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 blocks the effect of TPA on [3H]
thymidine incorporation and the entry of G1 cells into S-phase, but does not block the growth-inhibitory action of
thyrotropin-releasing hormone or
epidermal growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)