Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis

Two immortalized cell lines, sup (+) and sup (-), derived from mutagenized Syrian hamster embryo cells, were used to study the relationship and tempor al order between calcium and ceramide signals during apoptosis. The early p reneoplastic cells, termed sup (+), suppress tumorigenicity when hybridized with tumor cells, whereas later-stage sup (-) cells do not. In reduced ser um conditions, sup (+) cells cease proliferating and undergo apoptosis; in contrast, sup (-) cells continue slow growth and undergo necrosis. In sup ( +) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum treatment of sup (+) cells but are n ot found in sup (-) cells. Inhibiting ER calcium depletion in low serum-tre ated sup (+) cells by treating with high levels of calcium prevents both ce ramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup (-) cells by treating with low serum plus thapsigargin results in el evated ceramide levels and apoptosis. Furthermore, C-6-ceramide treatment i nduced apoptosis of sup (-) cells in low serum, a condition that does not n ormally cause apoptosis. C-6-ceramide treatment did not induce apoptosis in either sup (+) or sup (-) cells in 10% serum but did cause G(2)/M arrest. These studies show that ceramide production is downstream of ER calcium rel ease.