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Genomic variation at the MDR1 promoter and P-glycoprotein expression and activity in AML patients
Available online at www.sciencedirect.com
Leukemia Research 32 (2008) 976–979
Brief communication
Genomic variation at the MDR1 promoter and P-glycoprotein expression and activity in AML patients J.J. Lourenc¸o a,b , R.C. Maia c , M.A.M. Scheiner c , F.C. Vasconcelos c , M.A.M. Moreira a,∗ a
Instituto Nacional de Cˆancer, Coordena¸ca˜ o de Pesquisa, Genetics Division, Rio de Janeiro, RJ, Brazil b Universidade Federal do Rio de Janeiro, Genetics Department, Rio de Janeiro, RJ, Brazil c Instituto Nacional de Cˆ ancer, Laborat´orio de Hematologia Celular e Molecular, Hematology Service, Rio de Janeiro, RJ, Brazil Received 20 April 2007; received in revised form 25 September 2007; accepted 1 October 2007 Available online 14 November 2007
Abstract Sequence variation at the proximal MDR1 promoter of 72 patients with acute myeloid leukemia (AML) was investigated and its association with P-glycoprotein (Pgp) expression and activity using flow cytometry were analyzed. Two variants were found: −129T/C and a non-described A/T substitution at position +68 of intron 1 in one patient. Three different genotypes were identified for single nucleotide polymorphism (SNP) −129T/C: 60 patients TT, 11 individuals TC, and 1 CC. No significant association was found between SNP variants and Pgp activity and expression, at protein level. Our data also suggested that an evaluation of MDR1 promoter polymorphisms is of uncertain prognostic value. © 2007 Elsevier Ltd. All rights reserved. Keywords: Acute myeloid leukemia; P-glycoprotein; Multidrug resistance; MDR1; SNPs; Pgp expression; Pgp activity
1. Introduction In acute myeloid leukemia (AML), P-glycoprotein 1 (Pgp) over-expression and activity are prognostic factors associated to refractory disease, relapse after initial treatment, and drug dosage adjustment after relapse or refractory disease [1,2]. Approximately 35% of young patients (<60 years of age) and 71% of older patients express detectable Pgp levels in blast cells before treatment [3] while increased Pgp expression is detected in relapsed patients [4]. Pgp, encoded by the MDR1 gene, is a transmembrane protein capable of eliminating a variety of xenobiotic products, including antineoplastic and
∗ Corresponding author at: Centro de Pesquisa do Instituto Nacional de Cˆancer, Genetics Division, Rua Andr´e Cavalcanti 37, 4◦ andar, Rio de Janeiro, RJ CEP 20231-050, Brazil. Tel.: +55 21 3233 1469; fax: +55 21 3233 1423. E-mail address: [email protected] (M.A.M. Moreira).
0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2007.10.001
antiretroviral agents, by actively extruding these molecules [5]. Pgp activity and expression represent independent risk factors for treatment failure in acute myeloid leukemia [6,7]. Pgp activity depends on two parameters, its expression level, accounting for the amount of synthesized Pgp, and functionality [8]. In the last few years, attention has been focused on single nucleotide polymorphisms (SNPs), located in the MDR1 coding region, and their association with Pgp expression/function [9–11]. The proximal promoter region controls expression of most MDR1 transcripts, and several associations of SNPs variants and Pgp expression have been reported, although several of these findings are contradictory [12–16]. A correlation between the presence of different SNP variants at the proximal MDR1 promoter and placental Pgp expression was reported by Tanabe et al. by Western blotting [17], showing that −129T/C heterozygotes expressed a lower amount of Pgp with respect to −129T/T homozygotes. On the other hand, Calado et al. [18] did not find
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
this association in hematopoietic, CD34+ stem cells of normal blood donors. Additional data from Wang et al. [11] suggested that different promoter haplotypes produced different effects on the MDR1 promoter activity in different cell lines. In view of these contradictory reports and the relevance of Pgp as a prognostic factor in AML, we analyzed MDR1 promoter variants and Pgp expression/activity in a 665 bp region of the proximal promoter region, including the 5 UTR, in 72 AML patients.
2. Material and methods 2.1. Patients and leukemic cells Fresh bone marrow or peripheral blood samples from 72 unrelated patients, with different AML subtypes, registered at the Hematology Service of the Instituto Nacional de Cˆancer (INCA), Brazil, were analyzed. Patients had a mean age of 42.1 years (ranging from 6 to 86). AML diagnosis was based on clinical presentation, morphological criteria (French–American–British-FAB-classification), conventional surface marker analysis and cytogenetic analysis following WHO classification [19]. All procedures followed ethical guidelines approved by the local Ethics Committee (INCA). Bone marrow or peripheral blood samples were collected in heparinized tubes. Leukemic cells were isolated by density gradient centrifugation in Ficoll-Hipaque (Sigma), washed three times with PBS and re-suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum. Suspensions were adjusted to a concentration of 106 cells/ml. Blast cells represented more than 70% of the leukemic cells in all samples. 2.2. DNA isolation, PCR and sequencing DNA was isolated with “Genomic DNA Purification Kit” (Gentra System, Minnesota, USA) according to the instructions of the manufacturer. PCR amplifications of the proximal MDR1 promoter were carried out in final volumes of 25 l containing: 50 mM Tris pH 9, 1.5 mM MgCl2 , 40 mM KCl, 250 M of each DNTP, 50 pmol of each primer and 1 U of Taq DNA polymerase and approximately 100–200 ng of genomic DNA. Amplifications were carried out at 94 ◦ C for 5 min, followed by 35 cycles of 94 ◦ C (45 s), 61 ◦ C (35 s) and 72 ◦ C (45 s), followed by final extension at 72 ◦ C (3 min). The following primer pairs were used for the amplification of proximal promoter in two overlapping regions: 5 -TTTCCCTTAACTACGTCCTGTAG-3 /5 -TCACACTATCCACGCCTCAA-3 and 5 -CAACATGGTCCAGTGCCACTAC-3 /5 -AATGTCCCCAATGATTCAGC-3 . Amplified products were purified with GFX-PCR DNA and Gel Band Purification Kit (GE-Healthcare), according to the manufacturer’s guidelines. Purified PCR products were
977
sequenced in both directions using the DYEnamycTM ET Terminator Cycle Sequencing Premix Kit (GE-Healthcare). Reactions were run in an automatic sequencer (ABI Prism 377 DNA-Applied Biosystems). Heterozygous sites were confirmed by an additional, independent PCR reaction and sequencing of both strands. 2.3. Detection of functional Pgp status and expression, and flow cytometry analysis Pgp activity and expression were investigated in 64 and 47 patients, respectively, as described by Vasconcelos et al. [20]. For activity assays, 5 × 105 cells from each patient were re-suspended in RPMI 1640 with and without cyclosporine A (CSA; Novartis). Cells were incubated with 200 ng/ml rhodamine-123 for 45 min at 37 ◦ C, followed by one wash in cold PBS. A control cell sample, without rhodamine-123 or CSA was used to measure auto fluorescence. Results were expressed as the ratio of the mean fluorescence intensity (MFI) of cells treated with rhodamine-123 + CSA and the MFI of cells treated only with rhodamine. Ratios > 1.1 were considered positive for Pgp activity. Pgp expression was analyzed with 5 × 105 cells/patient which were washed twice in PBS/BSA, incubated with or without 10 l of 4E3 monoclonal antibody (anti-Pgp) for 30 min, washed twice in PBS/BSA and incubated with 10 l of rabbit anti-mouse (1:20) for 30 min. Subsequently, cells were suspended in PBS/BSA and analyzed by flow cytometry. Results were expressed as the ratio of the MFI of cells treated with 4E3 monoclonal antibody and the secondary antibody (labeled with FITC) divided by the MFI of cells treated only with the secondary antibody. Ratios > 1.1 were considered positive for Pgp expression. All assays were analyzed with a FACScan (Becton Dickinson, USA) using CellQuest software. Leukemic cells were gated by forward and side scatter characteristics. Acquisition was stopped when gated cells (10,000 cells) were acquired. 2.4. Statistical analysis The Chi-square (χ2 ) test was used for testing whether genotype frequencies were in Hardy-Weinberg equilibrium. The Fisher’s exact test (level of significance: p < 5%) was used to evaluate associations between patient genotype and Pgp expression and activity.
3. Results and discussion Sequence analysis of the MDR1 promoter region revealed two variable sites: a previously described SNP (−129T/C; SNP ID: rs3213619—NCBI), and a non-described substitution at position 68 of intron 1 (I-1/68A/T) in one patient that was not analyzed for Pgp expression/activity. The frequencies of the three −129 genotypes were: 83.3% TT
978
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
Table 1 Analysis of Pgp expression and activity in AML patients in respect to −129T/C polymorphism Genotype at −129
TT (n = 60)a
Expression
Activity
+ (n = 31)
+ (n = 20) − (n = 11) + (n = 3) − (n = 5) + (n = 5) − (n = 12)
− (n = 8) Not tested (n = 21) + (n = 7)
TC (n = 11)b
− (n = 1) Not tested (n = 3)
+ (n = 4) − (n = 3) + (n = 1) − (n = 0) + (n = 0) − (n = 0)
n: Number of patients; (+) positive expression or activity, (−) negative expression or activity. a Four samples were not tested for expression and activity. b Three samples were not tested for expression and activity.
Table 2 Analysis of Pgp expression and activity in AML patients accordingly to FAB subtypes Pgp activitya +
−
Pgp expressiona +
−
Subtype M0 M1 M2 M3 M4 M5 M6
3 0 5 2 6 4 0
0 1 4 4 6 5 1
3 1 7 1 7 6 0
0 0 2 1 1 0 1
Secondary AML-tb Biphenotypic Not sub-typed
2 2 1 8
1 4 0 5
2 2 1 8
1 1 0 2
33
31 64
38
9 47
Total
a Number of patients; (+) positive expression/activity; (−) negative expression/activity. b AML-t: Posttransplantation AML.
(60 patients), 15.7% T/C (11 patients), and 1.3% C/C (one patient). These genotypes were in Hardy-Weinberg equilibrium (p > 0.05) and frequencies were similar to those reported, in normal individuals, like African Americans (T/T = 91.3%; T/C = 8.7%; SNP ID: rs3213619—NCBI) or Caucasians (T/T = 96.9% and T/C = 3.1%) [16]. However, our data could be misinterpreted by variation in copy number involving the MDR1 locus and/or chromosome complex rearrangements, a common finding in AML [21]. The I-1/68A/T substitution has not been previously described, and a likely mutational event in tumor cells cannot be ruled out. The rhodamine-123 assay, carried out in 64 AML patients, showed the MDR functional phenotype (MFI-ratio > 1.1) in 33 patients (51.56%; Table 1). The MFI ratio ranged from 0.37 (in one AML M3 subtype) to 4.21 (in one AML M5 subtype). No significant statistical association was found between any genotype (−129T/T or −129T/C) and Pgp activity (p > 0.05; Fisher’s exact test). Pgp expression was analyzed in 47 of 72 AML patients and Pgp positivity was found in 38 of them (80.8%; Table 1). The antigenic density ranged widely from 1.0 to 24.0; the highest value being found in one patient with the M4-AML subtype (MFI = 24.0; −129T/T). Similarly, there was no significant association between any genotype and Pgp expression (p > 0.05; Fisher exact test). Additional data about Pgp expression and activity and AML subtypes are condensed in Table 2. Pgp expression and activity were not apparently affected by the −129T/C SNP in our AML patients, as was the case of CD34+ hematopoietic stem cells [18]. However, another study [16], working with transfected constructs, and a luciferase reporter gene assay, suggested that the C allele at position −129 was associated with a higher level of Pgp promoter activity (a 32% increase in MDR1 transcription), and that this position might be a binding site for transcription
factors. However, our analysis with SIGSCAN Version 4.05 (http://thr.cit.nih.gov/molbio/signal/) ruled out the presence of a transcription factor binding site at position −129T/C. These contradictory results pointed to a complex regulation of the MDR1 promoter activity and corroborated the findings of Wang et al. [11] who showed that promoter variants, in different cell lines, produced disparate effects on transcription, and that the effects of these polymorphisms might be restricted to cell specificity. Regardless our limited sample number, which did not give us a strong statistical power, our data suggested that an evaluation of MDR1 promoter polymorphisms (at least the 665 bp herein analyzed) is of uncertain prognostic value for disease progression and development of resistance in AML patients. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments This work was supported by SwissBridge Foundation, FAF (Fundac¸a˜ o Ary Frauzino-Brazil), CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico, Brazil) Fundac¸a˜ o Ary Frauzino, and Instituto Nacional de Cˆancer (Ministry of Health, Brazil). Contributions. Juliano Javert Lourenc¸o, Raquel C. Maia and Miguel Angelo M. Moreira wrote the manuscript. Juliano Javert Lourenc¸o contributed in sequencing of promoter regions and sequence analysis. Raquel C. Maia, Marcos A.M. Scheiner and Flavia C. Vasconcelos contributed in Flow Cytometry and Pgp expression and activity.
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
References [1] Pirker R, Wallner J, Geissler K, Linkesch W, Haas OA, Bettelheim P, et al. MDR1 gene expression and treatment outcome in acute myeloid leukemia. J Natl Cancer Inst 1991;83:708–12. [2] Wuchter C, Leonid K, Ruppert V, Schrappe M, Buchner T, Schoch C, et al. Clinical significance of P-glycoprotein expression and function for response to induction chemotherapy, relapse rate and overall survival in acute leukemia. Haematologica 2000;85:711–21. [3] Leith CP, Kopecky KJ, Chen IM, Eijdems L, Slovak ML, McConnell TS, et al. 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 1999;94:1086–99. [4] Guerci A, Merlin JL, Missoum N, Feldmann L, Marchal S, Witz F, et al. Predictive value for treatment outcome in acute myeloid leukemia of cellular Daunorubicin accumulation and P-glycoprotein expression simultaneously determined by flow cytometry. Blood 1995;85:2147–53. [5] Ambudkar SV, Kimchi DC, Sauna ZE, Gottesman MM. Pglycoprotein: from genomics to mechanism. Oncogene 2003;22:7468– 85. [6] Tsimberidou AM, Paterakis G, Androutsos G, Anagnostopoulos N, Galanopoulos A, Kalmantis T, et al. Evaluation of the clinical relevance of the expression and function of P-glycoprotein, multidrug resistance protein and lung resistance protein in patients with primary acute myelogenous leukemia. Leuk Res 2002;26:143– 54. [7] Mahadevan D, List F. Targeting the multidrug resistance-1 transporter in AML: molecular regulation a therapeutic strategies. Blood 2004;104:1940–51. [8] Hoffmeyer S, Burk O, Von Richter O, Arnold HP, Brockmo JL, Johne A, et al. Functional polymorphisms of the human multidrug resistance gene: multiple sequence variations and correlation of one allele with Pglycoprotein expression and activity in vivo. Proc Natl Acad Sci USA 2002;97:3473–8. [9] Kantharidis P, El-Osta S, Silva M, Lee G, Hu XF, Zalcberg J. Regulation of PGP gene expression: emerging concepts. Drug Resist Updat 2000;3:99–108. [10] Marzolini C, Paus E, Buclin T, Kim RB. Polymorphisms in human MDR1 (P-glycoprotein): recent advances and clinical relevance. Clin Pharmacol Ther 2004;75:13–33.
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[11] Wang B, Ngoi S, Wang J, Chong SS, Lee CGL. The promoter region of the PGP gene is largely invariant, but different single nucleotide polymorphism haplotypes affect PGP promoter activity differently in different cell lines. Mol Pharmacol 2006;70:267–76. [12] Ueda K, Clark DP, Chen CJ, Roninsonll IB, Gottesman MM, Pastan I. The human multidrug resistance (mdrl) gene. J Biol Chem 1987;262:505–8. [13] Kioka N, Yamano Y, Komano T, Ueda K. Heat-shock responsive elements in the induction of the multidrug resistance gene (MDRI). FEBS Lett 1992;301:37–40. [14] Cornwell MM, Smith DE. SP1 activates the MDRl promoter through one of two distinct G-rich regions that modulate promoter activity. J Biol Chem 1993;268:19505–11. [15] Taniguchi S, Mochida Y, Uchiumi T, Tahira T, Hayashi K, Takagi K, et al. Genetic polymorphism at the 5 regulatory region of multidrug resistance 1 (PGP) and its association with interindividual variation of expression level in the colon. Mol Cancer Ther 2003;2:1351–9. [16] Takane H, Kobayashi D, Hirota T, Kigawa J, Terakawa N, Otsubo K, et al. Haplotype-oriented genetic analysis a functional assessment of promoter variants in the PGP (ABCB1) gene. J Pharmacol Exp Ther 2004;311:1179–87. [17] Tanabe M, Ieiri I, Nagata N, Inoue K, Ito S, Kanamori Y, et al. Expression of P-glycoprotein in human placenta: relation to genetic polymorphism of the multidrug resistance (MDR)-1 gene. J Pharmacol Exp Ther 2001;297:1137–43. [18] Calado RT, Falc˜ao RP, Garcia AB, Gabellini SM, Zago MA, Franco RF. Influence of functional PGP gene polymorphisms on P-glycoprotein activity in CD34+ hematopoietic stem cells hematopoietic stem cells. Haematologica 2002;87:564–8. [19] Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, et al. The World Health Organization classification of neoplasms of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting—Airlie House 1997. Hematol J 2000;1:53–66. [20] Vasconcelos FC, Gattass CR, Rumjanek VM, Maia RC. Pomolic acid-induced apoptosis in cells from patients with chronic myeloid leukemia exhibiting different drug resistance profile. Invest New Drugs 2007;25:525–33. [21] Pedersen-Bjergaard J, Christiansen DH, Desta F, Andersen MK. Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukaemia. Leukemia 2006;20:1943–9.
Leukemia Research 32 (2008) 976–979
Brief communication
Genomic variation at the MDR1 promoter and P-glycoprotein expression and activity in AML patients J.J. Lourenc¸o a,b , R.C. Maia c , M.A.M. Scheiner c , F.C. Vasconcelos c , M.A.M. Moreira a,∗ a
Instituto Nacional de Cˆancer, Coordena¸ca˜ o de Pesquisa, Genetics Division, Rio de Janeiro, RJ, Brazil b Universidade Federal do Rio de Janeiro, Genetics Department, Rio de Janeiro, RJ, Brazil c Instituto Nacional de Cˆ ancer, Laborat´orio de Hematologia Celular e Molecular, Hematology Service, Rio de Janeiro, RJ, Brazil Received 20 April 2007; received in revised form 25 September 2007; accepted 1 October 2007 Available online 14 November 2007
Abstract Sequence variation at the proximal MDR1 promoter of 72 patients with acute myeloid leukemia (AML) was investigated and its association with P-glycoprotein (Pgp) expression and activity using flow cytometry were analyzed. Two variants were found: −129T/C and a non-described A/T substitution at position +68 of intron 1 in one patient. Three different genotypes were identified for single nucleotide polymorphism (SNP) −129T/C: 60 patients TT, 11 individuals TC, and 1 CC. No significant association was found between SNP variants and Pgp activity and expression, at protein level. Our data also suggested that an evaluation of MDR1 promoter polymorphisms is of uncertain prognostic value. © 2007 Elsevier Ltd. All rights reserved. Keywords: Acute myeloid leukemia; P-glycoprotein; Multidrug resistance; MDR1; SNPs; Pgp expression; Pgp activity
1. Introduction In acute myeloid leukemia (AML), P-glycoprotein 1 (Pgp) over-expression and activity are prognostic factors associated to refractory disease, relapse after initial treatment, and drug dosage adjustment after relapse or refractory disease [1,2]. Approximately 35% of young patients (<60 years of age) and 71% of older patients express detectable Pgp levels in blast cells before treatment [3] while increased Pgp expression is detected in relapsed patients [4]. Pgp, encoded by the MDR1 gene, is a transmembrane protein capable of eliminating a variety of xenobiotic products, including antineoplastic and
∗ Corresponding author at: Centro de Pesquisa do Instituto Nacional de Cˆancer, Genetics Division, Rua Andr´e Cavalcanti 37, 4◦ andar, Rio de Janeiro, RJ CEP 20231-050, Brazil. Tel.: +55 21 3233 1469; fax: +55 21 3233 1423. E-mail address: [email protected] (M.A.M. Moreira).
0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2007.10.001
antiretroviral agents, by actively extruding these molecules [5]. Pgp activity and expression represent independent risk factors for treatment failure in acute myeloid leukemia [6,7]. Pgp activity depends on two parameters, its expression level, accounting for the amount of synthesized Pgp, and functionality [8]. In the last few years, attention has been focused on single nucleotide polymorphisms (SNPs), located in the MDR1 coding region, and their association with Pgp expression/function [9–11]. The proximal promoter region controls expression of most MDR1 transcripts, and several associations of SNPs variants and Pgp expression have been reported, although several of these findings are contradictory [12–16]. A correlation between the presence of different SNP variants at the proximal MDR1 promoter and placental Pgp expression was reported by Tanabe et al. by Western blotting [17], showing that −129T/C heterozygotes expressed a lower amount of Pgp with respect to −129T/T homozygotes. On the other hand, Calado et al. [18] did not find
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
this association in hematopoietic, CD34+ stem cells of normal blood donors. Additional data from Wang et al. [11] suggested that different promoter haplotypes produced different effects on the MDR1 promoter activity in different cell lines. In view of these contradictory reports and the relevance of Pgp as a prognostic factor in AML, we analyzed MDR1 promoter variants and Pgp expression/activity in a 665 bp region of the proximal promoter region, including the 5 UTR, in 72 AML patients.
2. Material and methods 2.1. Patients and leukemic cells Fresh bone marrow or peripheral blood samples from 72 unrelated patients, with different AML subtypes, registered at the Hematology Service of the Instituto Nacional de Cˆancer (INCA), Brazil, were analyzed. Patients had a mean age of 42.1 years (ranging from 6 to 86). AML diagnosis was based on clinical presentation, morphological criteria (French–American–British-FAB-classification), conventional surface marker analysis and cytogenetic analysis following WHO classification [19]. All procedures followed ethical guidelines approved by the local Ethics Committee (INCA). Bone marrow or peripheral blood samples were collected in heparinized tubes. Leukemic cells were isolated by density gradient centrifugation in Ficoll-Hipaque (Sigma), washed three times with PBS and re-suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum. Suspensions were adjusted to a concentration of 106 cells/ml. Blast cells represented more than 70% of the leukemic cells in all samples. 2.2. DNA isolation, PCR and sequencing DNA was isolated with “Genomic DNA Purification Kit” (Gentra System, Minnesota, USA) according to the instructions of the manufacturer. PCR amplifications of the proximal MDR1 promoter were carried out in final volumes of 25 l containing: 50 mM Tris pH 9, 1.5 mM MgCl2 , 40 mM KCl, 250 M of each DNTP, 50 pmol of each primer and 1 U of Taq DNA polymerase and approximately 100–200 ng of genomic DNA. Amplifications were carried out at 94 ◦ C for 5 min, followed by 35 cycles of 94 ◦ C (45 s), 61 ◦ C (35 s) and 72 ◦ C (45 s), followed by final extension at 72 ◦ C (3 min). The following primer pairs were used for the amplification of proximal promoter in two overlapping regions: 5 -TTTCCCTTAACTACGTCCTGTAG-3 /5 -TCACACTATCCACGCCTCAA-3 and 5 -CAACATGGTCCAGTGCCACTAC-3 /5 -AATGTCCCCAATGATTCAGC-3 . Amplified products were purified with GFX-PCR DNA and Gel Band Purification Kit (GE-Healthcare), according to the manufacturer’s guidelines. Purified PCR products were
977
sequenced in both directions using the DYEnamycTM ET Terminator Cycle Sequencing Premix Kit (GE-Healthcare). Reactions were run in an automatic sequencer (ABI Prism 377 DNA-Applied Biosystems). Heterozygous sites were confirmed by an additional, independent PCR reaction and sequencing of both strands. 2.3. Detection of functional Pgp status and expression, and flow cytometry analysis Pgp activity and expression were investigated in 64 and 47 patients, respectively, as described by Vasconcelos et al. [20]. For activity assays, 5 × 105 cells from each patient were re-suspended in RPMI 1640 with and without cyclosporine A (CSA; Novartis). Cells were incubated with 200 ng/ml rhodamine-123 for 45 min at 37 ◦ C, followed by one wash in cold PBS. A control cell sample, without rhodamine-123 or CSA was used to measure auto fluorescence. Results were expressed as the ratio of the mean fluorescence intensity (MFI) of cells treated with rhodamine-123 + CSA and the MFI of cells treated only with rhodamine. Ratios > 1.1 were considered positive for Pgp activity. Pgp expression was analyzed with 5 × 105 cells/patient which were washed twice in PBS/BSA, incubated with or without 10 l of 4E3 monoclonal antibody (anti-Pgp) for 30 min, washed twice in PBS/BSA and incubated with 10 l of rabbit anti-mouse (1:20) for 30 min. Subsequently, cells were suspended in PBS/BSA and analyzed by flow cytometry. Results were expressed as the ratio of the MFI of cells treated with 4E3 monoclonal antibody and the secondary antibody (labeled with FITC) divided by the MFI of cells treated only with the secondary antibody. Ratios > 1.1 were considered positive for Pgp expression. All assays were analyzed with a FACScan (Becton Dickinson, USA) using CellQuest software. Leukemic cells were gated by forward and side scatter characteristics. Acquisition was stopped when gated cells (10,000 cells) were acquired. 2.4. Statistical analysis The Chi-square (χ2 ) test was used for testing whether genotype frequencies were in Hardy-Weinberg equilibrium. The Fisher’s exact test (level of significance: p < 5%) was used to evaluate associations between patient genotype and Pgp expression and activity.
3. Results and discussion Sequence analysis of the MDR1 promoter region revealed two variable sites: a previously described SNP (−129T/C; SNP ID: rs3213619—NCBI), and a non-described substitution at position 68 of intron 1 (I-1/68A/T) in one patient that was not analyzed for Pgp expression/activity. The frequencies of the three −129 genotypes were: 83.3% TT
978
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
Table 1 Analysis of Pgp expression and activity in AML patients in respect to −129T/C polymorphism Genotype at −129
TT (n = 60)a
Expression
Activity
+ (n = 31)
+ (n = 20) − (n = 11) + (n = 3) − (n = 5) + (n = 5) − (n = 12)
− (n = 8) Not tested (n = 21) + (n = 7)
TC (n = 11)b
− (n = 1) Not tested (n = 3)
+ (n = 4) − (n = 3) + (n = 1) − (n = 0) + (n = 0) − (n = 0)
n: Number of patients; (+) positive expression or activity, (−) negative expression or activity. a Four samples were not tested for expression and activity. b Three samples were not tested for expression and activity.
Table 2 Analysis of Pgp expression and activity in AML patients accordingly to FAB subtypes Pgp activitya +
−
Pgp expressiona +
−
Subtype M0 M1 M2 M3 M4 M5 M6
3 0 5 2 6 4 0
0 1 4 4 6 5 1
3 1 7 1 7 6 0
0 0 2 1 1 0 1
Secondary AML-tb Biphenotypic Not sub-typed
2 2 1 8
1 4 0 5
2 2 1 8
1 1 0 2
33
31 64
38
9 47
Total
a Number of patients; (+) positive expression/activity; (−) negative expression/activity. b AML-t: Posttransplantation AML.
(60 patients), 15.7% T/C (11 patients), and 1.3% C/C (one patient). These genotypes were in Hardy-Weinberg equilibrium (p > 0.05) and frequencies were similar to those reported, in normal individuals, like African Americans (T/T = 91.3%; T/C = 8.7%; SNP ID: rs3213619—NCBI) or Caucasians (T/T = 96.9% and T/C = 3.1%) [16]. However, our data could be misinterpreted by variation in copy number involving the MDR1 locus and/or chromosome complex rearrangements, a common finding in AML [21]. The I-1/68A/T substitution has not been previously described, and a likely mutational event in tumor cells cannot be ruled out. The rhodamine-123 assay, carried out in 64 AML patients, showed the MDR functional phenotype (MFI-ratio > 1.1) in 33 patients (51.56%; Table 1). The MFI ratio ranged from 0.37 (in one AML M3 subtype) to 4.21 (in one AML M5 subtype). No significant statistical association was found between any genotype (−129T/T or −129T/C) and Pgp activity (p > 0.05; Fisher’s exact test). Pgp expression was analyzed in 47 of 72 AML patients and Pgp positivity was found in 38 of them (80.8%; Table 1). The antigenic density ranged widely from 1.0 to 24.0; the highest value being found in one patient with the M4-AML subtype (MFI = 24.0; −129T/T). Similarly, there was no significant association between any genotype and Pgp expression (p > 0.05; Fisher exact test). Additional data about Pgp expression and activity and AML subtypes are condensed in Table 2. Pgp expression and activity were not apparently affected by the −129T/C SNP in our AML patients, as was the case of CD34+ hematopoietic stem cells [18]. However, another study [16], working with transfected constructs, and a luciferase reporter gene assay, suggested that the C allele at position −129 was associated with a higher level of Pgp promoter activity (a 32% increase in MDR1 transcription), and that this position might be a binding site for transcription
factors. However, our analysis with SIGSCAN Version 4.05 (http://thr.cit.nih.gov/molbio/signal/) ruled out the presence of a transcription factor binding site at position −129T/C. These contradictory results pointed to a complex regulation of the MDR1 promoter activity and corroborated the findings of Wang et al. [11] who showed that promoter variants, in different cell lines, produced disparate effects on transcription, and that the effects of these polymorphisms might be restricted to cell specificity. Regardless our limited sample number, which did not give us a strong statistical power, our data suggested that an evaluation of MDR1 promoter polymorphisms (at least the 665 bp herein analyzed) is of uncertain prognostic value for disease progression and development of resistance in AML patients. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments This work was supported by SwissBridge Foundation, FAF (Fundac¸a˜ o Ary Frauzino-Brazil), CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico, Brazil) Fundac¸a˜ o Ary Frauzino, and Instituto Nacional de Cˆancer (Ministry of Health, Brazil). Contributions. Juliano Javert Lourenc¸o, Raquel C. Maia and Miguel Angelo M. Moreira wrote the manuscript. Juliano Javert Lourenc¸o contributed in sequencing of promoter regions and sequence analysis. Raquel C. Maia, Marcos A.M. Scheiner and Flavia C. Vasconcelos contributed in Flow Cytometry and Pgp expression and activity.
J.J. Louren¸co et al. / Leukemia Research 32 (2008) 976–979
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