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Response to Chemotherapy and Expression of the Genes Encoding the Multidrug Resistance-associated Proteins MRP2, MRP3, MRP4, MRP5, and SMRP in Childhood Acute Myeloid Leukemia

University Children’s Hospital, 07740 Jena, Germany

	ABSTRACT

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Purpose: The family of multidrug resistance-associated proteins (MRPs) belongs to the ATP-binding cassette superfamily of transporters, which have the ability to function as outward pumps for chemotherapeutic drugs. Their structure, function, and substrate specificity have been studied intensively, but little is known about their clinical relevance in malignant diseases.

Experimental Design: In this study, the expression of the MRP2, MRP3, MRP4, MRP5, and SMRP genes was measured using TaqMan real-time PCR in 53 children with de novo acute myeloid leukemia. Nine patients were also analyzed in relapse.

Results: MRP3 gene expression was higher in patients who did not achieve remission (P = 0.023). Expression of MRP2 (P = 0.09) or MRP3 (P = 0.041) was associated with a lower rate of survival, and patients who expressed high levels of both genes had a particularly poor prognosis (P < 0.01). No significant association was found for overall survival or remission rate and the expression of MRP4, MRP5, and SMRP.

Conclusions: This study provides first data on the clinical relevance of five MRPs in acute myeloid leukemia patients. The results strongly suggest that MRP3 and possibly also MRP2 are involved in drug resistance in this disease. Those two proteins therefore represent interesting markers for risk-adapted therapy and possible targets for the development of specific drugs to overcome multidrug resistance.

	INTRODUCTION

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A major issue in the treatment of AML² is resistance to chemotherapeutic drugs. Many patients fail to respond to chemotherapy, and others relapse with resistant disease. Even with aggressive therapy, the survival rate in children with AML is only 50% (1 , 2) .

Several mechanisms of drug resistance have been identified. One of these is the overexpression of ATP-dependent membrane proteins that function as drug efflux pumps. The best characterized drug efflux pump is the P-gp, which is encoded by the MDR1. The expression of P-gp/MDR1 has been identified as an independent adverse prognostic factor for complete remission and survival in adult patients with AML (3 , 4) . Recent studies suggested that the results in the treatment of AML can be improved by combining chemotherapy with drugs that inhibit the function of P-gp (5 , 6) . However, the clinical relevance of P-gp seems to be much smaller in childhood than in adult AML (7 , 8) , and some adult patients too show multidrug resistance in the absence of P-gp expression.

The family of MRPs also belongs to the ATP-binding cassette superfamily of transporters (9) . Their structure, function, and substrate specificity have been studied intensively (10 , 11) , but little is known about their clinical relevance. Thus far, only MRP activity and the expression of MRP1 have been studied in larger groups of AML patients. It was found that MRP activity (12 , 13) , but not MRP1 expression (14, 15, 16) , is associated with a poor response to chemotherapy. Therefore, it seems possible that other members of the MRP family attribute to MRP activity and cause drug resistance in AML.

Van der Kolk et al. (17) studied the expression of MRP1, MRP2, MRP3, and MRP5 in 30 paired samples of de novo and relapsed AML in adult patients. They found that none of the genes was consistently elevated at relapse.

The aims of our study were to find out whether the more recently discovered members of the MRP family (MRP2, MRP3, MRP4, MRP5, and SMRP) are expressed in childhood AML and whether they are associated with a poor response to chemotherapy.

	PATIENTS AND METHODS

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Patients and Therapy.
All 53 patients were diagnosed with AML untreated previously. The main patient characteristics are summarized in Table 1 . The patients were treated according to four multicenter studies in Germany: (a) AML-I/82 (13 patients; Ref. 18 ); (b) AML-II/87 (17 patients; Ref. 18 ); (c) AML-BFM-93 (11 patients; Ref. 1 ); and (d) AML-BFM-98 (12 patients). All studies included induction therapies with cytosine-arabinoside, anthracycline, and thioguanine or etoposide, followed by bone marrow transplantation or intensive consolidation protocols with prophylactic central nervous system irradiation and maintenance therapy. Most (68%) of the patients (n = 36) achieved remission, and the overall survival was 38% after 5 years.

All samples were cryopreserved in liquid nitrogen. Total RNA was isolated using RNeasy Mini Kit, including DNase digestion (Qiagen, Hilden, Germany). The amount of RNA was measured by photometry, and a stock solution of 1 µg of RNA in 40 µl was prepared. RNA was transcribed into cDNA using Omniscript (Qiagen).

Quantitative Real-time PCR.
Quantitative PCR was performed using the ABI Prism 7700 Sequence Detector (Applied Biosystems, Weiterstadt, Germany). Primers and TaqMan probes for MRP2, MRP5, and SMRP were used as described previously (19) . Primers and TaqMan probes for MRP3 and MRP4 were:

MRP3: Forward 5'-GCACCATTGTCGTGGCTACA-3'; Reverse 5'-GCAGGACACCCAG GACCAT-3'; TaqMan probe 5'-CATCCTCTCCCACCTGTCCAAGCTCA-3'

MRP4: Forward 5'-TGGATTCTGTGGCTTTGAACAC-3'; Reverse 5'-AGCCAAAATGAG CGTGCAA-3'; TaqMan probe 5'-CGTACGCCTATGCCACGGTGCTG-3'

The final concentration of the primers was 900 nM (300 nM for MRP3 and SMRP); the final concentration of the TaqMan probes was 300 nM (200 nM for MRP3 and SMRP). All TaqMan probes were labeled with FAM and TAMRA.

The expression of the resistance genes was standardized for the expression of ß-2-microglobulin, which was measured using Pre-Developed Assay Reagents (Applied Biosystems).

The final volume for each PCR was 30 µl, including 1.5 µl of the investigated sample. Universal PCR Master Mix (Applied Biosystems) was used according to the manufacturer’s instructions.

Serial dilutions of cDNA of reference cell lines were used to generate standard curves. The reference cell lines were: MCF7/CH1000 (MRP2 and MRP3) and K562 (MRP4, MRP5, and SMRP). The expression of each gene in each sample was analyzed in duplicate. The regression coefficients of the standard curves ranged between 0.995 and 0.998.

Statistical Methods.
Kaplan-Mayer statistics and Log-rank tests were calculated to estimate the significance of differences between survival curves. To prevent a bias because of the different outcomes in the four studies, all tests were recalculated with the number of the study as a stratification variable. For all genes, we used the median as cutoff between high and low expression. The Mann-Whitney test, the Kruskal-Wallis test, and the Spearman’s correlation coefficient were used to investigate the association between gene expression and other findings. All Ps are given for two-sided tests.

	RESULTS

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Expression of MRPs in Childhood AML.
Measurable amounts of MRP2, MRP4, MRP5, and SMRP were found in all patients. MRP3 was not detectable in 8 patients. The variation from the 10^th percentile to the 90^th percentile was 5-fold for MRP2, 8-fold for MRP4, 10-fold for MRP5, and 13-fold for SMRP. The variation from the lowest measurable value for MRP3 to the 90^th percentile was 27-fold.

The strongest correlation was found between the expression of MRP5 and SMRP, which is a splicing variant of MRP5 and encoded by the same gene (Ref. 19 ; Spearman’s correlation coefficient = 0.87, P < 0.001). A significant correlation was also found between all other members of the MRP family but with smaller Spearman’s correlation coefficients (range from 0.27 to 0.53). None of the MRP genes was associated with the expression of the Breast Cancer Resistance Protein or MDR1, which were analyzed previously in the same group of patients (6 , 20) . All genes were investigated for their association with sex, age, FAB type, initial WBC, presence of Auer rods, and the chromosomal aberrations t(8;21), t(9;11), and inv (16) . Higher levels for MRP5 and SMRP were found in patients with high initial WBC (Spearman’s correlation coefficients: 0.34 and 0.29; Ps: 0.015 and 0.041). The expression of MRP3 was higher in patients with the FAB types M4 and M5 (Kruskal-Wallis test: P = 0.002). All other correlations were not significant.

MRP Expression and Remission.
The median expression of MRP3 was more than two times higher in patients who did not achieve remission (P = 0.023; Fig. 1 ). This finding was consistent throughout all four studies. MRP2, MRP4, MRP5, and SMRP were not significantly associated with a failure to achieve remission.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Box plots (10th, 25th, 50th, 75th, and 90th percentiles) of MRP3 gene expression in patients who achieved remission and in patients who failed to achieve remission. Mann-Whitney test: P = 0.023.

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Overall survival and expression of MRP2 and MRP3. The median was used as a cutoff for high and low expression. N, number of patients; OS, overall survival.

MRP Expression at the Time of Diagnosis and at Relapse.
In 9 patients, the expression of the five MRP genes was measured at the time of diagnosis, as well as in first relapse. The expression of all five genes was more often and to a higher degree elevated than reduced at the time of relapse. However, this trend was not statistically significant.

	DISCUSSION

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To our knowledge, this study provides first data on the association between the response to chemotherapy and the expression of MRP2, MRP3, MRP4, MRP5, and SMRP in AML patients. Our results strongly suggest that MRP3 is involved in drug resistance in this disease. It therefore represents an interesting marker for risk-adapted therapy and a possible target for the development of specific drugs to overcome multidrug resistance. Sirotnak et al. (21) found that the function of MRPs can be inhibited by probenecid and that the efficacy of chemotherapeutics in mice could be enhanced by the coadministration of probenecid.

Additionally in our study, MRP2 was associated with a poor prognosis, but the results were less convincing. It was not associated with a poor remission rate, and the impact on relapse-free survival was much smaller than the impact on overall survival. Patients who expressed high levels of both genes, MRP2 and MRP3, had a particularly poor prognosis.

Studies, including specific functional assays and the analysis of protein expression, are necessary to confirm our results. In a study on lung cancer cell lines, Young et al. (22) could demonstrate a good correlation between mRNA and protein levels for MRP2 and MRP3.

Our results do not indicate a clinical relevance of MRP4, MRP5, or SMRP in childhood AML. Either the expression of these genes in the leukemic cells is too small to cause a significant efflux of chemotherapeutic drugs or our group of patients was too small and heterogeneous to detect their clinical relevance. Another possible explanation is that the level of mRNA of these genes does not strongly correlate with the amount of functional protein in the cell membrane.

The expression levels of MRP5 and SMRP were closely correlated (Spearman’s correlation coefficient = 0.87, P < 0.001). This correlation does not support the hypothesis that the grades of transcription of these two splicing variants are regulated differently (19) .

	ACKNOWLEDGMENTS

 
We thank Dr. Douglas D. Ross from the University of Maryland Greenebaum Cancer Center for providing the cell line MCF7/CH1000, which was used to generate standard curves for MRP2 and MRP3.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at Klinikum der FSU Jena, Klinik für Kinder-und Jugendmedizin, Postfach, 07740 Jena, Germany. Phone: 0049 (0) 3641 938253; Fax: 0049 (0) 3641 938306; E-mail: daniel{at}steinba.ch

² The abbreviations used are: AML, acute myeloid leukemia; P-gp, Permeability glycoprotein; FAB, ; MRP, multidrug resistance-associated protein; MDR1, multidrug resistance gene 1.

Received 6/12/02; revised 10/ 7/02; accepted 10/21/02.

	REFERENCES

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1. Creutzig U., Ritter J., Zimmermann M., Hermann J., Gadner H., Sawatzki D. B., Niemeyer C. M., Schwabe D., Selle B., Boos J., Kaehl J., Feldges A. Idarubicin improves blast cell clearance during induction therapy in children with AML: results of study AML-BFM 93. AML-BFM Study Group. Leukemia, 15: 348-354, 2001.[CrossRef][Medline]
2. Stevens R. F., Hann I. M., Wheatley K., Gray R. G. Marked improvements in outcome with chemotherapy alone in paediatric acute myeloid leukemia: results of the United Kingdom Medical Research Council’s 10th AML trial. MRC Childhood Leukaemia Working Party. Br. J. Haematol., 101: 130-140, 1998.[CrossRef][Medline]
3. Wood P., Burgess R., MacGregor A., Yin J. A. P-glycoprotein expression on acute myeloid leukaemia blast cells at diagnosis predicts response to chemotherapy and survival. Br. J. Haematol., 87: 509-514, 1994.[Medline]
4. Pirker R., Wallner J., Geissler K., Linkesch W., Haas O. A., Bettelheim P., Hopfner M., Scherrer R., Valent P., Havelec L., Ludwig H., Lechner K. MDR1 gene expression and treatment outcome in acute myeloid leukemia. J. Natl. Cancer Inst. (Bethesda), 83: 708-712, 1991.[Abstract/Free Full Text]
5. List A. F., Kopecky K. J., Willman C. L., Head D. R., Persons D. L., Slovak M. L., Dorr R., Karanes C., Hynes H. E., Doroshow J. H., Shurafa M., Appelbaum F. R. Benefit of cyclosporine modulation of drug resistance in patients with poor-risk acute myeloid leukemia: a Southwest Oncology Group study. Blood, 98: 3212-3220, 2001.[Abstract/Free Full Text]
6. Tidefelt U., Liliemark J., Gruber A., Liliemark E., Sundman-Engberg B., Juliusson G., Stenke L., Elmhorn-Rosenborg A., Mollgard L., Lehman S., Xu D., Covelli A., Gustavsson B., Paul C. P-Glycoprotein inhibitor valspodar (PSC 833) increases the intracellular concentrations of daunorubicin in vivo in patients with P-glycoprotein-positive acute myeloid leukemia. J. Clin. Oncol., 18: 1837-1844, 2000.[Abstract/Free Full Text]
7. Sievers E. L., Smith F. O., Woods W. G., Lee J. W., Bleyer W. A., Willman C. L., Bernstein I. D. Cell surface expression of the multidrug resistance P-glycoprotein (P-170) as detected by monoclonal antibody MRK-16 in pediatric acute myeloid leukemia fails to define a poor prognostic group: a report from the Children’s Cancer Group. Leukemia (Baltimore), 9: 2042-2048, 1995.[Medline]
8. Steinbach D., Furchtbar S., Sell W., Hermann J., Zintl F., Sauerbrey A. Contrary to adult patients, expression of the multidrug resistance gene (MDR1) fails to define a poor prognostic group in childhood AML. Leukemia, 17: 470-471, 2003.[CrossRef][Medline]
9. Marie J. P. Drug resistance in hematologic malignancies. Curr. Opin. Oncol., 13: 463-469, 2001.[CrossRef][Medline]
10. Borst P., Evers R., Kool M., Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. J. Natl. Cancer Inst. (Bethesda), 92: 1295-1302, 2000.[Abstract/Free Full Text]
11. Kruh G. D., Zeng H., Rea P. A., Liu G., Chen Z. S., Lee K., Belinsky M. G. MRP subfamily transporters and resistance to anticancer agents. J. Bioenerg. Biomembr., 33: 493-501, 2001.[CrossRef][Medline]
12. Laupeze B., Amiot L., Drenou B., Bernard M., Branger B., Grosset J. M., Lamy T., Fauchet R., Fardel O. High multidrug resistance protein activity in acute myeloid leukaemias is associated with poor response to chemotherapy and reduced patient survival. Br. J. Haematol., 116: 834-838, 2002.[CrossRef][Medline]
13. Legrand O., Simonin G., Perrot J. Y., Zittoun R., Marie J. P. Pgp and MRP activities using calcein-AM are prognostic factors in adult acute myeloid leukemia patients. Blood, 91: 4480-4488, 1998.[Abstract/Free Full Text]
14. Leith C. P., Kopecky K. J., Chen I. M., Eijdems L., Slovak M. L., McConnell T. S., Head D. R., Weick J., Grever M. R., Appelbaum F. R., Willman C. L. Frequency and clinical significance of the expression of the multidrug resistance proteins MDR1/P-glycoprotein. MRP1, and LRP in acute myeloid leukemia: a Southwest Oncology Group Study. Blood, 94: 1086-1099, 1999.[Abstract/Free Full Text]
15. van der Kolk D. M., de Vries E. G., van Putten W. J., Verdonck L. F., Ossenkoppele G. J., Verhoef G. E., Vellenga E. P-glycoprotein and multidrug resistance protein activities in relation to treatment outcome in acute myeloid leukemia. Clin. Cancer Res., 6: 3205-3214, 2000.[Abstract/Free Full Text]
16. Filipits M., Suchomel R. W., Zochbauer S., Brunner R., Lechner K., Pirker R. Multidrug resistance-associated protein in acute myeloid leukemia: no impact on treatment outcome. Clin. Cancer Res., 3: 1419-1425, 1997.[Abstract]
17. van der Kolk D. M., de Vries E. G., Noordhoek L., van den Berg E., van der Pol M. A., Muller M., Vellenga E. Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia. Leukemia, 15: 1544-1553, 2001.[CrossRef][Medline]
18. Steinbach D., Dorffel W., Eggers G., Holfeld E., Kluba U., Krause I., Lauterbach I., Reiss T., Rieske K., Scharfe V., Schumacher R., Weigel H., Weinmann G., Zintl F., Hermann J. Improved results in the treatment of acute myeloid leukemia: results of study AML-BFM-93 in East Germany with comparisons to the preceding studies AML-I-82 and AML-II-87. Klin. Padiatr., 213: 162-168, 2001.[CrossRef][Medline]
19. Yoshida M., Suzuki T., Komiya T., Hatashita E., Nishio K., Kazuhiko N., Fukuoka M. 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, 2001.[CrossRef][Medline]
20. Steinbach D., Sell W., Voigt A., Hermann J., Zintl F., Sauerbrey A. BCRP gene expression is associated with a poor response to remission induction therapy in childhood acute myeloid leukemia. Leukemia (Baltimore), 16: 1443-1447, 2002.[CrossRef][Medline]
21. Sirotnak F. M., Wendel H. G., Bornmann W. G., Tong W. P., Miller V. A., Scher H. I., Kris M. G. Co-administration of probenecid, an inhibitor of a cMOAT/MRP-like plasma membrane ATPase, greatly enhanced the efficacy of a new 10-deazaaminopterin against human solid tumors in vivo. Clin. Cancer Res., 6: 3705-3712, 2000.[Abstract/Free Full Text]
22. Young L. C., Campling B. G., Cole S. P., Deeley R. G., Gerlach J. H. Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin. Cancer Res., 7: 1798-1804, 2001.[Abstract/Free Full Text]

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