The translational expression of ABCA2 and ABCA3 is a strong prognostic biomarker for multidrug resistance in pediatric acute lymphoblastic leukemia

Abstract

Purpose: The aim of this work was to study the correlation between the expressions of the ABCA2 and ABCA3 genes at the mRNA and protein levels in children with acute lymphoblastic leukemia (ALL) and the effects of this association on multidrug resistance (MDR).

Materials and methods: Sixty-nine children with de novo ALL and 25 controls were enrolled in the study. Mononuclear cells were isolated from the bone marrow. The mRNA levels of ABCA2 and ABCA3 were measured by real-time polymerase chain reaction (PCR). Samples with high mRNA levels were assessed for respective protein levels by Western blotting. Following the first year of treatment, persistent monoclonality of T-cell gamma receptors or immunoglobulin H (IgH) gene rearrangement was assessed and considered as the MDR. The tertiary structure of ABCA2 was predicted using Phyre2 and I-TASSER web systems and compared to that of ABCA3, which has been previously reported. Molecular docking was performed using DOCK 6.7.

Results: Real-time quantitative PCR (qRT-PCR) showed high levels of ABCA2 and ABCA3 mRNAs in 13 and 17 samples, respectively. Among them, five and eight individuals demonstrated high levels of ABCA2 and ABCA3, respectively. Response to chemotherapy was significantly decreased (P=0.001) when the mRNA and protein of both genes were overexpressed compared to individuals with high transcriptional levels of either ABCA2 or ABCA3 alone. Close similarity between ABCA2 and ABCA3 structures was revealed by protein tertiary structure prediction, whereas molecular docking analysis suggested similar binding of chemotherapy drugs and therefore a potentially similar role in determining the MDR.

Conclusion: Our findings suggested, for the first time, that quantification of the protein level of ABCA2 and ABCA3 transporters had a prognostic impact on pediatric ALL MDR. Furthermore, the tertiary structure of ABCA2 was predicted for the first time, and docking analysis revealed a possible compensatory effect between ABCA2 and ABCA3 transporters, which may contribute to the efflux of cytotoxic drugs and, ultimately, to chemoresistance.

Keywords: ABCA2 transporter; ABCA3 transporter; childhood acute lymphoblastic leukemia; molecular docking; multidrug resistance; tertiary structure.

Figure 1

Results obtained by RT-PCR amplification of ABCA2 and ABCA3 in a representative selection of ALL patients. Notes: Real-time PCR was performed in triplicate for each sample. To confirm the purity and correct size of the qRT-PCR products, the target and the housekeeping GAPDH gene, random amplicons were loaded on an agarose gel. The gel was stained with ethidium bromide and photographed. Abbreviations: ABC, ATP-binding cassette; ALL, acute lymphoblastic leukemia; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; M, molecular weight marker; NTC, nontemplate control; PCR, polymerase chain reaction; qRT-PCR; quantitative reverse transcriptase PCR.

Figure 2

ABCA2 and ABCA3 protein expression of samples with a high ABCA2/ABCA3 mRNA expression. Notes: Mononuclear cell lysis and extraction of total protein was performed on blood samples of de novo ALL patients with high expression of ABCA2 and ABCA3 at mRNA levels compared to controls. Accordingly, 21.5 μg of the extracted protein was loaded into the wells and Western blotting was performed following SDS-PAGE using goat anti-human ABCA2 and ABCA3 polyclonal antibodies, respectively. PVDF membranes were treated with related secondary antibody and exposed to ECL solution. Out of 12 MRD⁺ patients, seven showed visible bands corresponding to high expression levels of ABCA3 protein with an MWt of 191 kDa, and five showed visible bands corresponding to high expression levels of ABCA2 protein with an MWt of 270 kDa. One MRD patient (patient number 11) showed high expression levels of ABCA3. Each lane corresponds to an individual ABCA2 or ABCA3 mRNA⁺ patient and is labeled by numbers. Probing the blots with mouse anti-human GAPDH monoclonal antibody showed identical amounts of loaded proteins. P indicates a pool of 12 control samples. Abbreviations: ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MRD, minimal residual disease; MWt, molecular weight; PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3

Tertiary structures of ABCA2 and ABCA3. Notes: (A) Comparative structural analysis of ABCA2 (purple) and ABCA3 (yellow) models predicted by Phyre2 showed close similarity. (B) ABCA3 model obtained by I-TASSER ab initio method (orange) and the water molecules (cyan) within the active site. (C) ABCA2 active site used for grid calculations and docking analysis. ABCA2 model (yellow), ABCA2 molecular surface (green), water molecules (cyan) and ABCA2 active site (gray box).

Figure 4

Molecular docking analyses of ABCA2/A3 models shown in zoomed out (A) and zoomed in (B) images. Notes: Doxorubicin is depicted as a representative ALL antineoplastic drug and colored arbitrarily according to the heteroatom. ABCA2 (purple); ABCA3 (yellow). Abbreviation: ALL, acute lymphoblastic leukemia.