Millar, Jonathan B. A.; (1990) Regulation of mitogenetic signal transduction: Studies with peptides of the bombesin family. Doctoral thesis (Ph.D.), University College London.

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Abstract

Quiescent cultures of Swiss 3T3 cells have been used as a model system for elucidating the mechanisms of growth factor-induced cell proliferation. The amphibian tetradecapeptide bombesin and structurally related mammalian peptides, including gastrin releasing peptide (GRP), are potent mitogens for these cells in culture in serum free medium. The results in this thesis demonstrate that bombesin and related peptides induce the release of arachidonic acid and its cyclooxygenase product prostaglandin E2- This release is both dose- and time-dependent and mediated through the same single high affinity receptor previously described to mediate early signal transduction and mitogenic stimulation by bombesin. The neuropeptide vasopressin, which induces a similar array of early responses to bombesin such as monovalent ion fluxes, inositol polyphosphate production, Ca2+ mobilisation and activation of protein kinase C, does not induce the release of either arachidonic acid or prostaglandin E2 from quiescent cultures of Swiss 3T3 cells, and is unable to stimulate DNA synthesis in the absence of other growth promoting agents. Elevation of cAMP levels is a growth promoting signal for Swiss 3T3 cells in culture. Bombesin and structurally related mammalian peptides enhance the accumulation of cAMP in the presence of a variety of cAMP elevating agents. Enhancement of cAMP accumulation by bombesin is both dose- and time-dependent and mediated by a dual mechanism of action; through the release of E type prostaglandins and through activation of protein kinase C. Indomethacin, a specific cyclooxygenase inhibitor, abolishes E type prostaglandin production and partially attenuates enhancement of cAMP accumulation and DNA synthesis induced by bombesin without affecting Ca2+ mobilisation or activation of protein kinase C by this peptide. Pertussis toxin ADP-ribosylates a certain class of guanine nucleotide binding protein, but does not affect either the number or affinity of bombesin receptors or activation of phospholipase C or protein kinase C by bombesin in Swiss 3T3 cells. The results in this thesis demonstrate that pertussis toxin profoundly and selectively inhibits the mitogenic response to bombesin and structurally related peptides. Pertussis toxin also inhibits release of arachidonic acid, prostaglandin E2, enhancement of cAMP accumulation and mitogenic stimulation by bombesin in a similar dose-dependent manner. Cellular desensitization, an important and widely occuring mechanism of response regulation to external stimuli, has been investigated. The results in this thesis reveal that the mitogenic response to bombesin in Swiss 3T3 cells is sensitive to two distinct desensitization processes. Prolonged exposure of these cells to bombesin induces homologous desensitization to both early signal transduction and reinitiation of DNA synthesis, by progressive down-regulation of cell surface bombesin receptors. Down-regulation of receptors and homologous desensitization are induced by peptides structurally related to bombesin in an identical dose-dependent and reversible manner, and blocked by the specific bombesin antagonist [Leu13-(CH2NH)-Leu14]bombesin. Prolonged exposure of Swiss 3T3 cells to vasopressin induces heterologous mitogenic desensitization to peptides of the bombesin family. Onset of heterologous desensitization requires at least 10 hours exposure to vasopressin and is induced by various vasopressin agonists in a dose- dependent and reversible manner. Pretreatment with vasopressin does not alter the number, affinity or rate of internalisation of the bombesin receptor as measured by binding of 125I-GRP. Bombesin stimulates inositol polyphosphate production, mobilisation of intracellular Ca2+ and activation of protein kinase C but not arachidonic acid or prostaglandin E2 release in mitogenically desensitized cultures. Vasopressin-induced loss of mitogenic stimulation and release of prostaglandin E2 to bombesin requires on-going protein synthesis and is blocked by a specific V1-type vasopressin antagonist. These results provide compelling evidence for a role for arachidonic acid and its metabolites in mitogenic signal transduction by peptides of the bombesin family.

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