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Quantitative analysis of a MDR1 transcript for prediction of drug resistance in acute leukemia.

Although the development of new therapeutic agents and treatment regimens has improved the cure rate of acute leukemia, >50% of adult patients relapse and do not survive (1, 2). One of the major obstacles to successful treatment is the drug resistance of leukemic cells to chemotherapeutic agents. Once leukemic cells become resistant to antineoplastic agents, the choice of treatment becomes very limited because the range of drug resistance spreads widely, even to agents to which the leukemic cells have never been exposed (3, 4). Thus, to improve the clinical outcome of leukemia it is essential to develop an effective strategy to reverse this resistance, but the complicated mechanism by which this drug resistance is acquired poses a major problem.

This multidrug resistance has been shown to be partly mediated by the enhanced expression of the multidrug resistance 1 (MDR1) [3] gene, which encodes P-glycoprotein (5, 6), which is expressed widely in healthy tissues and physiologically functions as an ATP-dependent efflux pump (7, 8). Important agents in the treatment of leukemia, including anthracycline, Vinca alkaloids, and podophyllins, have been identified as substrates for P-glycoprotein (3, 4), so it is reasonable that overexpression of P-glycoprotein leads to excessive efflux and insufficient intracellular concentrations of these agents, leading to multidrug resistance. There is another drug resistance-associated gene, multidrug resistance-related protein 1 (MRP1), that is structurally similar to MDR1 and also functions as a transporter (9). Although MRP1 was originally cloned from a drug-resistant lung cancer cell line, it, like MDR1, has been implicated in multidrug resistance in leukemia (9-11).

To determine whether the overexpression of MDR1 and MRP1 is associated with the phenotype of multidrug resistance in clinical samples, the expression of these genes has been measured by different methods, such as Northern blot analysis (12), flow cytometric (FCM) analysis (13, 14), and competitive reverse transcription (RT)PCR analysis (15, 16). However, controversy remains because these methods are semiquantitative and the amount of expression can not be expressed as definite values. In this study, we established a quantitative system to measure the amounts of MDR1 and MRP1 transcripts by real-time quantitative RT-PCR. We determined the proper cutoffs for the copy numbers of MDR1 and MRP transcripts by comparing the biological drug sensitivity assay in human leukemia lines exhibiting various degrees of drug resistance. The cutoff for MDR1 reflected the clinical drug resistance, suggesting that this method may be applicable for the routine determination of drug resistance.

Materials and Methods

CELL LINES

The KG-1a, Kasumi-1, THP-1, U-937, and HL60 human myeloid lines; the Raji and Daudi B-cell lines; the Jurkat and CCRF-CEM T-cell lines; and the K562 and UT7 erythroid cell lines were obtained from the cell bank of Tohoku University. Cell cultures were maintained in RPMI-1640 (Life Technologies) supplemented with 100 mL/L fetal calf serum (Life Technologies), 100 IU of penicillin, and 100 mg/L streptomycin at 37 [degrees]C in a 5% CO2 atmosphere. K562/VCR, a vincristine (VCR)-resistant subline of K562 cells that overexpresses MDR1 (17), was generously provided by Dr. T. Tsuruo (University of Tokyo, Tokyo, Japan), and H69/VP, an etoposide (VP16)-resistant subline of small cell lung cancer cell line H69 that overexpresses MRP1 (18), was generously provided by Dr. N. Saijo (National Cancer Center, Tokyo, Japan). To establish variant sublines resistant to VCR, K562/VCR cells were maintained for 1 month in 0-100 nmol/L VCR (Sigma Chemical), and 200 [micro]l of the diluted cell suspension was then distributed to each well of a 96-well flat-bottomed tissue culture plate (Falcon; BD Labware). These subcloned cells were propagated into resistant sublines.

PATIENTS

After obtaining informed consent, we collected peripheral blood (PB) or bone marrow (BM) aspirates from 44 adult patients: 18 patients with acute lymphoblastic leukemia (ALL) and 26 patients with acute myelogenous leukemia (AML). The percentage of leukemic cells in samples was >70% in all cases. Each phenotype of leukemia was defined by the French-American-British Cooperative Group classification (19, 20). Twenty patients were at onset, whereas the others had previously received anticancer chemotherapy. Response to treatment was classified as follows: complete remission (CR; maintaining complete remission according to established criteria for >6 months: cellular marrow with <5% blast cells, neutrophil count [greater than or equal to]1.5 x [10.sup.9]/L, platelet count [greater than or equal to]100 x [10.sup.9]/L, and no evidence of leukemia in other sites); nonresponder (NR; cellular marrow with >5% blast cells or evidence of leukemia in other sites, after at least two courses of chemotherapy); and early relapse (ER; relapse within 6 months from remission) (21). For controls, PB from 20 healthy volunteers and BM aspirates from 6 patients with nonhematologic malignancies were obtained after informed consent. BM aspirates from these patients were normal in morphology and cellularity and did not show any cytogenetic abnormalities.

RNA EXTRACTION

Mononuclear cells from PB and BM samples were separated by Ficoll-Hypaque density gradient centrifugation (400g for 40 min at 20 [degrees]C) and suspended in phosphate-buffered saline. All samples contained at least 85% blasts after mononuclear cell isolation. The cellular viability, confirmed by trypan blue staining, was routinely 90-100%. Total cellular RNA was isolated from mononuclear cells by Isogen-LS (Nippon Gene), with a modified acid guanidinium thiocyanate-phenol-chloroform method. To ensure removal of chromosomal DNA from total RNA, total RNA was treated with DNase I (Takara), and the quality of RNA was estimated after migration on a 2% agarose gel. The concentration and purity of the RNA samples were determined spectrophotometrically, and they were stored at -80 [degrees]C until use.

REAL-TIME QUANTITATIVE RT-PCR FOR MDR1 AND MRP1

Quantitative RT-PCR was performed with the TaqMan EZ RT-PCR Kit (Perkin-Elmer Applied Biosystems) as described previously (22). A 1-[micro]g total RNA sample was used for RT-PCR in a 50-[micro]L amplification reaction solution containing 1x TaqMan EZ buffer; 4 mM manganese acetate; 200 [micro]M each of dATP, dCTP, and dGTP; 400 [micro]M dUTP; 0.75 U of rTth DNA polymerase; 0.5 U of AmpErase Uracil N-glycosylase; 200 nM each of the primers, and 100 nM TaqMan probe.

The thermal cycling conditions included 2 min at 50 [degrees]C, 30 min at 60 [degrees]C (reverse transcription reaction), and 10 min at 95 [degrees]C. Thermal cycling was performed with 50 cycles of 95 [degrees]C for 15 s and 60 [degrees]C for 1 min. All reactions were performed in the ABI PRISM [TM] 7700 Sequence Detection Systems (PE Applied Biosystems). For a control gene, we used a TaqMan glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Control Reagent (PE Applied Biosystems).

The sequences of the primers and probe for MDR1 were as follows:

Forward primer: 5'-TGC TCA GAC AGG ATG TGA GTT G-3' (nucleotides 2811-2832)

Reverse primer: 5'-TTA CAG CAA GCC TGG AAC CTA T-3' (nucleotides 2909-2930)

Probe: 5'-AGC ATT GAC TAC CAG GCT CG CCA A-3' (nucleotides 2860-2883)

The sequences of the primers and probe for MRP1 were as follows:

Forward primer: 5'-TCT ACC TCC TGT GGC TGA ATC TG-3' (nucleotides 1560-1583)

Reverse primer: 5'-CAC CTG ATA CGT CTT GGT CTT CAT-3' (nucleotides 1649-1672)

Probe: 5'-ATG GTC CTC ATG GTG CCC GTC AAT-3' (nucleotides 1613-1636)

After amplification, the product sizes were 120 bp for MDR1 transcripts and 113 bp for MRP1 transcripts. The probes were fluorescently labeled with 6-carboxyfluorescein (reporter) at the 5' end and 6-carboxytetramethylrhodamine (quencher) at the 3' end. The threshold cycle ([C.sub.t]) is defined as the fractional cycle number at which the reporter fluorescence generated by cleavage of the probe passes a fixed threshold above baseline. To establish a calibration curve, we synthesized a RNA calibrator by in vitro transcription. For in vitro transcription, MDR1, MRP1, and GAPDH cDNAs was amplified by PCR using reverse-transcribed total RNA from K562/VCR or H69/VP cells, purified using a Qiagen PCR Purification Kit (Qiagen), and cloned into the NotI (TAKARA) site of pGEM-T expression vector (Promega). After the sequence was verified, the plasmid was linearized and transcribed by T7 RNA polymerase (Roche Diagnostics). One ng of MDR1 RNA obtained by in vitro transcription corresponded to 9.7 x [10.sup.9] copies, 1 ng of MRP1 RNA corresponded to 1.0 x [10.sup.10] copies, and 1 ng of GAPDH RNA corresponded to 6.1 x [10.sup.9] copies, respectively. Using serial dilutions of the RNA calibrators, we then generated the calibration curves on the basis of the linear relationship between the [C.sub.t] value and the logarithm of the copy numbers. The absolute copy numbers of MDR1 and MRP1 in samples were calculated by [C.sub.t] using each of the calibration curves, and the results were standardized by the ratio of those copy numbers to the copy numbers for GAPDH.

DRUG SENSITIVITY ASSAY

The in vitro sensitivities of the parent and resistant cells to anticancer drugs were determined with the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (23). Briefly, 5 x [10.sup.4] viable cells were added to each well of 96-well culture dishes (Falcon), containing 0.2 mL of medium with graded concentrations of anticancer drugs, and incubated for 72 h at 37 [degrees]C in a 5% C[O.sub.2]-95% air atmosphere. After incubation, 10 [micro]L of MTT (Sigma) solution (4 g/L in phosphate-buffered saline) and 10 [micro]L of 0.1 mol/L sodium succinate (Sigma) were added to each well, and the cells were incubated at 37 [degrees]C for 4 h. After incubation, the plate was centrifuged at 650g for 5 min, and supernatants were then removed. The formazan crystals produced were dissolved by the addition of 150 [micro]L of dimethyl sulfoxide (Sigma), and the absorbance of each well was measured at 540 nm in a plate reader (ELSIA Reader; International Reagent). Cell growth inhibition-vs-drug concentration curves were obtained by plotting the percentage of viable cells, calculated based on the absorbance in drug-treated wells against that in control wells without any drug. The concentrations that produced 50% growth inhibition ([IC.sub.50]) in these cells were determined from the dose-response curves, and the relative resistance to each drug was determined as the ratio of the [IC.sub.50] of the resistant cells to that of the parent cells.

FCM ASSAY FOR P-GLYCOPROTEIN

P-Glycoprotein expression was analyzed with anti-human P-glycoprotein mouse monoclonal antibody MRK-16 (Kyowa Medicus) and a fluorescein isothiocyanate-labeled second antibody. The results were expressed as the ratios of the mean fluorescence of MRK-16-labeled cells divided by that of the isotype control-labeled cells (24). P-Glycoprotein function was measured by FCM determination of the increase in rhodamine 123 (Rh123) (25). Cells (2 _ 106) were incubated with 200 [micro]g/L Rh123 (Sigma Chemicals) for 1 h at 37 [degrees]C in RPMI-1640. After incubation, cells were washed twice in ice-cold phosphate-buffered saline and resuspended in fresh medium for 1 h at 37 [degrees]C, allowing efflux of the dye from the cells, and then analyzed on a Coulter EPICS ELITE flow cytometer. Cells exposed to Rh123 were used as controls.

Results

design of real-time quantitative rt-pcr assay To establish appropriate conditions for quantitative RTPCR, we prepared serial dilutions of synthetic RNA. MDR1 transcripts were detectable in 1 fg of synthetic RNA (9.7 x [10.sup.3] copies) in this system. The assay was linear from 9.7 x [10.sup.3] to 9.7 x [10.sup.8] copies (Fig. 1A). The calibration curve showed a good correlation between MDR1 RNA copy number and [C.sub.t] (r = -0.999; Fig. 1B). The within-run and day-to-day CVs for MDR1 RNA in this system were 9.5-25% (n = 10) and 15-39% (n = 10), respectively (data not shown). On the other hand, MRP1 transcripts were detectable in 0.1 fg of synthetic RNA (1.0 x [10.sup.3] copies). The assay for MRP1 was linear from 1.0 x [10.sup.3] to 1.0 x [10.sup.9] copies (Fig. 1C). The calibration curve also showed a good correlation between the MRP1 RNA copy number and [C.sub.t] (r = -0.998; Fig. 1D). The within-run and day-to-day CVs for MRP1 RNA in this system were 14-36% (n = 10) and 22-42% (n = 10), respectively (data not shown).

SPECIFICITY OF QUANTITATIVE RT-PCR FOR DETERMINING PHENOTYPE OF DRUG RESISTANCE

To confirm that the copy number obtained by quantitative RT-PCR correlates to drug sensitivity, we next examined the expression MDR1 mRNA in various cell lines, including drug-resistant K562/VCR cells, and in healthy BM and PB cells by quantitative RT-PCR. These results are shown as the ratio of MDR1 or MRP1 copy numbers to those of GAPDH as internal control in each sample. MDR1 expression was 0.21-6.7 in the K562/VCR sublines and H69/VP cells, but was <0.0041 in other drug-sensitive cell lines. MDR1 expression in healthy BM and PB cells was 0.024 [+ or ] 0.015 and 0.027 [+ or ] 0.011 (mean [+ or ] SD), respectively (Fig. 2). MRP1 expression was 0.053-0.37 in the examined cell lines, whereas in healthy BM and PB cells, it was 0.20 [+ or ] 0.15 and 0.23 [+ or ] 0.13, respectively (Fig. 2). On the basis of the expression values in healthy BM cells, we defined the cutoff for MDR1 as 0.0054 and that for MRP1 as 0.49 (mean + 2 SD), and the samples above this cutoff value were defined as positive.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The possible correlation between biological drug resistance and copy number of MDR1 transcripts was further examined by the in vitro MTT assay, based on the viability of cells exposed to the drug. For this purpose, sublines of K562/VCR with various degrees of drug resistance were established as described in Materials and Methods. Fig. 3 shows the relationship between the MDR1 mRNA expression in these sublines and the drug sensitivities for VCR examined by MTT. The [IC.sub.50]s in the MTT assay were 16-72 nmol/L in the VCR-resistant lines but <0.05 nmol/L in most sensitive sublines. The expression of MDR1 mRNA examined by quantitative RT-PCR clearly was related to the VCR [IC.sub.50] in the sublines. Interestingly, all sublines showing drug resistance were MDR1-positive, whereas all sensitive sublines were negative, indicating that the defined cutoff value reflected the biological drug sensitivity. On the other hand, expression of MRP1 and the [IC.sub.50] for VP16 did not correlate (data not shown).

CORRELATION BETWEEN EXPRESSION OF MDR1 AND MRP1 AND CLINICAL OUTCOMES

To date, several methods to detect drug resistance have been reported. We compared the sensitivity of quantitative RT-PCR with that of other methods, i.e., FCM analysis and the MTT assay. The drug resistance results in 14 leukemia samples (6 ALL and 8 AML) determined by MTT assay, FCM analysis, and quantitative RT-PCR are summarized in Table 1. No positive case of drug resistance in patients in CR was found by any of the methods. Some NRs were identified as positive by the MTT or FCM assays, but the number of NRs identified as positive by RT-PCR was higher than the numbers identified by either the MTT or the FCM assay. These results suggest that the sensitivity of the quantitative RT-PCR method is higher than that of the other two methods.

[FIGURE 3 OMITTED]

Finally, the expression of the MDR1 and MRP1 genes in clinical samples was analyzed according to clinical outcomes. Whereas all AML cases at relapse were MDR1-positive, 6 of 16 AML cases at onset were MDR1-positive, and the mean expression at onset was significantly lower than at relapse (0.16 [+ or -]0.32 vs 0.20[+ or -]0.21; P <0.05; Fig. 4). The differences in expression between subgroups of AML patients divided according to French-American-British classification did not differ from one another. On the other hand, 5 of 13 patients with ALL at onset were MDR1-positive, and 2 of 5 samples at relapse were MDR1-positive. The mean expression at onset was not significantly different from that at relapse (0.17 [+ or -] 0.35 vs 0.061 [+ or -] 0.094; Fig. 4). Fig. 5 shows the normalized numbers of MDR1 transcripts in the subgroups divided by the clinical drug resistance. In both AML and ALL at onset, all of the CR cases were negative for MDR1, and the mean level of MDRI transcription in positive cases increased with increasing clinical drug resistance. All MDR1-positive cases either failed to respond to chemotherapy (NR) or relapsed immediately after achieving remission (ER). The mean MRP1 values in ALL at onset and relapse and in AML at onset and relapse were 0.44 [+ or -]0.40, 2.13 [+ or -] 3.39, 0.93 [+ or -] 2.76, and 0.27 [+ or -] 0.18, respectively, and did not differ significantly from each other (data not shown). Moreover, different from MDR1, the numbers of MRP1 transcripts did not correlate with clinical drug resistance.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

Several mechanisms are thought to be involved in the development of resistance to chemotherapy, including overexpression of the membrane-associated ATP-dependent efflux pump, P-glycoprotein, which is encoded by MDR1 and MRP1 (8, 9). Because many kinds of antileukemic agents can be substrates for this efflux pump, overexpression of MDR1 leads to insufficient concentrations of agents in leukemic cells even at maximum doses. Thus, P-glycoprotein has been shown experimentally to be an attractive protein for explaining the mechanism of multidrug resistance; however, it is still controversial whether overexpression of the genes that encode for this protein are associated with prognosis for the disease (26). This is partly because of a lack of adequate laboratory methods to correctly assess cellular drug resistance. The MTT assay can directly determine biological sensitivity to drugs (23), but it has not been acceptable as a clinical assay because of the heterogeneity of leukemia cells and the requirements for a culture system and time to obtain results. Alternatively, the FCM assay has been applied for the detection of P-glycoprotein by use of a specific monoclonal antibody or the detection of the functional pump by fluorescent substrates. Because these methods can overcome the heterogeneity of leukemic cells by gating cells with leukemia cell-specific antibodies (24, 25), it could be promising if the sensitivity is improved. Although the positive rate of protein expression of the functional pump examined by FCM was relatively low in the present study, use of a sensitive secondary antibody or specific blockers may increase the sensitivity. A competitive RT-PCR assay is now widely used for the quantification of MDR1 gene expression (15, 16), but it is time-consuming and has a high risk of contamination because of the necessary post-PCR processing. In addition, a common and critical disadvantage of these methods is that they are not quantitative, so that the cutoff can not be determined.

Recently, a real-time PCR assay for leukemia has been developed as a marker for the monitoring of minimal residual disease with chimeric genes (27, 28). To overcome the disadvantage of competitive PCR, we have developed a real-time quantitative RT-PCR for MDR1 and MRP1 in this study. In this system, the MDR1 and MRP1 transcripts were detectable in 10_5 dilutions, corresponding to 10 pg of total RNA. The method is simple, rapid, and reliable for quantitative evaluation of these genes, and the dynamic quantification range may be satisfactory for clinical use. Furthermore, we confirmed a good correlation between drug sensitivity as assessed by the MTT assay and the copy number of MDR1 transcripts in leukemic cell lines, and the cutoff value clearly corresponded with the biological drug resistance.

When this quantitative system was applied to clinical samples, MDR1 expression was found to correlate with response to chemotherapy. The concentrations at relapse were obviously higher than those at onset. Furthermore, all cases who were MDR1-positive at onset did not respond to chemotherapy or relapsed after short remission periods. This relationship between MDR1 expression and clinical status was more evident in AML than in ALL. This is consistent with previous reports describing higher MDR1 expression detected in AML cases than in ALL cases (1, 2). On the other hand, MRP1 expression did not strongly correlate with clinical resistance in the present study. Taken together with the fact that most of the previous studies did not confirm the relationship between overexpression of MRP1 and clinical drug resistance in hematologic malignancies (1, 2), the detection of overexpression of MRP1 may not be helpful for the prediction of drug resistance.

In conclusion, we have developed a quantitative RT-PCR assay for MDR1 that supports the biological drug resistance assay. Because the cutoff determined in the RT-PCR assay can predict clinical drug resistance and prognosis, treatment can be modified according to the positivity of MDR1 at onset, especially in AML.

This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan. We thank Drs. K. Miyamura, O. Sasaki, M. Yamada, H. Yokoyama (Tohoku University Hospital, Sendai, Japan), T. Sugawara (Furukawa City Hospital, Furukawa, Japan), J. Kimura (Yamagata Saiseikan Hospital, Yamagata, Japan), and T. Shishido (Shishido Clinic, Sapporo, Japan) for providing clinical samples and helpful discussions. We also thank Drs. T. Tsuruo and N. Saijyo for generously providing drug-resistant cell lines.

Received January 18, 2002; accepted March 25, 2002.

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[3] Nonstandard abbreviations: MDR1, multidrug resistance-1; MRP1, multidrug resistance-related protein 1; FCM, flow cytometry; RT-PCR, reverse transcription-PCR; VCR, vincristine; PB, peripheral blood; BM, bone marrow; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; CR, complete remission; NR, nonresponder; ER, early relapse; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; [C.sub.t], threshold cycle; MTT, 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; [IC.sub.50], concentration that inhibits growth by 50%; and Rh123, rhodamine 123.

Shin-ichi Fujimaki, [1] Tadao Funato, [1] Hideo Harigae, [1] Junko Fujiwara, [1] Junichi Kameoka, [2] Kuniaki Meguro, [2] Mitsuo Kaku, [1] and Takeshi Sasaki [2] *

[1] Division of Molecular Diagnostics and

[2] Division of Rheumatology and Hematology, Department of Clinical Medicine, Tohoku University School of Medicine, Seiryoumachi 1-1, Aoba-ku, Sendai 980-8574, Japan.

* Author for correspondence. Fax 81-22-717-7168; e-mail takesa18@mail.cc.tohoku.ac.jp.
Table 1. Positivity of drug resistance and outcome of patients.

 Positive cases/total
 cases (%)

Detection methods for drug resistance CR ER NR

MTT assay ([IC.sub.50] of VCR) 0/3 1/3 2/8 (25)
FCM assay (expression of P-glycoprotein) 0/3 0/3 1/8 (13)
FCM assay (functional assay with Rh123) 0/3 1/3 2/8 (25)
Quantitative RT-PCR (MDR1 mRNA) 0/3 1/3 5/8 (63)
Quantitative RT-PCR (MRP1 mRNA) 0/3 1/3 5/8 63)
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Title Annotation:Molecular Diagnostics and Genetics
Author:Fujimaki, Shin-ichi; Funato, Tadao; Harigae, Hideo; Fujiwara, Junko; Kameoka, Junichi; Meguro, Kunia
Publication:Clinical Chemistry
Article Type:Clinical report
Date:Jun 1, 2002
Words:4586
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