[3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein.
[(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.
Orlov
S N
SN
Centre de recherche du Centre hospitalier de l'Université de Montréal (CHUM), PQ, Canada. sergei.n.orlov@umontreal.ca
Pchejetski
D V
DV
Sarkissian
S D
SD
Adarichev
V
V
Taurin
S
S
Pshezhetsky
A V
AV
Tremblay
J
J
Maximov
G V
GV
deBlois
D
D
Bennett
M R
MR
Hamet
P
P
D017500:Adenovirus E1A Proteins / Q000378:metabolism*; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D008858:Microscopy, Phase-Contrast; D051381:Rats; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013552:Swine; D013936:Thymidine / Q000737:chemistry* / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D000590:Amino Acid Chloromethyl Ketones; D002843:Chromatin; D016895:Culture Media, Serum-Free; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D004247:DNA; D000242:Cyclic AMP; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D000254:Sodium-Potassium-Exchanging ATPase; D013936:Thymidine