Multi-Agent Chemotherapy Overcomes Glucocorticoid Resistance Conferred by a BIM Deletion Polymorphism in Pediatric Acute Lymphoblastic Leukemia
Figure 1
Generation of isogenic CCRF-CEM cell lines with the BIM deletion polymorphism.
(A) Structure of the BIM gene and major splice isoforms. The BIM deletion polymorphism lies within intron 2 and upstream of exon 3, as indicated by the dashed line. Exon 4 contains the crucial BH3 domain required for apoptosis. Exon 3 (E3) and 4 (E4) are spliced in a mutually exclusive fashion, leading to the generation of either E4-containing isoforms with the BH3 domain (BIM EL, BIM L and BIM S) or E3-containing isoforms without the BH3 domain (BIM γ). When present, the deletion biases splicing towards E3-containing non-apoptotic isoforms. (B) Agarose gel of the products from a PCR reaction to detect the polymorphism in zinc finger nuclease (ZFN)-treated CCRF-CEM subclones, with the lower band indicating the presence of the deletion. Parental CCRF-CEM and KCL-22 cells (a CML cell line known to be heterozygous for the BIM deletion polymorphism) were included as controls. (C) The ratio of exon 3 to exon 4-containing transcripts (E3:E4) in CCRF-CEM BIMi2+/+, BIMi2+/− and BIMi2−/− clones as measured by qPCR. Error bars indicate mean ± SEM (n = 3). A student's t-test was performed for pairwise comparisons of E3:E4 ratio between genotypes. * indicates a significant difference with P<0.05.