Pozarowski Piotr, Halicka Dorota H, Darzynkiewicz Zbigniew
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Cytometry A. 2003 Aug;54(2):118-24. doi: 10.1002/cyto.a.10057.
Apoptosis and necrosis ("accidental cell death") are distinct modes of cell death. The feature that often distinguishes apoptotic from necrotic cells is preservation of the plasma membrane integrity, reflected by ability of the former cells to exclude cationic dyes such as propidium iodide (PI) for a certain length of time. During necrosis, the plasma membrane is rapidly ruptured and necrotic cells stain intensely with PI. While studying cytostatic effects of the anti-inflammatory sesquiterpene parthenolide (PRT), we have noticed that, concurrent with apoptosis, the cells were dying by necrosis in the same cultures. Furthermore, because apoptosis was atypical, reflected by rapid loss of plasma membrane integrity, it was difficult to distinguish apoptotic from necrotic cells based on this feature.
Three methods were used to distinguish apoptosis from necrosis: (a) HL-60 cells treated with PRT were subjected to analysis of caspases activation using antibody that detects activated (cleaved) caspase-3, (b) apoptotic cells were identified by binding of fluorochrome-labeled inhibitor of caspases FAM-VAD-FMK combined with the PI exclusion assay, and (c) cellular DNA and RNA were differentially stained with acridine orange (AO).
Apoptotic cells were characterized by (a) caspase-3 activation detected immunocytochemically and (b) binding of FAM-VAD-FMK followed by or concurrent with (c) loss of ability to exclude PI, (d) deficit in DNA content, and (e) relatively little changed RNA content. Necrotic cells showed (a) no evidence of caspase-3 activation, (b) no binding of FAM-VAD-FMK, (c) inability to exclude PI, (d) rapid loss of RNA, and (e) unchanged DNA content.
Identification of apoptotic cells versus necrotic cells was possible either based on the evidence of caspase-3 activation, by labeling with FAM-VAD-FMK combined with PI or by differential staining of cellular DNA and RNA with AO. The data indicate that plasma membrane appears to be one of the targets of PRT, because its integrity is lost very early during cell death, which is reflected by atypical apoptosis and by primary necrosis (lysis of the membrane).
凋亡和坏死（“意外性细胞死亡”）是不同的细胞死亡方式。通常区分凋亡细胞与坏死细胞的特征是质膜完整性的保留，这表现为前者细胞在一定时间内能够排斥阳离子染料，如碘化丙啶（PI）。在坏死过程中，质膜迅速破裂，坏死细胞被PI强烈染色。在研究抗炎倍半萜小白菊内酯（PRT）的细胞生长抑制作用时，我们注意到，在同一培养物中，细胞在发生凋亡的同时也在因坏死而死亡。此外，由于凋亡具有非典型性，表现为质膜完整性迅速丧失，基于这一特征很难区分凋亡细胞与坏死细胞。
采用三种方法区分凋亡与坏死：（a）用PRT处理的HL-60细胞，使用检测活化（裂解）的半胱天冬酶-3的抗体进行半胱天冬酶激活分析；（b）通过荧光染料标记的半胱天冬酶抑制剂FAM-VAD-FMK结合PI排斥试验鉴定凋亡细胞；（c）用吖啶橙（AO）对细胞DNA和RNA进行差异染色。
凋亡细胞的特征为：（a）免疫细胞化学检测到半胱天冬酶-3激活；（b）FAM-VAD-FMK结合，随后或同时出现（c）排斥PI能力丧失；（d）DNA含量不足；（e）RNA含量变化相对较小。坏死细胞表现为：（a）无半胱天冬酶-3激活的证据；（b）无FAM-VAD-FMK结合；（c）无法排斥PI；（d）RNA迅速丧失；（e）DNA含量不变。
基于半胱天冬酶-3激活的证据、用FAM-VAD-FMK结合PI标记或用AO对细胞DNA和RNA进行差异染色，都有可能区分凋亡细胞与坏死细胞。数据表明，质膜似乎是PRT的靶点之一，因为在细胞死亡早期质膜完整性就丧失了，这表现为非典型凋亡和原发性坏死（膜溶解）。
