Roberts, Sarah Anne; (1999) An investigation of protein kinase C in Swiss 3T3 cells using phorbol esters. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this thesis the literature relating to phorbol esters as potent activators of protein kinase C (PKC) has been reviewed. A programme of work has been performed to examine the PKC isotypes present in Swiss 3T3 cells and human keratinocytes; to monitor the changes in expression, subcellular distribution or activity of PKC isoforms during treatment with a variety of phorbol esters and to relate the activation of PKC in the cell to specific physiological effects. The work has identified that the alpha, delta, epsilon and zeta isotypes of protein kinase C are present in both Swiss 3T3 cells and human keratinocytes. The effect of five phorbol esters (TPA, Dopp, Doppa, Thy A and Rx) on inducing mitogenesis in Swiss 3T3 cells was examined. All of the phorbol esters tested apart from Rx were found to be capable of stimulating mitogenesis to varying degrees. In the case of both Dopp and Doppa where the stimulation of mitogenesis was equipotent, Doppa was found to be metabolising to the more potent Dopp in the cell system after four hours incubation. The phorbol esters were each shown to translocate the different isotypes of PKC with different potencies. Some of the phorbols tested were shown to be unable to translocate some of the isotypes examined. Using the mixed micellar assay, peaks of stimulatable activity were examined in partially purified PKC obtained from Swiss 3T3 cells. A peak of phorbol ester stimulated activity was shown to coincide with the elution of PKC alpha. Other peaks of activity uncovered could not be attributed to a PKC isoform present in this cell system. The phosphorylation of the myristoylated alanine rich c kinase (MARCKS) protein was also examined following phorbol ester treatment. High doses of all of the phorbol esters tested were capable of phosphorylating the MARCKS protein. Finally the morphology of TPA treated cells was examined using scanning electron microscopy.

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