Ohsawa Y, Isahara K, Kanamori S, Shibata M, Kametaka S, Gotow T, Watanabe T, Kominami E, Uchiyama Y
Department of Cell Biology and Anatomy, Osaka University Medical School, Suita, Japan.
Arch Histol Cytol. 1998 Dec;61(5):395-403. doi: 10.1679/aohc.61.395.
In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ultrastructures and immunoreactivity for these enzymes in PC12 cells cultured under serum deprivation. Laser scanning microscopy showed immunoreactivity for cathepsins B and D to be finely distributed in the cytoplasm of PC12 cells at the onset of culture under serum deprivation. At 3 h after the onset of culture, the immunoreactivity for cathepsin B slightly decreased in the cells, while immunodeposits for cathepsin D in the cells became more intense and larger in size than those at 0 h. Positive staining for TUNEL in nuclei of the cells appeared at 6 h, though fewer in number. Corresponding to the increase in the number of TUNEL-positive cells at 12 h and 24 h, the immunoreactivity for cathepsin B was drastically diminished in the cells, whereas that for cathepsin D was significantly augmented, especially in TUNEL-positive cells. Electron microscopically, autophagic vacuoles/autolysosomes appeared in the cytoplasm of the cells 3 h after the onset of culture. A distinct nuclear change showing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6 h. The number of PC12 cells having nuclei with chromatin condensation increased especially at 24 h, while these cells showed shrinkage of both their cytoplasm and nuclei. Dense bodies and autophagic vacuoles with limiting membranes were seen in these cells. These results showing the occurrence of autophagy and imbalance of protein amounts between cathepsins B and D during apoptosis may argue for our hypothesis that these enzymes are, in part, involved in the cell death cascade for PC12 cells following serum deprivation.
除了半胱天冬酶蛋白酶家族外，组织蛋白酶D（一种溶酶体天冬氨酸蛋白酶）也被认为在哺乳动物细胞中作为促凋亡介质发挥作用。为了进一步了解组织蛋白酶B和D在细胞凋亡中的作用，我们研究了血清剥夺培养条件下PC12细胞中这些酶的超微结构和免疫反应性的精确变化过程。激光扫描显微镜显示，在血清剥夺培养开始时，组织蛋白酶B和D的免疫反应性在PC12细胞的细胞质中分布良好。培养开始3小时后，细胞中组织蛋白酶B的免疫反应性略有下降，而细胞中组织蛋白酶D的免疫沉积物比0小时时变得更强且尺寸更大。细胞细胞核中的TUNEL阳性染色在6小时出现，尽管数量较少。与12小时和24小时TUNEL阳性细胞数量的增加相对应，细胞中组织蛋白酶B的免疫反应性急剧下降，而组织蛋白酶D的免疫反应性显著增强，尤其是在TUNEL阳性细胞中。电子显微镜观察显示，培养开始3小时后，细胞细胞质中出现自噬泡/自溶酶体。6小时时，细胞核外周部分首先出现明显的核变化，表现为染色质相对浓缩。具有染色质浓缩细胞核的PC12细胞数量尤其在24小时增加，而这些细胞的细胞质和细胞核均出现萎缩。在这些细胞中可见致密体和具有限制膜的自噬泡。这些结果表明，凋亡过程中自噬的发生以及组织蛋白酶B和D之间蛋白质含量的失衡可能支持我们的假设，即这些酶在一定程度上参与了血清剥夺后PC12细胞的细胞死亡级联反应。
