[3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein

S N Orlov, D V Pchejetski, S D Sarkissian, V Adarichev, S Taurin, A V Pshezhetsky, J Tremblay, G V Maximov, D deBlois, M R Bennett, P Hamet

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Abstract

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.
Original languageEnglish
Pages (from-to)199-208
Number of pages10
JournalApoptosis
Volume8
Issue number2
DOIs
Publication statusPublished - Mar 2003

Keywords

  • Adenovirus E1A Proteins
  • Amino Acid Chloromethyl Ketones
  • Animals
  • Apoptosis
  • Caspase 3
  • Caspases
  • Chromatin
  • Culture Media, Serum-Free
  • Cyclic AMP
  • DNA
  • Dogs
  • Dose-Response Relationship, Drug
  • Microscopy, Phase-Contrast
  • Rats
  • Signal Transduction
  • Sodium-Potassium-Exchanging ATPase
  • Swine
  • Thymidine

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