phorbol-12-13-didecanoate and Adenocarcinoma

By understanding the signal transduction pathways through which a cell responds to changes in environmental oxygen levels, we may be able to therapeutically exploit this response by manipulating these pathways.. The human adenocarcinoma cell line A549 was exposed to varying durations of hypoxia alone and then plated for survival, or treated with PKC activating agents for 1 h before plating for survival. Western blots were used to determine the kinetics of PKC epsilon and phospholipase C induction.. The level of hypoxic killing was directly related to the time of exposure and inversely related to the level of oxygen in the environment. Exposure of the cells to protein kinase C (PKC) activators for 1 h after chronic hypoxic exposure increased cell killing by at least an additional three logs beyond that found for hypoxia alone. Treatment of cells with an inactive phorbol ester 4 alpha-phorbol-12,13-didecanoate (PDA) resulted in no increase in hypoxic cell killing, even at the highest concentrations of PDA which produced no detectable toxicity under normal aerobic conditions. Using inhibitors of phospholipases A2 and C, we were able to completely inhibit the additional hypoxic cell killing induced by TPA, but not the uninduced hypoxic cell killing.. These studies suggest that accumulation of phospholipid breakdown products may be responsible for TPA induced cell killing, and that hypoxic cells differ from aerobic cells in their ability to tolerate these products.

Topics: Adenocarcinoma; Cell Hypoxia; Cell Survival; Enzyme Activation; Humans; Phorbol Esters; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Calcyclin is a member of the S-100 family of calcium-binding proteins, whose expression is enhanced when quiescent cells are exposed to mitogenic signals. The function of calcyclin is unknown, but it is thought to be involved in modulating the intracellular calcium concentration following mitogenic stimuli. Since activation of protein kinase C (PKC) also occurs following stimulation of quiescent cells by a variety of mitogens, we have investigated the relationship between calcyclin expression and PKC activation in three human endometrial adenocarcinoma cell lines. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cell cultures resulted in a change in cell morphology, an inhibition of proliferation, an increase in calcyclin transcription rate, and an increase in calcyclin mRNA and calcyclin protein levels. In contrast, PMA had no effect on cell morphology or cell proliferation in the Ishikawa adenocarcinoma cell line but enhanced calcyclin expression. Another bioactive phorbol ester had the same effect, whereas the calcium ionophore A23187 and the non-phorbol-ester-type tumor promoter thapsigargin had no effect on calcyclin expression. The effect of PMA on calcyclin expression was blocked by the simultaneous addition of the PKC inhibitor staurosporine and by protein synthesis inhibition with cycloheximide. RNase protection assays and primer extension analysis demonstrated that PMA enhanced transcription from all three of the previously identified transcription start sites in the calcyclin gene. These data clearly demonstrate a dissociation between calcyclin expression and cellular proliferation and suggest that the enhanced calcyclin expression which is seen in quiescent cells following mitogenic stimuli may result from activation of the PKC system.

Topics: Adenocarcinoma; Alkaloids; Calcimycin; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Division; Cycloheximide; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein A6; S100 Proteins; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Uterine Neoplasms