Aparna Gunda, Bhuyan Abani K, Sahdev Sudhir, Hasnain Seyed E, Kaufman Randal J, Ramaiah Kolluru V A
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.
Biochemistry. 2003 Dec 30;42(51):15352-60. doi: 10.1021/bi0349423.
Spodoptera frugiperda (Sf9) ovarian cells, natural hosts for baculovirus, are good model systems to study apoptosis and also heterologous gene expression. We report that uninfected Sf9 cells readily undergo apoptosis and show increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in the presence of agents such as UVB light, etoposide, high concentrations of cycloheximide, and EGTA. In contrast, tunicamycin, A23187, and low concentrations of cycloheximide promoted eIF2alpha phosphorylation in Sf9 cells but without apoptosis. These findings therefore suggest that increased eIF2alpha phosphorylation does not always necessarily lead to apoptosis, but it is a characteristic hallmark of stressed cells and also of cells undergoing apoptosis. Cell death induced by the above agents was abrogated by infection of Sf9 cells with wild-type (wt) AcNPV. In contrast, Sf9 cells when infected with vAcdelta35, a virus carrying deletion of the antiapoptotic p35 gene, showed increased apoptosis and enhanced eIF2alpha phosphorylation. Further, a recombinant wt virus vAcS51D expressing human S51D, a phosphomimetic form of eIF2alpha, induced apoptosis in UVB pretreated Sf9 cells. However, infection with vAcS51A expressing a nonphosphorylatable form (S51A) of human eIF2alpha partially reduced apoptosis. Consistent with these findings, it has been observed here that caspase activation has led to increased eIF2alpha phosphorylation, while caspase inhibition by z-VAD-fmk reduced eIF2alpha phosphorylation selectively in cells exposed to proapoptotic agents. These findings therefore suggest that the stress signaling pathway determines apoptosis, and caspase activation is a prerequisite for increased eIF2alpha phosphorylation in Sf9 cells undergoing apoptosis. The findings also reinforce the conclusion for the first time that the "pancaspase inhibitor" baculovirus p35 mitigates eIF2alpha phosphorylation.
草地贪夜蛾（Sf9）卵巢细胞是杆状病毒的天然宿主，是研究细胞凋亡和异源基因表达的良好模型系统。我们报告称，在诸如紫外线B（UVB）照射、依托泊苷、高浓度环己酰亚胺和乙二醇双乙胺醚（EGTA）等因素存在的情况下，未感染的Sf9细胞很容易发生凋亡，并显示真核起始因子2（eIF2α）的α亚基磷酸化增加。相比之下，衣霉素、A23187和低浓度环己酰亚胺可促进Sf9细胞中的eIF2α磷酸化，但不会导致细胞凋亡。因此，这些发现表明，eIF2α磷酸化增加并不一定总是导致细胞凋亡，但它是应激细胞以及正在经历凋亡的细胞的一个特征性标志。上述因素诱导的细胞死亡可通过野生型（wt）苜蓿银纹夜蛾核型多角体病毒（AcNPV）感染Sf9细胞而被消除。相比之下，当Sf9细胞感染携带抗凋亡p35基因缺失的病毒vAcdelta35时，细胞凋亡增加，eIF2α磷酸化增强。此外，表达人S51D（一种eIF2α的磷酸模拟形式）的重组野生型病毒vAcS51D在紫外线B预处理的Sf9细胞中诱导细胞凋亡。然而，感染表达人eIF2α不可磷酸化形式（S51A）的vAcS51A可部分减少细胞凋亡。与这些发现一致，在此观察到半胱天冬酶激活导致eIF2α磷酸化增加，而z-VAD-fmk对半胱天冬酶的抑制在暴露于促凋亡因素的细胞中选择性地降低了eIF2α磷酸化。因此，这些发现表明应激信号通路决定细胞凋亡，并且半胱天冬酶激活是正在经历凋亡的Sf9细胞中eIF2α磷酸化增加的先决条件。这些发现还首次强化了“泛半胱天冬酶抑制剂”杆状病毒p35减轻eIF2α磷酸化这一结论。
