epidermal-growth-factor and bryostatin-1

The anti-proliferative activity of the DNA-interactive anti-cancer agent cis-diamminedichloroplatinum(II) (cDDP) can be modulated by intracellular signaling systems. We have investigated the effects of growth factors on the sensitivity of human cervical carcinoma (HeLa) cells to cDDP. A 24-hr pretreatment of HeLa cells with 10 ng/ml epidermal growth factor (EGF) or transforming growth factor-alpha increased the anti-proliferative activity of cDDP by 2- to 4-fold. A similar pretreatment of HeLa cells with EGF did not alter cellular sensitivity to doxorubicin or vincristine. A brief exposure (15 min) to growth factors was not sufficient for cDDP sensitization. EGF caused a modest and transient increase in cellular diacylglycerol, the endogenous activator of protein kinase C. Bryostatin I, a partial agonist of protein kinase C, antagonized phorbol ester-mediated cDDP sensitization but had no effect on EGF-mediated sensitization to cDDP. Both EGF and phorbol 12,13-dibutyrate (PDBu) enhanced the rate of [195mPt]cDDP uptake but had no effect on the rate of [195mPt]cDDP efflux in HeLa cells. Bryostatin I reversed the increase in [195mPt]cDDP content by PDBu but failed to block EGF-induced increase in [195mPt]cDDP accumulation. Therefore, although the mechanism of cDDP sensitization by both EGF and phorbol ester appears to involve enhanced drug uptake, they may utilize distinct signal transduction pathways.

Topics: Bryostatins; Cell Division; Cell Survival; Cisplatin; Diglycerides; Doxorubicin; Epidermal Growth Factor; HeLa Cells; Humans; Lactones; Macrolides; Protein Kinase C; Transforming Growth Factor alpha; Vincristine

The growth of human-derived A549 lung carcinoma cells is inhibited by activators of protein kinase C (PKC) such as 12-O-tetradecanoylphorbol- 13-acetate (TPA). In this study, the effect of serum deprivation on TPA-induced growth retardation has been investigated. Cells cultured with 10% FCS and TPA (10(-8) M) stopped growing for 6 days, whereas inhibition of DNA synthesis caused by TPA in cells which were grown in medium containing the serum substitute ultraser lasted for less than 48 hr. The ability of cells to respond to the growth-inhibitory potential of TPA decreased with decreasing amounts of FCS in the cellular medium. Addition of fetuin or epidermal growth factor (EGF) to incubates with serum-deprived cells increased the ability of TPA to affect growth, but addition of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) or retinoic acid (RA) was without effect. Growth arrest caused by bryostatin I, another PKC activator, was equally transitory in serum-supplemented and serum-deprived cells. Cytosol of serum-deprived cells contained only 32% of specific phorbol ester binding sites compared to cells grown with FCS; PKC enzyme activity and immunodectable protein were similarly reduced in cells grown without FCS. There was no difference in rate of TPA-induced down-regulation of PKC activity and cytosolic phorbol ester receptor sites between cells grown with or without serum.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Agents; Blood; Bryostatins; Cattle; Cell Division; Culture Media; DNA Replication; Enzyme Activation; Epidermal Growth Factor; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin

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

The bryostatins, a group of macrocyclic lactones isolated on the basis of their antineoplastic activity, protein kinase C in vitro and block phorbol ester binding to this enzyme. In some cellular systems, bryostatins mimic phorbol ester action. In other systems, however, the bryostatins display only marginal agonistic action and, instead, inhibit phorbol ester-induced responses. At least in primary mouse epidermal cells, a transient duration of action of bryostatin 1 could rationalize these differences. To determine whether this model of transient activation could explain the dual actions of bryostatin 1 in other cell systems, we have examined the effects of bryostatin 1 on short-term responses in C3H 10T1/2 mouse fibroblasts. Even at very short exposures (30 min), bryostatin 1 blocked phorbol ester-induced arachidonic acid metabolite release and induced only minimal release when assayed alone. In contrast, epidermal growth factor binding was markedly and rapidly decreased in bryostatin 1-treated C3H 10T1/2 cells, and this decrease showed only limited reversal 16 h after initial exposure. Bryostatins 2, 3, 4, 10, and several of their derivatives caused variable arachidonic acid metabolite release (10 to 60% of phorbol ester control) and correspondingly variable inhibition of phorbol ester action. Our findings on arachidonic acid metabolite release argue against transient activation of the protein kinase C pathway as the sole explanation of bryostatin 1 action. They indicate, moreover, differences in the structure-activity relations of the bryostatins for the phorbol ester-mimetic and phorbol ester-inhibitory actions.

Topics: Animals; Arachidonic Acids; Bryostatins; Calcimycin; Calcium; Cell Line; Epidermal Growth Factor; ErbB Receptors; Lactones; Macrolides; Mice; Phorbol Esters; Protein Kinase C; Structure-Activity Relationship; Time Factors

Bryostatin 1, a macrocyclic lactone, functions like the phorbol esters biochemically in binding to and activating protein kinase C. Biologically, however, although it induces some phorbol ester responses such as mitogenesis in Swiss 3T3 cells, it paradoxically blocks the effects of the phorbol esters on differentiation in HL-60 promyelocytic leukemia cells and Friend erythroleukemia cells. Since the phorbol esters induce proliferation and terminal differentiation in distinct subpopulations of epidermal basal cells, we have now examined the action of bryostatin 1 in that system. Bryostatin 1 decreased epidermal growth factor binding and induced ornithine decarboxylase activity, the latter a marker of proliferation. The magnitude of the maximal induction of ornithine decarboxylase was less than for phorbol 12,13-dibutyrate. Bryostatin 1 only transiently caused the morphological change typical of phorbol ester treatment and did not induce transglutaminase or cornified envelope production, markers of the differentiative pathway. Combined treatment with bryostatin 1 and phorbol 12,13-dibutyrate gave similar results to treatment with bryostatin 1 alone, i.e., slight reduction to complete inhibition of phorbol ester action, depending on the response. The mechanism may reflect time dependent block of the protein kinase C pathway by bryostatin 1 in this system; although bryostatin 1 inhibited epidermal growth factor binding at short incubation times (1-2 h), by 4 h of incubation its inhibition was markedly reduced and it correspondingly blocked inhibition of epidermal growth factor binding by phorbol 12,13-dibutyrate. Since induction of terminal differentiation is proposed to be an essential component of phorbol ester mediated tumor promotion in skin, our findings suggest that bryostatin 1 may function as an inhibitor of phorbol ester promotion.

Topics: Animals; Bryostatins; Cells, Cultured; Cycloheximide; Dactinomycin; Enzyme Activation; Epidermal Growth Factor; Epidermis; Lactones; Macrolides; Mice; Ornithine Decarboxylase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transglutaminases