bryostatin-2 and Lung-Neoplasms

Activators of protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatins 1 and 2, inhibit the growth of A549 cells. At high concentrations the bryostatins do not affect cell growth. Here the hypothesis has been tested that modulation of A549 cell growth is the consequence of agent-induced changes in location or extent of cellular PKC activity. PKC activity was measured after semi-purification with nondenaturing polyacrylamide gel electrophoresis in the cytosol and the particulate fraction of A549 cells. When cells were exposed to TPA or mezerein, PKC activity underwent rapid and concentration-dependent translocation from the cytosol to the membrane. TPA at 0.1 microM or mezerein at 1 microM caused almost complete translocation within 30 min. Incubation with bryostatins 1 or 2 also led to enzyme translocation, which was, however, much weaker than that observed with the tumor promoters. Neither 4 alpha-phorboldidecanoate nor the synthetic diacylglycerols 1,2-sn-dioctanoylglycerol or 1-oleoyl-2-acetyl-sn-glycerol mimicked TPA in this way. Exposure of cells to TPA or the bryostatins for longer than 30 min caused the gradual disappearance of total cellular PKC activity. PKC downregulation was concentration dependent and complete after 24 h. A549 cells which had acquired temporary resistance toward the growth-arresting potential of TPA were completely devoid of any measurable PKC activity. The bryostatins were potent inhibitors of the binding of [3H]phorbol-12,13-dibutyrate to its receptors in intact cells, and the inhibition was dependent on bryostatin concentration. The results support the contention that PKC is involved in the mediation of growth inhibition caused by TPA or the bryostatins. However, the relationship between growth arrest and PKC translocation or downregulation seems to be a complex one.

Topics: Antineoplastic Agents; Bryostatins; Cell Line; Cytosol; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Membranes; Protein Kinase C; Tetradecanoylphorbol Acetate

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit the growth of A549 human lung carcinoma cells at non-toxic concentrations, whereas 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol, synthetic analogues of the physiological ligands of protein kinase C (PKC), do not. Experiments were conducted to test the hypothesis that other activators of PKC are capable of interfering with A549 cell growth. The non-phorboid tumour promotor mezerein mimicked the growth-inhibitory effect of TPA in that it arrested growth for 5 days, after which cells proliferated again in the continued presence of the agent. TPA was 20 times more potent as a growth inhibitor than was mezerein. Bryostatin 1 at 10 nM and bryostatin 2 at 100 nM also arrested A549 cell growth and inhibited DNA replication as measured by incorporation of [methyl-3H]-thymidine into cells. Inhibition of DNA synthesis to between 90 and 75% of control values developed during the first hour of incubation of the cells with TPA, mezerein or the bryostatins. The extent of inhibition changed little during the subsequent 5 hr of incubation, after which it increased further to reach maximal values within 12 hr. At concentrations above those which caused maximal growth inhibition, the bryostatins abolished both their own inhibition of DNA synthesis and the anti-replicative effect of TPA and mezerein. The results show that activators of PKC other than phorbol esters are capable of inhibiting the growth of A549 cells. The bryostatins not only interfere with A549 cell growth but can also counter the growth-inhibitory effect of PKC activators, presumably via interaction with a target separate from the phorbol ester receptor site.

Topics: Animals; Bryostatins; Cell Division; Diterpenes; DNA Replication; Enzyme Activation; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate; Time Factors