Maellaro E, Bello B D, Comporti M
Institute of General Pathology, University of Siena, Siena, I-53100, Italy.
Exp Cell Res. 1996 Jul 10;226(1):105-13. doi: 10.1006/excr.1996.0208.
Apoptosis can be triggered in thymocytes with stimuli (6alpha-methylprednisolone, thapsigargin, and etoposide) acting by different mechanisms. In each of these instances cell death is extensively prevented until 5 h of incubation when cells are preincubated with 250 microM ascorbic acid (AA) for 1 h, then washed, and incubated in fresh medium containing the above mentioned apoptotic stimuli. In addition, the degree of spontaneous apoptosis of untreated thymocytes is somewhat lower in the AA-preincubated cells. The protection against apoptosis does not seem to be dependent on the intracellular enrichment of AA, as measured at the end of the preincubation period. On the contrary, such a protection is strictly related to a partial loss of ascorbate in the medium (possibly due to its autooxidation), is catalase-inhibitable, and is reproduced by a preincubation of the cells with nontoxic concentrations of hydrogen peroxide. The AA-supplemented cells show a remarkable decrease in NAD+ levels and a significant increase of poly(ADP-ribose) polymerase (PARP) activity. Consistently with these results, the addition of PARP inhibitors, such as thymidine and 3-aminobenzamide, during the preincubation with AA, prevents NAD+ depletion and abolishes the protective effect of AA against apoptosis. The possibility is discussed that an early activation of PARP by stimuli which are nontoxic per se makes the cells able to withstand subsequent apoptotic stimuli which are otherwise lethal.
凋亡可由通过不同机制起作用的刺激因素（6α-甲基泼尼松龙、毒胡萝卜素和依托泊苷）在胸腺细胞中触发。在每种情况下，细胞死亡都会被广泛抑制，直到培养5小时，前提是细胞先用250微摩尔的抗坏血酸（AA）预孵育1小时，然后洗涤，并在含有上述凋亡刺激因素的新鲜培养基中孵育。此外，在AA预孵育的细胞中，未处理胸腺细胞的自发凋亡程度略低。对凋亡的保护作用似乎不依赖于预孵育期结束时所测定的细胞内AA的富集情况。相反，这种保护作用与培养基中抗坏血酸的部分损失密切相关（可能是由于其自身氧化），可被过氧化氢酶抑制，并且通过用无毒浓度的过氧化氢对细胞进行预孵育也能重现这种保护作用。补充了AA的细胞显示NAD⁺水平显著降低，聚（ADP - 核糖）聚合酶（PARP）活性显著增加。与这些结果一致，在与AA预孵育期间添加PARP抑制剂，如胸苷和3 - 氨基苯甲酰胺，可防止NAD⁺耗竭，并消除AA对凋亡的保护作用。本文讨论了一种可能性，即本身无毒的刺激因素对PARP的早期激活使细胞能够耐受随后否则会致命的凋亡刺激因素。
