Figure 2
Figure 2. The t(3;5)-encoded GCR-lincRNA-3q fusion protein is associated with glucocorticoid resistance. (A) Schematic representation of the genomic organization surrounding the normal 3q and der(3) breakpoint regions, indicating the resulting fusion transcript NR3C1-lincRNA-3q. The blue filled circle denotes the GATA2 superenhancer. In black, 3q sequences. In gray, 5q sequences. The NR3C1-lincRNA-3q fusion transcript likely derives from a splicing event between NR3C1 exon 2 and a cryptic splice acceptor site located in the antisense orientation in lincRNA-3q exon 2 (chromosome 3:129,832,478). (B) Schematic representation of the structure of wild-type GCR (top left) and predicted GCR-FP (bottom left). GCR and ACTIN western blot analyses showing expression of an abnormal GCR isoform (GCR-FP) exclusively in GEN2.2 cells compared with t(3;5)-negative cell lines, as indicated (right); * denotes nonspecific band. (C) Western blot using anti-GCR and anti-ACTIN antibodies in the parent CAL-1 cell line (nontransduced; NT) compared with CAL-1 cells transduced with empty GFP vector (−) or with the GFP-tagged GCR-FP expression vector (+), as indicated (n = 2). (D) Western blot using anti-GCR and anti-ACTIN antibodies in the parent CAL-1 cell line (NT) compared with CAL-1 cells transduced with shCtrl or shGCR (n = 2 independent hairpins, shGCR1 and 2), as indicated (right). Affymetrix NR3C1 messenger RNA expression in shGCR1 compared with shCtrl-transduced cells (experimental haploinsufficiency, n = 3) (left). (E) Evaluation of drug sensitivity of CAL-1 cells overexpressing GCR-FP compared with control CAL-1 cells after 16 hours of treatment with 10 μM etoposide (Eto), 72 hours of treatment with 10 μM dexamethasone (Dex), or a combination of both treatments. Specific apoptosis was measured by AnnexinV/propidium iodide staining and flow cytometry, and calculated as follows: (% drug-induced cell death − % control cell death) ÷ (100 − % control cell death) × 100. P value determined by Wilcoxon test; n = 3. (F) Evaluation of drug sensitivity of CAL-1 cells shGFP compared with shCtrl as described in panel E. aa, amino acid; AF, activation function domain; cen, centromere; DBD, DNA binding domain; LBD, ligand binding domain; ex, exon; NTD, N-terminal domain; tel, telomere; TSS, transcription start site; ZnF, zinc finger domain.

The t(3;5)-encoded GCR-lincRNA-3q fusion protein is associated with glucocorticoid resistance. (A) Schematic representation of the genomic organization surrounding the normal 3q and der(3) breakpoint regions, indicating the resulting fusion transcript NR3C1-lincRNA-3q. The blue filled circle denotes the GATA2 superenhancer. In black, 3q sequences. In gray, 5q sequences. The NR3C1-lincRNA-3q fusion transcript likely derives from a splicing event between NR3C1 exon 2 and a cryptic splice acceptor site located in the antisense orientation in lincRNA-3q exon 2 (chromosome 3:129,832,478). (B) Schematic representation of the structure of wild-type GCR (top left) and predicted GCR-FP (bottom left). GCR and ACTIN western blot analyses showing expression of an abnormal GCR isoform (GCR-FP) exclusively in GEN2.2 cells compared with t(3;5)-negative cell lines, as indicated (right); * denotes nonspecific band. (C) Western blot using anti-GCR and anti-ACTIN antibodies in the parent CAL-1 cell line (nontransduced; NT) compared with CAL-1 cells transduced with empty GFP vector (−) or with the GFP-tagged GCR-FP expression vector (+), as indicated (n = 2). (D) Western blot using anti-GCR and anti-ACTIN antibodies in the parent CAL-1 cell line (NT) compared with CAL-1 cells transduced with shCtrl or shGCR (n = 2 independent hairpins, shGCR1 and 2), as indicated (right). Affymetrix NR3C1 messenger RNA expression in shGCR1 compared with shCtrl-transduced cells (experimental haploinsufficiency, n = 3) (left). (E) Evaluation of drug sensitivity of CAL-1 cells overexpressing GCR-FP compared with control CAL-1 cells after 16 hours of treatment with 10 μM etoposide (Eto), 72 hours of treatment with 10 μM dexamethasone (Dex), or a combination of both treatments. Specific apoptosis was measured by AnnexinV/propidium iodide staining and flow cytometry, and calculated as follows: (% drug-induced cell death − % control cell death) ÷ (100 − % control cell death) × 100. P value determined by Wilcoxon test; n = 3. (F) Evaluation of drug sensitivity of CAL-1 cells shGFP compared with shCtrl as described in panel E. aa, amino acid; AF, activation function domain; cen, centromere; DBD, DNA binding domain; LBD, ligand binding domain; ex, exon; NTD, N-terminal domain; tel, telomere; TSS, transcription start site; ZnF, zinc finger domain.

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