Variation of enzyme activities in cultured Chinese hamster Kupffer cells

Clark, Julian Maxwell (1976) Variation of enzyme activities in cultured Chinese hamster Kupffer cells. PhD thesis, University of Glasgow.

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The purpose of this study was to examine the variation of enzyme activities between cell lines which were either adult, foetal or Simian Virus 40 (SV40) transformed and possessing a similar genetic and epigenetic background. Utilizing Kupffer cells from the Chinese hamster (Cricetulus griseus), it was possible to isolate cell lines which expressed Kupffer cell functions and survived at least 70 population doublings in culture. A large number of cell lines, each originating from a single Kupffer cell, could be isolated from a single animal. Three adult siblings provided material for the isolation of 130 primary adult Kupffer cell lines and one mid-term foetus was used to initiate 24 primary foetal Kupffer cell lines. At various stages in culture the Kupffer cell lines were assayed for the following enzyme activities: catalase, arginase, microsomal haem oxygenase, beta-glucuronidase, peroxidase, alcohol dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. The following points emerged from a study of the primary Kupffer cell lines. (a) Enzyme activities rapidly declined when Kupffer cells were cultured. (b) After 26 population doublings for primary foetal and 40 population doublings for primary adult cell lines, all enzyme activities were stable until at least the stage of 70 population doublings. The enzyme activities were less in the primary foetal cell lines. (c) Each enzyme activity demonstrated highly significant variation between the cell lines. The variation was greatest between the primary adult cell lines. Both primary adult and primary foetal Kupffer cell lines were transformed by SV40 and assayed for the above enzyme activities. Transformation of 65 primary adult Kupffer cell lines by SV40 resulted in the loss of Kupffer cell functions and all enzyme activities, with the exception of beta-glucuronidase and glucose-6-phosphate dehydrogenase were rapidly reduced to a fraction of those observed in primary adult Kupffer cell lines. The method of transformation resulted in the emergence of the transformed phenotype within a few cell divisions after infection with SV40. All enzyme activities in SV40-transformed Kupffer cell lines were stable for at least 90 population doublings after transformation. Accompanying the change in enzyme activities after transformation by SV40 was an increase in variation between cell lines which, prior to transformation were isolated from material with a genetically identical origin. Four SV40-transformed foetal cell lines were indistinguishable from the SV40-transformed adult cell lines. A study of lactate dehydrogenase (LDH) isoenzyme patterns in primar adult, primary foetal and SV40-transformed Kupffer cell lines revealed that complexities may underlie a total enzyme activity. While culturing of primary adult or primary foetal Kupffer cells resulted in a decline in total LDH activity, the relative proportions of LDH A and LDH B gene products changed Primary cell lines demonstrated a decreased proportion of LDH B gene product when compared with freshly isolated Kupffer cells. After transformation of adult or foetal Kupffer cells by SV40 the polypeptide coded by the LDH B was not detected. The variation in enzyme activities between primary Kupffer cell lines of similar or identical origin is interpreted to be an example of epigenetic variation arising as a result of each Kupffer cell's individual response to the culture environment. The increase in enzyme activity variation between cell lines after transformation by SV40 is suggested to be the result of a change in the differentiated state. The phenotypes of primary adult, primary foetal and SV40-transformed Kupffer cell lines are discussed in terms of the differentiated state and in the light of the theory of foetalism in neoplasia. The heterogeneity in enzyme activities was not paralleled by karyo-typic variation. Although there was a gradual transition towards heteroploidy, all cell lines, whether primary or transformed, possessed a diploid karyotype during the stages when variation in enzyme activities was apparent The normal karyotype possessed by cells which demonstrated properties of transformation suggests that transformation by SV40 is not the result of karyotypic change. Analysis of the data revealed quantitative correlations between severa enzyme activities. Primary adult, primary foetal and SV40-transformed cell lines demonstrated differences in the pattern of the quantitative correlations. The correlations provide evidence for the existence of a mechanism which regulates the activity of two or more unrelated enzymes. Possible metabolic and regulatory bases for the enzyme activity correlations are considered.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J A Pateman
Keywords: Biochemistry
Date of Award: 1976
Depositing User: Enlighten Team
Unique ID: glathesis:1976-72955
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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