DISCRIMINATION OF LATE APOPTOTIC NECROTIC CELLS (TYPE-III) BY FLOW-CYTOMETRY IN SOLID TUMORS/

A method is described for the discrimination of Type III, late apoptot ic, and necrotic cells, to improve the accuracy of proliferation and p loidy determinations of breast tumors, We selected an immunological pr obe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, la te apoptotic, and necrotic tells, This study utilized MDA-MB-175-VII b reast carcinoma cells deprived of oxygen for up to 11 d to simulate in tratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means, After 24 h under hypoxic conditions, the MDA cells displayed characteristic s associated with both apoptosis and necrosis, Approximately 50% of da y 1 cells showed membrane permeability by trypan blue and absence of D NA laddering; however, by day 3-4 characteristic apoptotic DNA ladderi ng by gel electrophoresis was evident. Substantial DNA content loss, f urther evidenced by a reduction in PI staining and fluorescent microsc opy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were po sitive for antitubulin antibody staining, When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showe d a range of 8%-74% with a median value of 11.5%. This study demonstra tes that a large percentage of cells in tumors and hypoxic cell popula tions have significantly reduced DNA content, such that conventional l ive/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investiga tions. (C) 1997 Wiley-Liss, Inc.