Actinomycin D upregulates proapoptotic protein Puma and downregulates Bcl-2 mRNA in normal peripheral blood lymphocytes

Anticancer Drugs. 2007 Aug;18(7):763-72. doi: 10.1097/CAD.0b013e3280adc905.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibiotics, Antineoplastic / administration & dosage
  • Antibiotics, Antineoplastic / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / metabolism
  • Blotting, Western
  • Cell Survival
  • DNA Damage / drug effects
  • DNA, Ribosomal / drug effects
  • Dactinomycin / administration & dosage
  • Dactinomycin / pharmacology*
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Flow Cytometry
  • Humans
  • Lymphocytes / drug effects
  • Mitochondria / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Suppressor Protein p53 / metabolism
  • Up-Regulation / drug effects

Substances

  • Antibiotics, Antineoplastic
  • Apoptosis Regulatory Proteins
  • BBC3 protein, human
  • DNA, Ribosomal
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Messenger
  • Tumor Suppressor Protein p53
  • Dactinomycin