Bacsó Z, Everson R B, Eliason J F
Barbara Ann Karmanos Cancer Institute, Wayne State School of Medicine Detroit, Michigan 48201, USA.
Cancer Res. 2000 Aug 15;60(16):4623-8.
Jurkat leukemia cells induced to undergo apoptosis by treatment with an antibody against the Fas receptor have two annexin V (AV)-binding subpopulations: (a) single-positive cells that bind AV but not propidium iodide (PI); and (b) double-positive cells that bind AV and PI. The single-positive population is thought to represent an early stage of apoptosis. We have examined the relationship between AV binding and a classical characteristic of apoptosis, DNA fragmentation. Time course studies with Jurkat cells treated for 1, 2, or 4 h with anti-Fas indicated that the proportion of AV-binding cells was increased after 2 h. A significant increase in DNA fragmentation was observed only at 4 h as measured by the mean tail moment determined with the alkaline single cell gel electrophoresis (comet) assay. This correlation suggests a temporal relationship between the two parameters, but does not provide direct evidence of what happens in individual cells. We developed a method to measure fluorescent markers of cellular structure or function with a laser scanning cytometer and then perform the comet assay on the same cells. Cells in each AV-binding subpopulation were re-examined before and after electrophoresis. Most AV-/PI- cells had no DNA damage, although a few cells showed a pattern of damage characteristic for apoptosis. Double-positive cells all had damaged DNA; approximately half had the apoptotic pattern, and the rest had a pattern typical for necrosis. Nearly all of the single-positive cells had damaged DNA with the apoptotic pattern. Both AV-positive populations contained cells with little or no detectable DNA after electrophoresis, indicating that the DNA was highly fragmented. These results indicate that AV binding is an excellent marker for apoptotic cells, but that these cells already have fragmented DNA.
用抗Fas受体抗体处理诱导凋亡的Jurkat白血病细胞有两个膜联蛋白V（AV）结合亚群：（a）结合AV但不结合碘化丙啶（PI）的单阳性细胞；（b）结合AV和PI的双阳性细胞。单阳性群体被认为代表凋亡的早期阶段。我们研究了AV结合与凋亡的一个经典特征——DNA片段化之间的关系。用抗Fas处理Jurkat细胞1、2或4小时的时间进程研究表明，2小时后AV结合细胞的比例增加。通过碱性单细胞凝胶电泳（彗星）试验测定的平均尾矩，仅在4小时时观察到DNA片段化显著增加。这种相关性表明这两个参数之间存在时间关系，但没有提供单个细胞中发生情况的直接证据。我们开发了一种方法，用激光扫描细胞仪测量细胞结构或功能的荧光标记，然后对同一细胞进行彗星试验。在电泳前后对每个AV结合亚群中的细胞进行重新检查。大多数AV-/PI-细胞没有DNA损伤，尽管有少数细胞显示出凋亡特征性损伤模式。双阳性细胞均有DNA损伤；约一半具有凋亡模式，其余具有坏死典型模式。几乎所有单阳性细胞都有凋亡模式的DNA损伤。两个AV阳性群体中都有电泳后几乎没有或没有可检测到DNA的细胞，表明DNA高度片段化。这些结果表明，AV结合是凋亡细胞的一个优秀标记，但这些细胞已经有了片段化的DNA。
