Buschle, Michael Joachim; (1993) Analysis of normal and malignant human B cell growth using gene transfer. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this thesis was to investigate the effects of individual oncogenes on human B lymphocytes. To permit these studies, a gene transfer and expression technique for normal B lymphocytes and cells from patients with B cell chronic lymphocytic leukaemia (B-CLL) was developed. Of the numerous techniques tested, only electroporation proved to have reasonable efficiency and in due course, a powerful promoter/enhancer element containing cytomegalovirus and human T cell leukaemia virus I sequences was identified. Several eukaryotic expression vectors based on this promoter/enhancer element were constructed: a cassette vector was designed and used to subclone the oncogenes c-myc, v-Ha-ras, v-fos, v-raf and v-mos. As the malignant B lymphocytes B-CLL constitute a homogeneous, monoclonal B cell population with large number of cells available for experiments, gene transfer studies were initiated with material derived from patients with this disease. Transfection of B-CLL samples with the constructs alone or in combination did not result in cell transformation, however, prolonged cell survival was consistently observed in c-myc transfected samples. Subsequent analysis revealed that interferon-y was secreted at high levels following c-myc gene transfer. Addition of recombinant IFN-γ to cultured B-CLL cells demonstrated that this cytokine promotes B-CLL cell survival by preventing programmed cell death, or apoptosis, and may therefore be responsible for the slow and progressive accumulation of the Go arrested malignant B lymphocytes observed in vivo. Furthermore, IFN-γ was shown to be produced by B-CLL cells in an autocrine manner by analysis of IFN-γ transcripts and double colour flow cytometry. Synthesis of IFN-γ by B-CLL cells may also explain why high levels of this cytokine were found in serum samples from patients with this disease. In conclusion, IFN-γ may be an important growth factor contributing to the development of this common form of leukaemia. Inhibition of IFN-γ mediated effects may lead to novel therapeutic approaches.

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