Flow cytometric analysis of apoptosis and bcl-2 in human solid neoplasms.
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.
Steck
K D
KD
Department of Pathology, M.D. Anderson Cancer Center, Houston, Texas, USA.
McDonnell
T J
TJ
el-Naggar
A K
AK
D017209:Apoptosis / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2