APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model

Abstract

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested 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 permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive 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% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.