Protein phosphorylation is a key regulatory pathway for nutogenesis and apoptosis. The marine toxin, calyculin A (CAL A), is a potent SER and THR protein phosphatase (types 1 & 2A) inhibitor with potential antitumor activity. The cytotoxocity of CAL A was, therefore, investigated on a lung cancer cell line derived from human non-small cell carcinoma (201-T). CAL A induced a concentration- and time-dependent detachment of cells from monolayer cultures measured by méthylène blue binding. CAL A ( 1 nM) produced approximately 50% and 100% cell detachment by 4 hrs and 8 hrs, respectively, in sparsely plated cells under serum-free conditions, hi contrast,' confluent cells showed no cell loss 8 hrs after treatment and 40% of the cells remained after 24 hrs of drug exposure. CAL A-induced cell detachment could be differentially modulated by serum-derived factors. Fetal bovine serum (FBS) (10%) delayed drug-induced detachment, while 1% FBS potentiated CAL A effects. Subsequent to detachment lung cancer cells stained with Hoescht 33342 showed cell shrinkage, chromatin condensation and nuclear fragmentation. While 180-200 bp internucleosomal DNA fragments typical of apoptosis in some cells were not observed, higher molecular weight (approximately 50 kb) DNA fragments were seen 24 hrs after 1 nM CAL A by field inversion gel electrophoresis. These data show that CAL A induces apoptosis in lung cancer cells. This process is preceded by cell detachment that can be modulated by cell-cell interactions and unidentified serum-derived factors.
|Original language||English (US)|
|State||Published - Dec 1 1996|