Li X, Traganos F, Darzynkiewicz Z
Cancer Research Institute, New York Medical College, Valhalla 10595.
Cancer Res. 1994 Aug 15;54(16):4289-93.
A new method is described which combines the identification of DNA replicating and apoptotic cells in a single measurement by flow cytometry. The detection of DNA replicating cells is based on incorporation of 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine, followed by selective photolysis at the site of incorporation of the halogenated DNA precursors. Single-strand breaks in DNA resulting from the photolysis are subsequently labeled with digoxygenin or biotin-conjugated dUTP in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The double-stranded DNA breaks in apoptotic cells resulting from activation of the endonuclease can be labeled in this reaction as well. However, in contrast to the photolysed DNA, the low molecular weight fraction of DNA of apoptotic cells is extractable from the cells, and the degree of DNA elution can be modulated by cross-linking with formaldehyde. Thus, apoptotic cells can be distinguished and quantified by virtue of their fractional DNA content. Replication of less than 1% of a genome of a cell in the presence of 5-bromo-2'-deoxyuridine (equivalent of a 5-min 10 microM 5-bromo-2'-deoxyuridine pulse) can be detected by the selective photolysis method. The method was applied to study apoptosis and proliferation of human leukemic HL-60 cells and normal, mitogen-stimulated lymphocytes. Whereas apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin was selective to DNA replicating cells, apoptosis induced by hyperthermia showed no such selectivity. Lymphocytes that preferentially underwent apoptosis in cultures stimulated by phytohemagglutinin did not initiate DNA replication. By offering the possibility for identification of both DNA replicating and apoptotic cells in a single measurement, the method may find an application in studies of the prognostic value of both cell proliferation and death in human tumors and the apoptotic response of DNA replicating vs. nonreplicating cells to different treatments.
本文描述了一种新方法，该方法通过流式细胞术在单次测量中结合鉴定DNA复制细胞和凋亡细胞。DNA复制细胞的检测基于5-溴-2'-脱氧尿苷或5-碘-2'-脱氧尿苷的掺入，随后在卤化DNA前体掺入位点进行选择性光解。光解产生的DNA单链断裂随后在外源末端脱氧核苷酸转移酶催化的反应中用洋地黄毒苷或生物素偶联的dUTP标记。凋亡细胞中由核酸内切酶激活导致的双链DNA断裂也可在此反应中标记。然而，与光解的DNA不同，凋亡细胞的低分子量DNA部分可从细胞中提取，并且DNA洗脱程度可通过与甲醛交联来调节。因此，凋亡细胞可根据其DNA含量分数进行区分和定量。在存在5-溴-2'-脱氧尿苷的情况下（相当于5分钟10 microM 5-溴-2'-脱氧尿苷脉冲），细胞基因组中小于1%的复制可通过选择性光解方法检测到。该方法应用于研究人类白血病HL-60细胞以及正常的、有丝分裂原刺激的淋巴细胞的凋亡和增殖。DNA拓扑异构酶I抑制剂喜树碱诱导的HL-60细胞凋亡对DNA复制细胞具有选择性，而热诱导的凋亡则无此选择性。在植物血凝素刺激的培养物中优先发生凋亡的淋巴细胞未启动DNA复制。通过提供在单次测量中鉴定DNA复制细胞和凋亡细胞的可能性，该方法可能在研究人类肿瘤中细胞增殖和死亡的预后价值以及DNA复制细胞与非复制细胞对不同治疗的凋亡反应方面找到应用。
