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

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.

Na + /K + pump and endothelial cell survival: [Na + ] i /[K + ] i -independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na + ] i

“…5b). This finding is consistent with previously reported data [32] and indicates that the decreased content of attached cells due to induction of apoptosis is a major mechanism of decreased MTT catabolism in [ 3 H]-thymidine-treated PAEC.…”

“…Second, caspase-mediated damage is a common mechanism for the irreversible triggering of the apoptotic machinery in the majority of cells studied so far [35]. Similar to VSMC transfected with E1A adenoviral protein and highly susceptible to apoptosis [32], PAEC death triggered by 3 H decay-induced DNA damage was sharply attenuated by the pan-caspase inhibitor z-VAD.fmk (Table 3). In contrast to ouabain, we failed to detect any protection of PAEC by this compound against the toxic action of ouabain (Fig.…”

“…Previously, we have reported that extensive DNA labelling with [ 3 H]-thymidine sharply attenuates the growth of renal epithelial cells, VSMC and PAEC due to the induction of apoptosis triggered by 3 H decay-induced DNA damage [32]. We used this approach to study the effect of Na + /K + pump inhibition on apoptosis.…”

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

“…3d). In previous studies, the same cell fragments were detected morphologically in MDCK cells treated with classic apoptotic stimuli, such as actinomycin D [26], partial ATP-depletion [27], [ 3 H]-decay-induced DNA damage [28] and hyperosmotic shrinkage [29]. Note: ND, because of the low content of floating cells in control conditions, this parameter was not measured.…”

Apoptosis VS. Oncosis: Role of Cell Volume and Intracellular Monovalent Cations

“…17,18,22 With this well-established cell culture model of apoptosis, we noted that pretreatment with ouabain, a highly selective Na + -K + pump inhibitor, delayed VSMC death triggered by distinct apoptotic stimuli such as serum deprivation, inhibition of serine-threonine phosphatases, cytochrome c release, and DNA damage triggered by extensive labeling with [ 3 H]-thymidine at a step upstream of caspase-3. 21,23 Later, protection against apoptosis by Na + -K + pump inhibitors was documented in neuronal cells, 24 and renal epithelial cells. 25 The antiapoptotic action of ouabain might be caused by rapid membrane depolarization due to electrogenicity of the Na 26 abolished the antiapoptotic action of ouabain in VSMC-E1A.…”