Cytosine arabinoside induces programmed endothelial cell death through the caspase-3 pathway.

The anti-cancer effects of cytosine arabinoside (ARA-C) are well known. However, effects on nonmalignant cells have not been elucidated and may be important to understanding treatment-related toxicity. The purpose of this study was to examine the effect of ARA-C on nondividing vascular endothelial cells. The objectives were to determine the effects of ARA-C on cell viability and to ascertain whether ARA-C caused apoptosis in cultured vascular endothelial cells and hydrocortisone blunted caspase-3-induced apoptosis. Endothelial cells were cultured until confluent and mitotically quiescent then exposed to ARA-C (10(-7)to 10(-3) M) for 1 to 4 days. Some experiments involved cotreatment with hydrocortisone (10(-11),10(-10),10(-4), and 10(-3) M). Light microscopy and the colorimetric MTS assay were used to measure viability. Fluorescent annexin-V and DNA fragmentation assays were used to measure apoptosis, and a fluorescence-based enzymatic assay was used to measure caspase-3 activity, which is one pathway involved in the apoptosis cascade. Two-way ANOVA or the appropriate nonparametric test was used to determine statistical significance in studies of viability and apoptosis. Oneway ANOVA was used to determine statistical significance for caspase-3 activity. Viability was decreased with higher concentrations of ARA-C and increased days of treatment. The percentage of apoptotic cells increased with higher concentrations of ARA-C and increased days of treatment. ARA-C-treated samples showed DNA fragmentation, indicative of apoptosis. Caspase-3 activity increased after ARA-C addition; hydrocortisone blunted this increase. ARA-C caused apoptosis in nondividing endothelial cells in culture. Hydrocortisone may protect against ARA-C-induced apoptosis by reducing caspase-3 activity.

In vitro age-related responses of endothelial cells to breast cancer cell addition.

AIM: The purpose of this study was to determine if the in vitro age of endothelial cells alters endothelial response(s) to breast cancer cells. METHOD: After characterizing lower passage ("young"; passages 10-16) and higher passage ("old"; passages 30-36) bovine pulmonary artery endothelial cells (BPAECs), fluorescently labeled MCF-7 breast cancer cells were added to confluent monolayers of young and old BPAECs. RESULTS: Transient gaps that peaked in size by 12 h and closed by 48h occurred between the young BPAECs, while large persistent gaps formed between the old BPAECs. Gap formation did not occur when 184A1 cells, a non-malignant mammary epithelial cell line, were added in place of MCF-7 cells, suggesting that the age-related responses of the endothelial cells to MCF-7 cell addition were specific to the tumor cell addition. Additionally, more MCF-7 cells migrated through old BPAEC monolayers, than young BPAEC monolayers, grown on Matrigel-coated filters. Finally, DNA fragmentation and fluorescent annexin-V binding assays suggested increased MCF-7 cell-induced apoptosis in older BPAECs, though results from a caspase-3 activation assay were equivocal. CONCLUSIONS: In sum, our findings support the notion that aged endothelial cells are more susceptible to breast cancer-induced injury, perhaps due to increased apoptosis.