bryostatin-1 and Pituitary-Neoplasms

The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate [TPA) or phorbol 12-myristate 13-acetate), directly activates the calcium- and phospholipid-dependent protein kinase C (protein kinase C), which, in turn, generates a number of cellular responses. The bryostatins, a family of macrocyclic lactones isolated from marine bryozoans, also bind to and active protein kinase C. However, they differ from TPA in the selectivity of their responses in that they behave either as agonists or antagonists of protein kinase C actions. We used several bryostatins and TPA to examine the role of protein kinase C in the regulation of GH4C1 rat pituitary tumor cell proliferation. TPA inhibited [3H]thymidine incorporation in GH4 cells in a stereoselective and concentration-dependent manner. Examination of cell cycle distribution by flow cytometry revealed that TPA decreased the percentage of cells in S-phase and proportionally increased the percentage of G1-phase cells. Bryostatin 1 alone did not affect cell proliferation, but prevented the TPA inhibition of cell proliferation. Bryostatin 1 treatment from 30 min to 6 h after TPA treatment also prevented the growth-inhibitory action of TPA, suggesting that prolonged stimulation of protein kinase C is necessary for growth inhibition. Both bryostatin 1 and TPA down-regulated protein kinase C, indicating that down regulation of the enzyme cannot account for the growth inhibitory action of TPA. Bryostatin 2, which differs from bryostatin 1 by a hydroxyl substitution for the acetyl group at the C-7 carbon of the macrocyclic lactone ring (R1), inhibited cell proliferation and did not reduce the growth-inhibitory action of TPA. Bryostatins 3 and 8 (each of which has an ester group in the R1 position, yet contains other structural modifications) are antagonists for TPA inhibition of GH4 cell proliferation like bryostatin 1. We next examined the effect of bryostatins 3 and 8 on cell-substratum adhesion, a cellular response observed after GH4 cells are treated with growth-inhibitory agents. Bryostatin 8 (like bryostatin 1) did not enhance cell-substratum adhesion and blocked the action of TPA. In contrast, bryostatin 3 enhanced cell-substratum adhesion. Because bryostatin 3 blocked TPA inhibition of cell proliferation, yet did not block TPA-enhanced cell-substratum adhesion, these responses are not interdependent. We next examined the effect of bryostatin on other growth-inhibitory agents for GH4 cells. Bryostatin 8 blo

Topics: Animals; Antineoplastic Agents; Bryostatins; Cell Adhesion; Cell Cycle; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; Kinetics; Lactones; Macrolides; Pituitary Neoplasms; Protein Kinase C; Rats; Structure-Activity Relationship; Tetradecanoylphorbol Acetate; Thymidine; Thyrotropin-Releasing Hormone

Phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol acetate (TPA) activate the calcium- and phospholipid-dependent protein kinase C and enhance three biological responses (prolactin release, prolactin synthesis, and cell stretching) in GH4C5 rat pituitary cells. We have examined several actions on GH4C5 cells of TPA and two other classes of protein kinase C activators, synthetic cell permeant dioleins and bryostatins isolated from the marine bryozoan Bugula neritina. Bryostatins 1 and 2 (B1 and B2, respectively) competed for [3H]phorbol 12,13-dibutyrate binding to the protein kinase C complex in intact cells nearly equipotently with TPA. B1 and B2, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoylglycerol (Di8) as well as TPA each activated partially purified protein kinase C from GH4C5 cells. B1, B2, and TPA each enhanced the acute release of prolactin from GH4C5 cells to a similar maximal extent. B1, B2, and TPA also enhanced prolactin synthesis. However, B1 and B2 were only partial agonists because they enhanced prolactin synthesis to a lesser maximal extent than did TPA and, given in combination, they reduced TPA-enhanced prolactin synthesis. OAG and Di8 stimulated prolactin release (to a lesser maximal extent than TPA) and did not stimulate prolactin synthesis. Pretreatment with OAG did not reduce TPA-stimulated prolactin release or synthesis. B2 and TPA induced cell stretching in GH4C5 cells, whereas B1, OAG, and Di8 induced little if any stretching. B1, but not B2, given in combination with TPA antagonized TPA-induced stretching but did not reduce thyrotropin-releasing hormone- or epidermal growth factor-induced stretching. We conclude that the bryostatins, phorbol esters, and dioleins bind to the same site on the protein kinase C complex to activate the enzyme, but they alter three biological responses in GH4C5 cells with selectivities and efficacies that differ. We propose that different activators of protein kinase C (such as bryostatins, dioleins, and phorbol esters) may elicit different cellular responses by altering the substrate specificity or activating multiple forms of the kinase.

Topics: Animals; Binding Sites; Binding, Competitive; Bryostatins; Cell Line; Diglycerides; Enzyme Activation; Eukaryota; Glycerides; Kinetics; Lactones; Macrolides; Phorbol 12,13-Dibutyrate; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Tetradecanoylphorbol Acetate