MDR1 gene Polymorphisms Are Related to Efflux-Independent P-Glycoprotein Overexpression in Brazilian AML Patients

Introduction: In acute myeloid leukemia (AML) P-glycoprotein (Pgp) overexpression and activity are prognostic factors associated to refractory disease and relapse. MDR1 gene codifies to Pgp, an efflux pump that extrudes chemotherapeutic drugs commonly used in AML. Drugs dosage adjustment may depend on the MDR1 gene status. Polymorphisms in the MDR1 gene, mainly in the exons 12 and 26, can alter Pgp expression and/or activity and therefore, may determine the multidrug resistance (MDR) phenomenon.

Objective: To verify MDR1 gene variants, in exons 12 and 26, and their relationship to Pgp activity and expression in a group of AML patients.

Materials and Methods: Blastic cells from 83 (exon 12) and 80 (exon 26) Brazilian AML patients were analyzed. 4E3 monoclonal antibody was used to determine Pgp expression and Rhodamine-123/cyclosporine to determine Pgp activity. They were detected by flow cytometry. Results were expressed as the ratio of the mean fluorescence intensity (MFI). The same cut-off (< 1.1 = MDR negative; ≥1.1 = MDR positive) was used for both Pgp expression and activity analysis. MDR1 C1236T and C3435T were genotyped by PCR-RFLP technique and the fragments were detected in 12% acrylamide gel stained with silver nitrate. Patients were gathered according to the type of variant and Pgp status (expression and activity). Results were analysed in the Prisma 5.0 software by the Qui-square test, Mann-Whitney test and Kruskal-Wallis test and p value less than 0.05 was considered as statistically significant.

Results: The exon 12 variants were: C1236T = 43 samples (0.52), C1236C = 30 samples (0.36) and T1236T = 10 samples (0.12). The exon 26 variants were: C3435T = 54 samples (0.675), C3435C = 16 samples (0.20) and T3435T = 10 samples (0.125). For the exon 12, 0.62 and 0.38 represented C and T phenotypes and for the exon 26, 0.54 and 0.46 represented C and T phenotypes, respectively. The median of Pgp expression was 2.1 for the exon 12 and 2.29 for the exon 26 (range: 0.86 to 23.04) for both. The median of Pgp activity was 1.19 (range: 0.37 to 4.21) for exon 12, and 1.18 (range: 0.39 to 4.21) for exon 26. Positive Pgp expression was found in 47 out of 58 samples (81%) and 44 out of 54 samples (81.5%) for the exons 12 and 26, respectively. Positive Pgp function was found in 42 out of 71 (59%) samples and 41 out of 69 (59.4%) samples in the exons 12 and 26, respectively. The number of samples showing positive Pgp expression in both exons was statistically different (p < 0.001). The Pgp expression compared to Pgp activity for exons 12 and 26 in all variants were statistically different (p < 0.001). The number of samples showing Pgp positive expression in the exon 12 in all variants was statically different from the samples showing Pgp positive activity in the exon 26 and their variants (p=0.02). In the same way, the number of samples showing Pgp positive expression in the exon 26 in all variants was statically different from the samples showing Pgp positive activity in the exon 12 and their variants (p=0.03).

Conclusions: We observed differences among samples with Pgp expression and activity, inside both exons 12 and 26 groups in the MDR1 gene, in the studied variants. In the two exons and their variants, the number of samples positive for Pgp expressions was superior in relation to the number of samples positive for Pgp activity. Our study shows a correlation between Pgp overexpression, but not a correlation between Pgp activity, and MDR1 gene variants for exons 12 and 26. Efflux-independent Pgp expression may also contribute to clinical drug resistance by preventing cell death.