We have examined the ability of actinomycin D to induce apoptosis in human peripheral blood lymphocytes. Run-On assays were performed to specify the primary molecular damage, reverse transcription-PCR, Western blots and flow cytometry studies were performed to ascertain which proteins of the apoptosis machinery were affected to cause actinomycin D-induced cell death. Expression of 23 apoptosis-related genes was investigated. The down-regulation of ribosomal RNA synthesis caused by actinomycin D induced a mitochondria-dependent apoptosis. Although the expression of the majority of examined genes remained indifferent against actinomycin D activity, the cellular level of p53 protein increased, subsequently upregulating both Puma mRNA and protein. Puma-mediated mitochondrial apoptosis was accompanied by nucleolin cleavage and Bcl-2 mRNA destabilization. The stability of the cellular level of Bcl-2 protein independent of a mRNA decrease suggests that protection of Bcl-2 protein against proteasomal degradation can moderate the apoptotic process. In peripheral blood lymphocytes cultured in vitro, the apoptosis induced by a low concentration of actinomycin D (10 nmol/l) is dependent on p53 and Puma activation. This apoptotic pathway is demonstrated in peripheral blood lymphocytes for the first time. A different apoptotic pathway induced in peripheral blood lymphocytes using this drug has, however, been previously revealed by other authors. The combination of cell specificity and dose-dependent effects can likely play a decisive role in apoptosis observed in peripheral blood lymphocytes after genotoxic drug application.

Department of Cellular Biochemistry Institute of Hematology and Blood Transfusion U Nemocnice 1 128 20 Prague Czech Republic

References provided by Crossref.org

Decitabine and SAHA-induced apoptosis is accompanied by survivin downregulation and potentiated by ATRA in p53-deficient cells

Combined treatment with low concentrations of decitabine and SAHA causes cell death in leukemic cell lines but not in normal peripheral blood lymphocytes

Generation of reactive oxygen species during apoptosis induced by DNA-damaging agents and/or histone deacetylase inhibitors

Decitabine-induced apoptosis is derived by Puma and Noxa induction in chronic myeloid leukemia cell line as well as in PBL and is potentiated by SAHA