American Society of Hematology

Figure 1

$Figure 1. Response of Eμ-Myc cell lines to DNA-damaging drugs in vitro. (A) Eμ-Myc cell lines with functional p53 (Eμ-Myc, n = 6 independently derived lymphomas; black circles), harboring either mutant p53 (Eμ-Myc/p53 mutant, n = 3; red diamonds) or a targeted p53-deleted allele (with subsequent loss of heterozygosity of wild-type allele) (Eμ-Myc/p53−/−, n = 4; red triangles), or retrovirally overexpressing BCL-2 (Eμ-Myc/rvBCL-2, n = 3; blue squares) were left untreated or treated for 24 hours with etoposide (0-1 μg/mL). Cell death expressed as percent specific apoptosis. Mean ± SEM from 3-6 independent cell lines for each genotype, from 3 independent experiments per cell line. (B) Eμ-Myc/p53+/+ cell line AF47-T2 treated with etoposide (1 μg/mL) in the presence or absence of QVD-OPH (25μM) with cell death assessed at 2, 4, and 6 hours. Mean ± SEM from 3 independent experiments. Similar results for 2 further Eμ-Myc/p53+/+ cell lines (AH29 and AH54). (C) The Eμ-Myc/p53+/+ cell line, AF47-T2, was treated with etoposide (E; 1 μg/mL) in the presence or absence of QVD-OPH (QVD, 25μM) for 6 hours, prior to the assessment for caspase processing by Western blot analysis. Comparable results for the Eμ-Myc cell line AH29. NT = not treated. (D) Subcellular redistribution assay demonstrating, for Eμ-Myc cell line AF47-T2, the movement of BAX to the mitochondrial fraction (“M”) and concomitant translocation of cytochrome c from mitochondria into the cytosol (“C”) after treatment with etoposide (1 μg/mL) for 6 hours. BAK and HSP70 provide loading controls. Comparable results were obtained with the Eμ-Myc cell line, AH29. (E) Eμ-Myc/p53+/+ and Eμ-Myc/p53−/− cell lines (n = 3 for each genotype) were treated with etoposide (1 μg/mL) for 3 hours and assessed by RT-PCR for BH3-only gene mRNA expression. Mean fold change ± SEM from 3 independent experiments. (F) Eμ-Myc/p53+/+ and Eμ-Myc/p53−/− cell lines (n = 2 for each genotype) were treated with etoposide (1 μg/mL) for 6 hours and assessed by Western-blot analysis for levels of PUMA and p53 proteins.$

Response of Eμ-Myc cell lines to DNA-damaging drugs in vitro. (A) Eμ-Myc cell lines with functional p53 (Eμ-Myc, n = 6 independently derived lymphomas; black circles), harboring either mutant p53 (Eμ-Myc/p53 mutant, n = 3; red diamonds) or a targeted p53-deleted allele (with subsequent loss of heterozygosity of wild-type allele) (Eμ-Myc/p53^−/−, n = 4; red triangles), or retrovirally overexpressing BCL-2 (Eμ-Myc/rvBCL-2, n = 3; blue squares) were left untreated or treated for 24 hours with etoposide (0-1 μg/mL). Cell death expressed as percent specific apoptosis. Mean ± SEM from 3-6 independent cell lines for each genotype, from 3 independent experiments per cell line. (B) Eμ-Myc/p53^+/+ cell line AF47-T2 treated with etoposide (1 μg/mL) in the presence or absence of QVD-OPH (25μM) with cell death assessed at 2, 4, and 6 hours. Mean ± SEM from 3 independent experiments. Similar results for 2 further Eμ-Myc/p53^+/+ cell lines (AH29 and AH54). (C) The Eμ-Myc/p53^+/+ cell line, AF47-T2, was treated with etoposide (E; 1 μg/mL) in the presence or absence of QVD-OPH (QVD, 25μM) for 6 hours, prior to the assessment for caspase processing by Western blot analysis. Comparable results for the Eμ-Myc cell line AH29. NT = not treated. (D) Subcellular redistribution assay demonstrating, for Eμ-Myc cell line AF47-T2, the movement of BAX to the mitochondrial fraction (“M”) and concomitant translocation of cytochrome c from mitochondria into the cytosol (“C”) after treatment with etoposide (1 μg/mL) for 6 hours. BAK and HSP70 provide loading controls. Comparable results were obtained with the Eμ-Myc cell line, AH29. (E) Eμ-Myc/p53^+/+ and Eμ-Myc/p53^−/− cell lines (n = 3 for each genotype) were treated with etoposide (1 μg/mL) for 3 hours and assessed by RT-PCR for BH3-only gene mRNA expression. Mean fold change ± SEM from 3 independent experiments. (F) Eμ-Myc/p53^+/+ and Eμ-Myc/p53^−/− cell lines (n = 2 for each genotype) were treated with etoposide (1 μg/mL) for 6 hours and assessed by Western-blot analysis for levels of PUMA and p53 proteins.

This Feature Is Available To Subscribers Only