A novel non-spreading variant of transformed hamster fibroblasts

Abu-Zayyad, Amineh Naji (1997) A novel non-spreading variant of transformed hamster fibroblasts. PhD thesis, University of Glasgow.

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Abstract

Using repeated plating on fibronectin-coated surfaces as a selection procedure, I isolated three different mutants unresponsive to fibronectin, from thioguanine resistant Py-BHK (TG) cells in a simple assay of cell spreading. All three were recloned on soft agar. Two had morphologies similar to lines which had been selected previously, FI and F2. The mutant F3, then a unique isolate, had a different morphology in culture. Whereas F1 colonies contain exclusively rounded cells, and F2 have a few partially spread cells, more scattered than F1, F3 has a spread epithelial-like morphology in culture. I selected F3 from TG cells, on the basis that this mutant is non- responsive to fibronectin. However, in spreading assays, I found that F3 spreads on fibronectin, when the simple saline in the assay (HEPES-buffered Hanks') is supplemented with foetal bovine serum, or replaced by Ham's F10. Unexpectedly, I found that the active component stimulating spreading of F3 was pyruvate. To explain the pyruvate requirement, I searched for an abnormality of glucose utilisation in F3. Glucose uptake, studied using 14C- labelled glucose, suggested there could be some such abnormality. However, separating metabolites from TG and F3 by one and two dimensional paper chromatography gave inconclusive results. The activity of mitochondrial dehydrogenases of TG and F3, measured using MTT, responded similarly to glucose, suggesting that pyruvate in F3 is more likely needed to supply metabolites than as an energy source. The possible identity of such metabolites and explanation for how F3 came to be selected, are discussed.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Adam Curtis
Keywords: Genetics
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-71296
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71296

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