Steck K D, McDonnell T J, el-Naggar A K
Department of Pathology, M.D. Anderson Cancer Center, Houston, Texas, USA.
Cytometry. 1995 Jun 1;20(2):154-61. doi: 10.1002/cyto.990200208.
Apoptosis (programmed cell death) and the genes regulating this process (e.g., bcl-2), have recently become a focus of interest in the study of cancer development and progression. We adapted flow cytometric techniques for measuring apoptosis and bcl-2 protein in solid tissues and concomitantly determined both parameters in 65 malignant solid neoplasms. Four different fixation methods were evaluated for bcl-2 analysis using a lymphoma cell line possessing a t(14;18), and the Daudi cell line as positive and negative controls, respectively; optimal fixation was achieved using 70% ethanol. Apoptosis was determined using the terminal deoxynucleotidyl transferase (TdT) end-labeling method of (Gorczyca et al.: Cancer Res 53:1945-1951, 1993). Treated (5Gy radiation, 4 h) and untreated portions of a murine cell line were used as positive and negative controls for apoptosis induction. For tumor specimens, bcl-2 positivity ranged from 0 to 89.5% (15.8 +/- 22.9), and apoptosis (TdT end-labeling) ranged from 0.4 to 84.3% (15.9 +/- 17.0). Generally, tumors with high bcl-2 expression (> or = 20.0%) showed significantly lower numbers of apoptotic cells than those with low (< 20.0%) bcl-2 (P = 0.05). A subset of tumors, however, exhibited low values for both parameters. We also observed that the proliferative fractions of tumors with high apoptosis (> or = 15.0%) were significantly different from those with low (< 15.0%) apoptosis (P = 0.005); higher proliferative rates were associated with high apoptosis. We conclude that optimal bcl-2 analysis is achieved using ethanol fixation and that flow cytometry provides a rapid and reliable technique for the measurement of these parameters. Concurrent analysis of these markers provides detailed information on the biological characteristics of tumor subpopulations and may assist in categorizing tumors for different management strategies.
细胞凋亡（程序性细胞死亡）以及调控这一过程的基因（如bcl-2），最近已成为癌症发生和发展研究中的一个关注焦点。我们采用流式细胞术来检测实体组织中的细胞凋亡和bcl-2蛋白，并同时测定了65例恶性实体肿瘤中的这两个参数。使用一株具有t(14;18)的淋巴瘤细胞系评估了四种不同的固定方法用于bcl-2分析，分别以Daudi细胞系作为阳性和阴性对照；使用70%乙醇可实现最佳固定。采用末端脱氧核苷酸转移酶（TdT）末端标记法（Gorczyca等人：《癌症研究》53:1945 - 1951, 1993）测定细胞凋亡。将一株鼠细胞系经处理（5Gy辐射，4小时）和未处理的部分分别用作诱导细胞凋亡的阳性和阴性对照。对于肿瘤标本，bcl-2阳性率范围为0至89.5%（15.8±22.9），细胞凋亡（TdT末端标记）范围为0.4至84.3%（15.9±17.0）。一般来说，bcl-2高表达（≥20.0%）的肿瘤显示凋亡细胞数量明显低于bcl-2低表达（<20.0%）的肿瘤（P = 0.05）。然而，有一部分肿瘤这两个参数的值都很低。我们还观察到，细胞凋亡率高（≥15.0%）的肿瘤增殖分数与细胞凋亡率低（<15.0%）的肿瘤有显著差异（P = 0.005）；较高的增殖率与高细胞凋亡率相关。我们得出结论，使用乙醇固定可实现最佳的bcl-2分析，并且流式细胞术为测量这些参数提供了一种快速且可靠的技术。同时分析这些标志物可提供关于肿瘤亚群生物学特性的详细信息，并可能有助于对肿瘤进行分类以制定不同的管理策略。
