The object of the proposed studies is to examine in detail the biochemical and molecular mechanisms underlying the differentiation of epithelial cells. Mammalian keratinocyte cultures are dependent on extracellular calcium (Ca++) levels with respect to degree of differentiation. Keratinocytes in low Ca++ medium (O.O3mM) grow as a proliferative relatively undifferentiated monolayer. Elevation of medium Ca2+ to physiologic level ( 1.OmM) induces differentiation of formation of a stratified squamous epithelium. Well-defined morphologic and biochemical criteria serve as markers of differentiation in these in vitro systems, but the controlling factors in the differentiative program are unknown. All work will employ cultures of mammalian keratinocytes. The early (Phase I) studies will entail: 1) further investigation of the control of cellular ornithine decarboxylase (ODC) activity by extracellular Ca2+ concentration, which we have previously described, and 2) preliminary examination of the mechanisms by which Ca++ levels control differentiation. ODC activity is stimulated by sequential deprivation and restoration of extracellular Ca++ and will be assayed in cytosolic preparations. The point along the biosynthetic and activation pathway ODC at which Ca++ is regulatory will be assessed, as will the possible involvement of the Ca++ dependent regulator proteins, calmodulin and C-kinase. Ca++ induced differentiation of keratinocytes will be examined. Using DNA probles, the level of expression of various oncogenes, before and after Ca++ induced differentiation, will be assessed. The longer term (Phase II) investigations involve application of recombinant DNA technology to the study of Ca2+ induced differentiation in cultured human epidermal keratinocytes. The objective of these studies is to identify mRNA species which are expressed at different levels in undifferentiated and differentiated cultures. This problem will be approached in 2 ways: 1) Respective cDNA libraries will be constructed from cytoplasmic poly (A)+ RNA present in undifferentiated and differentiated cultures and will be cloned in bacteriophage Lambda vectors. The libraries will be screened for cDNA elements which hybridize to mRNA species whose abundance differs in undifferentiated and differentiated cells. 2) High molecular weight cDNA derived from differentiated cultures will be exhaustively hybridized with total poly(A)+ RNA from undifferentiated cultures. Unhybridized cDNA, representing a fraction of genomic elements enriched for differentially expressed species, will be cloned in pBR322. Clones will be screened as above.
| Langdon, R C; Cuono, C B; Birchall, N et al. (1988) Reconstitution of structure and cell function in human skin grafts derived from cryopreserved allogeneic dermis and autologous cultured keratinocytes. J Invest Dermatol 91:478-85 |
