Methods

The study included all children who were diagnosed to be at risk for leukemia in the Seyed-ol-Shohada Hospital, Isfahan, during the period from 2012 to 2016. Philadelphia-positive leukemia, infantile leukemia, mixed lineage leukemia and Burkitt-type leukemia were excluded from the research because of the nonsimilar type of treatment protocol applied. This research was permitted and approved by the ethics committee of Isfahan University of Medical Sciences (protocol number 192018). Informed written consents were obtained from the children’s parents prior to participation in the study. Following diagnosis, 1–2 mL of heparinized bone marrow was sent to the Cellular and Molecular Biology Laboratory of University of Isfahan on ice for further studies. Bone marrow samples from children without evidence of any cancer (including leukemia) were selected as controls. Extraction of mononuclear cells was performed using a protocol based on the Lymphoprep™ Kit. The total RNA was extracted using the RNX-Plus kit and 2 μg of total RNA was used for cDNA synthesis by a cDNA synthesis kit, in accordance with the specific temperature protocol (sequentially, 5 minutes at 25°C, 60 minutes at 42°C, and 5 minutes at 70°C). The cDNA was then stored at −20°C for further studies. Patients were treated based on the Australian and New Zealand Children’s Cancer Study Group’s ALL Study 8 protocol (http://www.anzctr.org.au/trial_view.aspx?ID=1568). Bone marrow samples were evaluated for monoclonality (IgH or T-cell gamma receptor gene rearrangement) at diagnosis and again at the end of the first year of treatment using PCR analysis. Persistent monoclonality of T-cell gamma receptors or IgH gene rearrangement after 1 year of treatment, or observation of any sign of leukemia relapse before this time, was considered as MDR. Twenty-five children, with no evidence of malignancy in subsequent studies, were used as controls.

The housekeeping gene GAPDH was used as an endogenous control to study the relative expression of ABCA2 and ABCA3 mRNAs. The sequences of the primers used in this study are reported in Table 1. Real-time PCR was performed using the real-time PCR kit (Applied Biosystems Step One™ Real-Time PCR system). Briefly, 1 μL of 0.01 μg/mL of the synthesized cDNA, 0.4 μL of 100 nM forward and reverse primers, 5 μL of SYBR green Master Mix and 3.2 μL of ddH₂O were added to the real-time tubes. Then, qRT-PCR was performed in triplicate according to the protocol that included a preincubation at 95°C (5 minutes), denaturation at 95°C (20 seconds), annealing at 60°C (30 seconds) and product expansion at 72°C (25 seconds). Relative expression levels were calculated using the 2^−ΔΔCt method.

Primer sequences of ABCA2, ABCA3 and GAPDH

Abbreviations: F, forward; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; R, reverse; Tm, melting temperature.

After assessing the expression level of ABCA2 and ABCA3 mRNAs of the patients’ and controls’ samples, cells belonging to patients with high levels of one or both mRNAs were chosen for Western blotting analysis. Twelve randomly selected control samples were pooled and used as a negative control sample for the aforementioned assay. Cell lysis was performed using RIPA lysis buffer and protease inhibitor cocktail after freezing and thawing cells three times. To conduct freezing and thawing, 30 μL PBS solution was added to the cells and cooled at −70°C for 10 minutes. Frozen tubes were then placed in 37°C water until the cells melted completely. Cells were centrifuged for 10 minutes at 250× g and 4°C; the PBS supernatant was discarded and the cells were incubated for 30 minutes on ice (4°C) in cold lysis buffer (using 1 mL RIPA buffer and 10 μL protease inhibitor cocktail for every 10⁷ cells). Tubes were then centrifuged at 8,000× g for 10 minutes at 4°C, and the supernatant was stored in small volumes at −70°C after determining the protein concentration using the Bradford technique.