APO2.7 DEFINES A SHARED APOPTOTIC-NECROTIC PATHWAY IN A BREAST-TUMOR HYPOXIA MODEL

A breast tumor hypoxia model used to simulate conditions which may exi st within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondria l membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma ce lls in tightly capped centrifuge tubes for up to 10 days. Cells were h arvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabiliz ation prior to staining) for all time points and processed cells (perm eabilized prior to staining) for only the first 24 h, Cell viability p robes trypan blue and anti-tubulin. antibody show ed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specifi c annexin V and DNA fragmentation by flow cytometry (range of 60-81% p ositive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% stain ing for all other methods tested. When APO2.7 antigen expression was e xamined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fe wer cells stained and cells showed fewer apoptotic features on an ultr astructural level than cells at the cell media interface. The study in dicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cel ls, traversing a heterogeneous pathway to cell death induced by hypoxi a. Cytometry 33:324-332, 1998, (C) 1998 Wiley-Liss, Inc.