Regulation of proteasome activity in adriamycin-treated leukemic blasts from patients with AML.
(A) Nuclei from human leukemic blasts in the absence (▪) or presence (●) of 1 mM 3-ABA and nuclei from human leukemic blasts transiently transfected with the asPARP vector (▴) were isolated and lysed after treatment with 10−7 M adriamycin for the time points indicated, respectively. Proteasome activity is expressed as lactacystin-sensitive suc-LLVY-MCA degradation. Data are given as mean ± SD (n = 3) from one representative patient typical for all AML patients (n = 4) investigated. (B) Western blot control of PARP protein expression in leukemic blasts of an AML patient and human leukemic blasts of the same patient transiently transfected with the asPARP vector (asPARP blasts) and the control vector (control vector blasts), respectively, without impairment of proteasome protein levels. Transient transfection of the blasts is documented by the GFP blot, and the actin immunoblot demonstrated equal loading. (C) Human leukemic blasts from AML patients were treated with 10−7 M adriamycin in the absence or presence of 1 mM 3-ABA for the time points indicated. Thereafter, the nuclei were isolated, lysed, and immunoprecipitated with a polyclonal rabbit anti-PARP antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transblotted, and visualized with a polyclonal anti-PARP and a polyclonal antiproteasome antibody, respectively. A representative set of data is presented from one AML patient out of all AML patients (n = 4) investigated.
