Detection of the leukemic clone in bone marrow SP cells of patients with CML, and expression of ABCA3 in the stem cell compartment of patients with CML. Bone marrow samples of CML patients were stained with Hoechst 33342 and subsequently sorted into cells with the SP phenotype (A upper panel, as boxed) and the remaining non-SP cells. In the samples from patients with untreated CML (n=11, blast crisis excluded), ABCA3 transcript levels, as detected by quantitative RT-PCR were higher in the SP cell fraction than in the non-SP cell fraction (A, lower panel, Wilcoxon’s signed rank test). As for whole marrow, ABCA3 transcripts were detected by quantitative RT-PCR in 25 bone marrow samples from patients with CML (19 cases in chronic phase, 4 cases in acceleration, and 2 cases in blast crisis), with significantly higher levels of expression in leukemic samples than in bone marrow cells of healthy volunteers (B, middle panel, Kruskal-Wallis test with Dunn’s multiple comparison post-test, ** and *** indicate p<0.01 and p<0.001, respectively). In bone marrow from healthy volunteers, immunocytological detection of the ABCA3 protein revealed a small fraction of positive cells (17%) in sorted CD34-positive cells, and no ABCA3-positive cells in the CD34-negative fraction (D, left column, quantification in C). The scale bars represent 10 μm, the respective isotype control stains are shown as inserts. In the marrow samples of CML patients, ABCA3-positive cells were found in both the CD34-positive and in the CD34-negative fractions, with a significantly higher proportion of positive cells in the CD34-positive fraction (D, right column, quantification in C). One hundred cells each from three representative slide sectors of three different individuals were counted.

ABCA3 modulates susceptibility of CML cell lines to imatinib and imatinib-mediated inhibition of Crkl phosphorylation. Clonogenic growth of BCR-ABL-positive cell lines exposed to imatinib was measured in colony-forming-unit (CFU) assays after specific siRNA-mediated suppression of ABCA3 expression levels, or addition of scrambled siRNA. Incubation with increasing doses of imatinib reduced the number and size of colonies, with markedly smaller colonies after specific knock-down of ABCA3 (A, middle and lower panels of the right column). Over an imatinib dose range of 100 to 500 nM, ABCA3 knock-down significantly reduced the number of colonies obtained from the K562, LAMA84 and BV173 cell lines (A, right panel). Correspondingly, ABCA3 knockdown sensitized K562 and LAMA84 to the suppressive effects of imatinib on viability as measured by the MTT test (B). Vice versa, increasing ABCA3 levels by ectopic expression protected K562 and LAM84 cells from the suppressive effects of imatinib on clonogenicity (C). Differences were tested for statistical significance by two-way ANOVA with Bonferroni’s post-test (*p<0.05). Forty-eight hours after transfection with siRNA against ABCA3 or a scrambled siRNA control, K562 cells were exposed to imatinib for 16 h at the concentrations indicated (D). The phosphorylation status of the BCR-ABL adaptor protein Crkl (Tyr207) was visualized by western blot with a phosphorylation-specific antibody. Knock-down of ABCA3 shifted the threshold of suppression in Crkl phosphorylation to lower imatinib concentrations.

Localization of ABCA3 to the limiting membrane of lysosomes and multivesicular bodies. Ultramicroscopy following immunogold staining of ABCA3 (black triangles) and the lysosomal membrane protein LAMP-1 (white triangles) revealed that the transporter was localized in the limiting membrane of lysosomes (L), (A) and multivesicular bodies (MVB), B) of K562 cells. Ectopic expression of ABCA3 significantly increased the number of lysosomes (L) and multivesicular bodies (MVB) in HEK293-ABCA3 cells (C), compared to in the HEK293 wild-type control cells (D).

Release of imatinib from the lysosomal lumen. Following fractionation by density centrifugation, the pooled lysosomal fractions were left untreated (solo), or treated with either Triton-X100 (+Triton) or Tris (+Tris), and then centrifuged again. Supernatant (S) and membrane pellet (P) were then analyzed for imatinib and luminal marker β-hexosaminidase. Disrupting the membrane with Triton-X100, and thereby lysing the lysosomes, released both imatinib and β-hexosaminidase in all cell lines, whereas using Tris as a strong washing buffer, active on the outside of the lysosome, had no such effect (A–D). Importantly, there were no qualitative differences in imatinib release between ABCA3-positive and ABCA3-negative cells (C, D). Western blot of LAMP-1 served as a positive control for the purity of membrane preparations. Differences were analyzed using a two-tailed Student’s t test (*p<0.05).

Biochemical confinement of imatinib to the lysosomal fractions. Following 2 hours of exposure to [¹⁴C]-imatinib, cells were homogenized and subcellular fractions of the post-nuclear supernatant separated by density centrifugation. [¹⁴C]-imatinib was recovered mainly from the lysosomal fractions (19–22), with an identical pattern of imatinib distribution in the K562, LAMA84, HEK293 and HEK293-ABCA3 cell lines (A). The lysosomal fractions were identified by the lysosomal markers β-hexosaminidase and LAMP-1, and were negative for the early endosome marker EEA1 and the plasma membrane marker Na/K-ATPase (A, lower panel). The ABCA3/GFP fusion protein was also detected mainly in the lysosomal fractions of HEK293-ABCA3 cells (A, lower panel). Comparison of pooled lysosomal (19–23=L+) and non-lysosomal (1–18=L−) fractions confirmed the predominant localization of imatinib to this compartment as a consistent phenomenon in all cell lines (B). Accordingly, most [¹⁴C]-imatinib was detected in the post-nuclear supernatant of HEK293 and HEK293-ABCA3 cells (C), and was subsequently co-purified with the intact membranes of organelles, and not in the free cytosol (D).

Impact of ABCA3 on uptake and subcellular sequestration of imatinib. Following exposure to [¹⁴C]-imatinib for 2 h at the concentrations indicated, HEK293-ABCA3 cells (gray columns) retained larger amounts of imatinib than did the wild-type HEK293 control cells (black columns) (A). Note the different scale bars between left and right panels, indicating that total cellular [¹⁴C]-imatinib is reduced by approximately 50% within 24 h. As shown by using the fluorescent dye Lysotracker red as a marker for lysosomal storage capacity, HEK293-ABCA3 cells retained this substance more efficiently than wild-type HEK293 cells (B). Following incubation of isolated lysosomal fractions with [¹⁴C]-imatinib for 2 h (F) at 4°C, uptake was 43% versus 61% of radio-labeled imatinib in the lysosomes of HEK293 and HEK293/ABCA3 cells, respectively (C, sample 1). Incubation at 37°C without ATP (sample 2), with ATP (sample 3), or with ATP and a competitor (sample 4), did not change the ratio of greater imatinib accumulation in ABCA3-positive lysosomes than in ABAC3-negative lysosomes. Differences were tested using a two-tailed Student’s t test (*p<0.05). Representative experiments of four independent replicates are shown.