Growth factors are polypeptides that initiate mitogenesis in quiescent fibroblasts and other cell types. Ultimate control of DNA synthesis initiated by growth factors must occur in the nucleus, probably by specific but as yet unknown gene products. However, because growth factors are initially received by specific receptors at the cell surface, it may reasonably be hypothesized that cytoplasmic signals are necessary as informational intermediaries between plasma membrane and nucleus. The proposed experiments address the possible role of cytoplasmic Ca++ and pH as cytoplasmic signals for growth factors and certain cell cycle events. Intracellular free calcium ion concentration ([Ca++]i) will be measured using the calcium sensitive photoprotein, aequorin; intracellular pH (pHi) using the fluorescein fluorescence ratio technique. Moreover, several techniques for loading otherwise impermeant molecules into living cell cytoplasm will be put to novel experimental use, and a new technique for bulk-loading will be developed. Some of the questions asked are: 1) is a rise in [Ca++]i necessary and/or sufficient for initiating DNA synthesis; 2) can DNA synthesis be synergistically activated in quiescent cells by raising [Ca++]i and activating protein kinase C; 3) does inositol 1,4,5-triphosphate (IP3) release Ca++ from internal stores of living, quiescent fibroblasts and is IP3 mitogenic; 4) what are the intracellular sites of growth factor- and IP3-induced changes in [Ca++]i; 5) do changes in [Ca++]i and pHi occur during fibroblast transit through the cell cycle and, if so, how do they compare in timing; 6) do experimentally induced rises in pHi result in initiation of DNA synthesis by quiescent cells; and 7) are there differences between resting and/or growth factor-stimulated [Ca++]i and pHi of normal versus transformed fibroblasts. The proposed studies address problems fundamental to understanding the mitogenic response of mammalian cells, including the chemical identity of possible cytoplasmic signals for mitogenesis, the concentrations involved and location in the cytoplasm, whether or not these putative signals are sufficient and/or necessary by themselves for mitogenesis, and activity of these signal molecules in transformed cells not responsive to growth factors.

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National Cancer Institute (NCI)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Harvard University
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Paimela, H; Goddard, P J; Carter, K et al. (1993) Restitution of frog gastric mucosa in vitro: effect of basic fibroblast growth factor. Gastroenterology 104:1337-45
Clarke, M S; Khakee, R; McNeil, P L (1993) Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle. J Cell Sci 106 ( Pt 1):121-33
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Clarke, M S; McNeil, P L (1992) Syringe loading introduces macromolecules into living mammalian cell cytosol. J Cell Sci 102 ( Pt 3):533-41
McNeil, P L; Khakee, R (1992) Disruptions of muscle fiber plasma membranes. Role in exercise-induced damage. Am J Pathol 140:1097-109
Muthukrishnan, L; Warder, E; McNeil, P L (1991) Basic fibroblast growth factor is efficiently released from a cytolsolic storage site through plasma membrane disruptions of endothelial cells. J Cell Physiol 148:1-16
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McNeil, P L; Ito, S (1989) Gastrointestinal cell plasma membrane wounding and resealing in vivo. Gastroenterology 96:1238-48
Swanson, J A; McNeil, P L (1987) Nuclear reassembly excludes large macromolecules. Science 238:548-50

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