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

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.

BimEL‐dependent apoptosis induced in peripheral blood lymphocytes with n‐butyric acid is moderated by variation in expression of c‐myc and p21(WAF1)

“…The construction of specific primers (Invitrogen) shown in Table 1 has been described earlier 23 as well as the RT-PCR program. Amplified products were detected following electrophoresis on 2% agarose gels containing ethidium bromide (0.5 mg ml À1 ) followed by examination under UV light.…”

Deregulated expression of A1, Bcl-2, Bcl-xL, and Mcl-1 antiapoptotic proteins and Bid, Bad, and Bax proapoptotic genes in polycythemia vera patients

“…Actinomycin D acts inhibiting the synthesis of RNA and it seems to potentiate Fas-mediated apoptosis, prevent binding of NF-kb to DNA (Wang et al, 2007), decrease the expression of the extrinsic antiapoptotic pathway, c-Flip, and stimulate Puma and Noxa expression of p53 and APAF-1 pathway (Kalousek et al , 2007). ACT-D and cycloheximide conduct cells to apoptosis via the TRAIL protein or TNF-a receptor.…”

Variations in c‐Myc and p21WAF1 expression protect normal peripheral blood lymphocytes against BimEL‐mediated cell death

“…Semiquantitative RT-PCR was performed using 2 mg of total RNA and One-Step RT-PCR kit (Qiagen). The design of specific primers (invitrogen) in Table 1 as well as the thermal cycler conditions has been described earlier 20 . Amplified products were detected on electrophoretic agarose gels containing 0.5 mg/mL of ethidium bromide.…”

“…Following the procedure described earlier 20 the absorbance of reporter substrate was measured on an ELISA reader (MTX Lab Systems, Inc., Vienna, VA, USA).…”