Orlov S N, Pchejetski D V, Sarkissian S D, Adarichev V, Taurin S, Pshezhetsky A V, Tremblay J, Maximov G V, deBlois D, Bennett M R, Hamet P
Centre de recherche du Centre hospitalier de l'Université de Montréal (CHUM), PQ, Canada.
Apoptosis. 2003 Mar;8(2):199-208. doi: 10.1023/a:1022931028235.
[(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(+)/K(+) ratio.
[³H]胸腺嘧啶核苷常用于分析凋亡细胞中[³H]标记的染色质片段的积累情况。本研究表明，DNA内[³H]胸腺嘧啶核苷的掺入本身就足以抑制犬肾上皮细胞和猪主动脉内皮细胞的生长并诱导其凋亡。尽管[³H]胸腺嘧啶核苷 - DNA标记水平很高，但与其他细胞类型相比，大鼠血管平滑肌细胞（VSMC）仅表现出适度的生长抑制和凋亡诱导。与血清剥夺类似，[³H]胸腺嘧啶核苷标记引发的凋亡通过用c - myc的功能类似物E1A - 腺病毒蛋白（VSMC - E1A）转染VSMC而显著增强，并通过用福斯可林刺激cAMP信号传导以及用哇巴因抑制钠钾泵而受到抑制。在[³H]胸腺嘧啶核苷处理的VSMC - E1A中观察到的凋亡诱导和生长抑制均被泛半胱天冬酶抑制剂z - VAD.fmk降低。因此，我们的结果表明，凋亡机制的不同效率决定了具有[³H]胸腺嘧啶核苷标记DNA的细胞中生长的细胞类型特异性减弱。它们还表明，[³H]胸腺嘧啶核苷处理和血清剥夺的VSMC采用凋亡机制的共同中间体，包括被E1A - 腺病毒蛋白增强、被cAMP信号激活以及细胞内[Na⁺]i/[K⁺]i比值倒置抑制的步骤。
