Hall, Peter Anthony; Proliferative and differentiative heterogeneity in epithelial cell populations. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

This thesis will describe studies of cellular proliferation and differentiation in two contrasting epithelial systems. Epidermal keratinocytes have been investigated as a model of a continually renewing tissue. It is shown that functionally distinct sub-populations can be defined using a colony morphology assay. These sub-populations have differing proliferative capacities and probabilities of terminal differentiation. The relative proportions of the colony subtypes can be altered by treatment with phorbol esters or by suspension culture. These procedures induce differentiation and allow the enrichment of those cells that have the highest proliferative capacity and lowest probability of differentiation. The sub-populations have differing adhesive properties and the most adhesive cells have the highest proliferative capacity. It is demonstrated that the most adhesive cells include the population resistant to differentiation induction by phorbol ester or suspension culture. Cultured keratinocytes have been employed in an experimental test of Cairns immortal strand hypothesis. Keratinocytes have been labelled with tritiated thymidine and autoradiography used in an attempt to identify asymmetric segregation of labelled DNA. While such asymmetric daughter pairs have been identified these may represent artefacts thus no support for the immortal strand hypothesis has been obtained. The clonal architecture of keratinocytes has been investigated using mixtures of male and female cells grown in nude mice in which Y chromosome specific sequences were demonstrated by in-situ hybridisation. Retroviral lineage markers have also been employed as independent markers of clonal architecture. Both these methods have proven difficult to interpret but do not conflict with Potten's notion of the epidermal proliferative unit. The exocrine pancreas has been used as a model of a conditionally renewing epithelial population. Cell proliferation in the pancreas has been assessed by pulse labelling and by immunohistochemical methods. Clonal architecture has been investigated using XX/XY chimaeras probed by in situ-hybridisation and pancreatic islets have been shown to be polyclonal. Immunophenotypic markers of cellular differentiation have been characterised and employed in developing and adult pancreas, and in pathological tissues. A novel pattern of differentiation has been identified in areas of ductal epithelial damage using immunohistochemistry and in-situ hybridisation for mRNA. The immunophenotypic markers have been employed in two in vitro models. First, a human pancreas culture system has been developed. Pancreatic acinar cells transdifferentiate rapidly to a duct phenotype indicating considerable phenotypic plasticity. Second, a series of human pancreatic carcinoma cell lines have been studied and their phenotypes defined. It is shown that some cell lines can respond to culture in extracellular matrix by forming duct like structures. This property correlates with the state of differentiation of the cell lines, including their integrin profile. The phenomenon is inhibited in part by exogenous RGD peptide indicative of a role for integrins in this process. Finally, it is shown that the cell line PSN-1 can respond to growth as a nude mouse xenograft by showing immunophenotypic evidence of endocrine differentiation: direct experimental support for the Unitarian origin of pancreatic epithelial populations.

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