Actinomycin D upregulates proapoptotic protein Puma and downregulates Bcl-2 mRNA in normal peripheral blood lymphocytes.
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.
Kalousek
Ivan
I
Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128-20 Prague, Czech Republic. kal@uhkt.cz
Brodska
Barbora
B
Otevrelova
Petra
P
Röselova
Pavla
P
D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D015153:Blotting, Western; D002470:Cell Survival; D004249:DNA Damage / Q000187:drug effects; D004275:DNA, Ribosomal / Q000187:drug effects; D003609:Dactinomycin / Q000008:administration & dosage / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005434:Flow Cytometry; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D004275:DNA, Ribosomal; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D003609:Dactinomycin