the 5′ end was quenched by the second fluorescence dye (6-carboxy-tetramethyl-rhodamine, TAMRA) at the 3′ end. The increasing amount of reporter dye emission was detected by an automated sequence detector combined with a software (Gene Amp 5700 Sequence Detection System; Perkin-Elmer). The algorithm normalized the reporter signal (Rn) to a passive reference. Next, the algorithm multiplied the standard deviation of the background Rn in the first few cycles (in most PCR systems cycles 3–15, respectively) by a default factor of 10 to determine a threshold. The cycle at which this baseline level was exceeded was defined as threshold cycle (CT). CT has a linear relation with the logarithm of the initial template copy number. Its absolute value additionally depends on the efficiency of both DNA amplification and cleavage of the TaqMan probe. The CT values of the samples were interpolated to an external reference curve constructed by plotting the relative or absolute amounts of a serial dilution of a known template vs. the corresponding CT values. The housekeeping gene glyceraldehyde-3-phosphate (GAPDH) was used to normalize mRNA concentration. The following primers and TaqMan probes were used:
GAPDH: Forward 5′-CCC ATG TTC GTC ATG GGT GT-3′; Reverse 5′-TGG TCA TGA GTC CTT CCA CGA TA-3′; TaqMan probe 5′(FAM)-CTG CAC CAC CAA CTG CTT AGC ACC C-(TAMRA)3′
MDR-1: Forward 5′-GCT CAG ACA GGA TGT GAG TTG G-3′; Reverse 5′-ATA GCC CCT TTA ACT TGA GCA GC-3′; TaqMan probe 5′(FAM)-AAA ACA CCA CTG GAG CAT TGA CTA CCA GGC T-(TAMRA)3′
MRP 1: Forward 5′-TAC CTC CTG TGG CTG AAT CTG G-3′; Reverse 5′-CCG ATT GTC TTT GCT CTT CAT G-3′; TaqMan probe 5′(FAM)-CCG TCA ATG CTG TGA TGG CGA TGA T-(TAMRA)3′
MRP2: Forward 5′-TTC AGC GAG ACC GTA TCA GGT T-3′; Reverse 5′-TTC TGG TTG GTG TCA ATC CTC A-3′; TaqMan probe 5′(FAM)-7TGC CTT TGA GCA CCA GCA GCG ATT T-(TAMRA)3′
MRP5: Forward 5′-CCC AGG CAA CAG AGT CTA ACC-3′; Reverse 5′-CGG TAA TTC AAT GCC CAA GTC-3′; TaqMan probe 5′ (FAM)- TGA CGG AAA TCG TGC GGT CTT GGT-(TAMRA)3′
SMRP: Forward 5′-TCC TTC CAC TGT ATA GCC TGA TGT-3′; Reverse 5′-ACA CTG GAT CAA GAC CAG GGA-3′; TaqMan probe 5′(FAM)- TTT TGC TAC GTG AGT GTA CGC CCT AGG C-(TAMRA)3′
LRP: Forward 5′-GCC TGA CTT CTT CAC AGA CGT C-3′; Reverse 5′-TCA AAG TGC CAG TTG TAG GCC-3′; TaqMan probe 5′(FAM)-TCA CCA TCG AAA CGG CGG ATC ATG-(TAMRA)3′
The thermocycler parameters were 95°C for 10 min (for initial denaturation) followed by denaturation at 95°C for 15 sec and annealing and extension at 60°C for 1 min.
RNase protection assay
For the measurement of mRNA levels of MRP5 and SMRP by RNase protection assay in the lung cancer cells, we used a 32P-labeled probe for MRP5. By PCR amplification of MRP5 cDNA, a 225-bp fragment corresponding to nucleotides 1345–156912 (Gene Bank accession number AB005659) was used as a probe. Total RNA (20 μM) was hybridized with radioactive probes at 45°C overnight and then digested with RNase A (10 μg/ml) and RNase T1 (1000 U/ml) for 1 hr at 37°C. Protected probes were visualized by electrophoresis through a denatured 6% acrylamide gel, followed by autoradiography. In all experiments, a probe for β-actin (120 bp) was included as an internal control. The amount of MRP5 mRNA relative to that of β-actin was calculated using a laser imaging analyzer.
Total RNA was prepared from the cell lines by the acid guanidinium thiocyanate-phenylchloroform extraction method. Approximately 20 μg of total RNA was electrophoresed and transferred to nylon (Hybond-N) filters as described previously.16 We probed with the E-1 fragment of MRP5 and SMRP.12, 21 The probe was labeled with α-32P dCTP to a specific activity of 2 × 108 cpm μg-1 DNA using the Rediprime II DNA Labelling System (Amersham Pharmacia Biotech, Tokyo, Japan). The filters were prehybridized in 5× SSC [1× SSC: 0.15 M NaCl and 15 mM sodium citrate (pH 7.0)], 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml sonicated salmon sperm DNA and 50% foramide at 42°C for 3 hr. Hybridization with 32P-labeled E-1 was performed overnight in the same buffer containing 10% dextran sulfate. The hybridized membrane was washed in 2× SSC, 0.1% SDS at room temperature for 30 min and 15 min, followed by washing with 0.1× SSC, 0.1% SDS at 55°C for 10 min. Filters were exposed to a BioMAX MS film (Amersham) at −80°C. The spots on autoradiography were semiquantified densitometrically with Photoshop 5.0 (Adobe Systems, Tokyo).
Total cell lysates were made as described elsewhere.22 Protein concentrations were determined with a Bio-Rad protein assay (Bio-Rad, Richmond, CA). Ten to 40 μg of cell lysates or crude or purified fusion proteins were fractionated on a 7.5% polyacrylamide slab gel and transferred onto a nitrocellulose membrane by electroblotting. After blocking, the membrane was incubated for 1.5 hr with anti-MRP5 human monoclonal antibody (M5II-54, Alexis, in 1:20 dilution. Biotinylated anti-rat serum (1:1,000; Dako) was used as a secondary antibody. Enhanced chemiluminescence (Amersham, Buckinghamshire, UK) was used to detect monoclonal antibody binding.
Contingency table analysis based on analysis of variance (ANOVA) statistics was used to determine the significance of association between categorical variables in the TaqMan PCR experiment. Overall, statistical differences between the levels of expression of each gene in the samples exposed and not exposed to with or without different concentration of Adriamycin were analyzed using repeated-measures ANOVA. Test for linear trend of Bonferroni multiple comparison was performed as posttests following repeated-measures ANOVA to determine dose-dependent gene induction ability. The statistical calculations and tests were performed using Stat View J4.11 Software (Abacus Concepts, Berkeley, CA) and an IBM computer. All the statistical tests were 2-sided, the data were expressed as means and SEM; differences at p < 0.05 were considered significant.
Induction of MRP1 and expression of MRP5 and SMRP by Adriamycin
To determine whether Adriamycin induces expression of transporter gene, we monitored the MRP1, MRP2, MRP5, SMRP and LRP expression levels in lung cancer cells using TaqMan real-time RT-PCR. Normalization was performed using GAPDH as internal control (Fig. 1). We exposed lung cancer cells to different concentrations of Adriamycin for various periods (6–48 hr). Differences in the grade of induction at 48 hr were observed between MRP5 and SMRP, speculating that the grade of transcription of MRP5 and SMRP might be regulated differently. However, the overall induction of MRP5 and SMRP was significantly induced by Adriamycin exposure, as determined by repeated ANOVA. The levels of MRP1, MRP2 and LRP mRNA were not significantly different between Adriamycin-treated and nontreated PC-14 cells (Table I, Fig. 2).
|Exposure period (hr) ADM (μM)||Gene||PC-14||PC-14/CDDP|
To confirm the induction of MDR-1, MRP5 and SMRP gene expressions, the same experiments were repeated using PC-14/CDDP cells (Table I, Fig. 2). Consistent with the PC-14 cells, expression of these 2 genes was significantly increased in PC-14/CDDP cells after a 48-hr exposure. The increase in expression of MRP5 and SMRP by Adriamycin exposure was larger in PC-14/CDDP cells than in PC-14 cells. These results suggest that Adriamycin induces MDR1, MRP5, and SMRP expression in human lung cancer cells. In contrast, decreased expression of MRP1 and LRP was observed in the Adriamycin-treated PC-14 and PC-14/CDDP cells, suggesting different mechanisms of induction between these 2 pairs of genes (MDR-1, MRP5 and SMRPvs. MRP1 and LRP).
MDR1 is a stress-response gene and therefore, it was not surprising that MDR1 mRNA is induced by Adriamycin exposure. However, to date nothing is known about the induction of MRP5 and SMRP expressions by Adriamycin. Increased mRNA expression of MDR-1, MRP5 and SMRP was observed only from 48 hr, suggesting that induction occurs at the subacute phase, unlike known early response genes.
MRP5 and SMRP overexpression in Adriamycin-resistant cells
To elucidate the biologic significance of MRP5 and SMRP induction in drug resistance, the mRNA levels of MRP5 and SMRP were examined in the Adriamycin-resistant cells by Northern blot analysis. Steady-state mRNA expression of MRP5 and SMRP in 3 Adriamycin-resistant cells was compared with that of the respective parental cells (Fig. 3a). SBC-3/ADM, K562/ADM and AdrR MCF-7 cells showed markedly higher MRP5 expression than their respective parental cells, with 1.47-, 1.57- and 2.36-fold increases in expression, respectively (Fig. 3b). The overexpression of MRP5 and SMRP in these Adriamycin-resistant cells was confirmed by RNA protection assay (Fig. 3c,d). These results suggest that MRP5 and SMRP are overexpressed in Adriamycin-resistant cells and may be related with Adriamycin resistance. Increased expression levels of MRP5 in 3 Adriamycin-resistant cells were also observed as compared with respective parental cells. This finding strengthens our hypothesis, that MRP5 is 1 candidate for conferring Adriamycin resistance (Fig. 3e).
We previously reported that SMRP is 1 of the spicing variant of MRP5.21 Therefore we examined the levels of MRP5 and SMRP expression separately in the TaqMan RT-PCR experiment. Although the overall expressions of MRP5 and SMRP were significantly induced by exposure to Adriamycin, there was some difference in the grade of MRP5 and SMRP transcripts that led us to believe they might be regulated in a different manner. Although the physiologic function of MRP5 remains unknown, MRP5 may play a role in cellular drug resistance such as MRP1 and MRP2(cMOAT), because MRP5 has topologic homology with MRP1 and MRP2.10, 22, 23 Expression of MRP5 has been observed in tissues including brain and skeletal muscle,10, 12, 24 whereas overexpression of MRP1 has been noted in the lung, bladder, spleen, thyroid, testis and adrenal gland and MRP2 is expressed in the liver.25, 26 These results suggest that different GS-X pumps may play major roles in different organs.
Our present results showed that the levels of expression MRP5 and SMRP are significantly induced by Adriamycin exposure in human cancer cells. Previously we demonstrated a lack of rapid induction of MRP5 by CDDP in PC-14 cells and the possibility that prolonged retention of platinum may induce the MRP5 gene in a clinical setting.27 These results suggest that MRP5 and SMRP may be induced only by specific drugs.
Two different clones of human lung cancer cells PC-14 were used in this study. PC-14/CDDP cells are a CDDP-resistant subclone derived from PC-14.14 The chemosensitivity test revealed no cross-resistance of PC-14/CDDP to Adriamycin (data not shown). Adriamycin-resistant cell lines, including the resistant cell lines we used, overexpress P-glycoprotein.16, 17 In that case, decreased intracellular accumulation of Adriamycin directly influences the lack of gene induction by Adriamycin. Therefore we selected the CDDP-resistant clone that does not overexpress P-glycoprotein. The results using PC-14/CDDP could confirm the Adriamycin-induced gene expression of MDR-1, MRP5 and SMRP.
Increased expression of MRP5 and SMRP was observed after a 48-hr exposure of cancer cells to Adriamycin, which was probably mediated by induction of specific genes. Elevated expression of MRP5 and SMRP was also observed in 3 Adriamycin-resistant cell lines, suggesting that Adriamycin treatment can induce MRP5- and SMRP-mediated drug resistance in lung cancer cells either by induction of gene expression or by selection of resistant clones.
MRP family genes may be induced by various stimuli. MRP3 is relatively easily induced by stress.28, 29 We recently reported that MRP5 and SMRP gene expression was not induced by short-term exposure to CDDP.27 However, Adriamycin induced more MRP5 and SMRP expression in PC-14/CDDP compared with PC-14 cells. Therefore acquired resistance to CDDP did not reduce the potency of MRP5- and SMRP-mediated resistance. This discussion may be extended to other transporters including MDR-1, MRP1 and MRP2, the expression of which was also affected by Adriamycin exposure in PC-14/CDDP and PC-14 cells.
The LRP gene was reported to be related to drug resistance in lung cancer and other malignant cells,30, 31, 32 so we also measured LRP expression. There was no marked induction of LRP expression by Adriamycin, consistent with a previous study.33
MDR1 expression was induced by Adriamycin in PC-14 and PC-14/CDDP cells under the same conditions that MRP5 and SMRP was induced. Induction of MDR1 by various stresses including Adriamycin has been widely observed in various cell types.34, 35, 36 However, it is interesting that coinduction of MDR-1, MRP5 and SMRP but not with MRP1 or LRP. Furthermore overexpression of MRP5 and SMRP was observed in all 3 Adriamycin-resistant cell lines. Because these Adriamycin-resistant cells overexpress MDR-1,16, 17MDR-1, MRP5 and SMRP are probably coexpressed. We previously reported co-overexpression of GS-X pump and γ-glutamylcysteine synthetase in human lung cancer cells.37 Kuo et al.38 demonstrated the co-overexpression of γ-glutamylcysteine synthetase and MRP1. Cooverexpression of MDR1 and MRP5 (or SMRP) may also be related to drug resistance. The molecular mechanisms of co-overexpression should be clarified in future studies.
In conclusion, Adriamycin exposure induced expression of MRP5 and SMRP genes in lung cancer cells and overexpression of MRP5 and SMRP were observed in Adriamycin-resistant cell lines. MRP5 and SMRP are thus candidates for Adriamycin resistance.
- 10Kool M, de Haas M, Scheffer G.L, et al. Analysis of expression of cMOAT (MRP2), MRP3, MRP4 and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res 1997;57: 3537–47.