epidermal-growth-factor and phorbol-12-13-didecanoate

EGF has been reported to promote oocyte maturation in several species, although the mechanism of action is not yet known. The present study is designed to determine the pathway used by EGF to enhance porcine oocyte maturation. Oocytes were aspirated from 2-5 mm follicles and cultured with various treatments in Medium-199 at 37 degrees C, 100% relative humidity, and 5% CO2 for 48 hr for the maturation study and 3 hr for intracellular cAMP measurement. Although treatment with 100 IU/ml hCG stimulated both intracellular cAMP formation and oocyte maturation, 10 ng/ml EGF stimulated oocyte maturation only. Dibutyryl cAMP (dbcAMP) inhibited oocyte maturation at 10(-5), 10(-4), and 10(-3) M concentration s in the control medium. However, in the presence of 10 ng/ml EGF, dbcAMP inhibited oocyte maturation only at a concentration of 10(-3) M. Increasing concentrations of EGF (i.e., 25 and 50 ng/ml) were ineffective in overcoming the inhibitory effect of dbcAMP at 10(-3) M. In contrast, EGF reversed the decreased maturation rate caused by transforming growth factor-beta. Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, enhanced the spontaneous maturation rate; 4 alpha-phorbol dideconoate, an inactive phorbol ester, did not show this effect. PMA- and EGF-stimulated porcine oocyte maturation is reversed by calphostin-C, a PKC inhibitor. In conclusion, EGF's promotional activity on porcine oocyte maturation is independent of the cAMP pathway and probably mediated by the PKC pathway.

Topics: Animals; Bucladesine; Cyclic AMP; Dose-Response Relationship, Drug; Epidermal Growth Factor; Mice; Naphthalenes; Oocytes; Oogenesis; Phorbol Esters; Swine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

Primary cultures of 9-d-gestation Syrian hamster embryo (E9) cells are distinct from primary cultures of later gestational age in terms of their growth and differentiation. First, primary E9 cell cultures express multiple mesenchymal differentiation lineages (e.g., adipocyte, myoblast) only rarely seen in cultures of 13-d-gestation fetal (F13) cells. Second, although most primary E9 cultures have a limited in vitro proliferative life span and exhibit cellular senescence similar to primary cultures of F13 cells, E9 cultures seem to have higher frequency of escape from senescence and conversion to continuous cell lines compared to F13 cells. Moreover, this frequency can be further increased 4- to 5-fold by continuous exposure of the E9 cells to tumor promoters or epidermal growth factor. Eleven continuous cell lines have been isolated from untreated, promoter-treated, or epidermal growth factor-treated primary E9 cultures. Seven of these are neoplastic or preneoplastic. However, the remaining four do not show any evidence of being in neoplastic progression and three of these continue to express the same differentiated phenotype observed in ther parental primary cell cultures.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cricetinae; Embryo, Mammalian; Epidermal Growth Factor; Gestational Age; Mesocricetus; Neoplasms, Experimental; Phorbol Esters; Ploidies; Stem Cells; Tetradecanoylphorbol Acetate

The phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-didecanoate, which activate the enzyme protein kinase C, stimulated resorption in fetal rat long-bone cultures at concentrations of 1 and 10 microM. This effect appeared specific for active phorbol esters, since the inactive analogue 4-alpha-phorbol-12,13-didecanoate was without effect. The resorptive responses of fetal rat long-bone cultures to active phorbol esters differed from those previously described in newborn mouse calvaria cultures, since resorption stimulated by TPA in the rat long bones was not inhibited by either indomethacin (10 microM) or flufenamic acid (10 microM). However, calcitonin, an inhibitor of osteoclastic resorption, did decrease the response to TPA. There were some similarities between the response of fetal rat long-bone cultures to TPA and their response to epidermal growth factor (EGF). Like EGF, TPA stimulated DNA synthesis in the bones (measured as the incorporation of [3H]-thymidine) at concentrations below those necessary to stimulate resorption. TPA also did not stimulate resorption in the presence of aphidicolin (10 microM), an inhibitor of DNA synthesis that has been previously shown to block the resorptive response of these cultures to EGF. However, the responses of the cultures to TPA and EGF were not identical, since, unlike the effects of EGF, the stimulatory effects of TPA on DNA synthesis were biphasic. These results demonstrate that active phorbol esters stimulate bone resorption in fetal rat long-bone cultures through mechanisms that do not require prostaglandin synthesis but do appear to be mediated by osteoclasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aphidicolin; Bone and Bones; Bone Resorption; Calcium; Diterpenes; DNA Replication; Epidermal Growth Factor; Flufenamic Acid; Indomethacin; Organ Culture Techniques; Parathyroid Hormone; Phorbol Esters; Prostaglandins; Rats; Tetradecanoylphorbol Acetate; Thymidine

Cell cultures derived from human neonatal foreskins (HF cells) are susceptible to phorbol-12,13-didecanoate- (PDD) induced inhibition of queuine uptake, but this inhibition is pronounced only in early passage HF cells. The present analysis of five different primary cultures demonstrated that, between 10 and 30 population doublings beyond the primary cultures, HF cells gradually became refractile to PDD-induced inhibition of queuine uptake, after which PDD begins to stimulate queuine uptake. Treating late passage HF cells with conditioned medium from early passage HF cells partially restored the PDD-induced inhibition of queuine uptake. This indicates the existence of a factor produced by early passage HF cells that permits PDD to inhibit queuine uptake. The tumor promoter, teleocidin, mimics the effects of PDD on queuine uptake. Both PDD and teleocidin are known to activate protein kinase C; therefore, this kinase may be an intermediary in tumor promoter-induced effects on queuine uptake. Epidermal growth factor, platelet-derived growth factor, and transforming growth factor beta stimulated queuine uptake in both early and late passage HF cells. Growth factor stimulation of uptake was enhanced by PDD in late passage cells but inhibited by PDD in early passage cells. Polyinosinic polycytidylic acid treatment of late passage HF cells partially restored PDD-induced inhibition of queuine uptake. Human recombinant beta-interferon, plus or minus PDD, had no effect on queuine uptake. PDD did not inhibit queuine uptake in the immortal human and non-human cell lines examined.

Topics: Cells, Cultured; Epidermal Growth Factor; Fibroblasts; Guanine; Humans; Kinetics; Lyngbya Toxins; Peptides; Phorbol Esters; Platelet-Derived Growth Factor; Poly I-C; Transforming Growth Factors

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) caused partial inhibition of the binding of [125I]epidermal growth factor ([125I]EGF) to intact mouse epidermal cells (HEL/37). The proportion of [125I]EGF binding which was insensitive to TPA inhibition decreased with increasing EGF concentration. The partial inhibition of [125I]EGF binding was not due to destruction of TPA or to covalent linkage of EGF to receptor sites. The binding of [125I]EGF to HEL/37 cells showed a curvilinear Scatchard plot; this was converted into a linear plot in the presence of TPA. We conclude that TPA acts to inhibit interactions between EGF receptors or between EGF receptors and some other membrane component(s).

Topics: Animals; Carcinogens; Depression, Chemical; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Mice; Phorbol Esters; Protein Binding; Tetradecanoylphorbol Acetate