3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Effects of IL-2,IL-6,IL-7 and IFN on the proliferation,survival,induction and reduction of spontaneous in-vitro apoptosis of B CLL cells(2007-02-14T11:50:19Z) Seahloli, Michael SelloB chronic lymphocytic leukaemia (B-CLL) is a monoclonal haematopoietic disorder with expansion of small lymphocytes of B-cells. B-CLL cells accumulate in blood, bone marrow, lymph nodes and spleen, resulting in enlargement of these organs and decreased bone marrow function. B-CLL is the most common leukaemia, with an annual incidence of 1.8 to 3.0 per 100 000 population in the United States. It is characterised by the accumulation of long-lived monoclonal CD5+ B lymphocytes. In vivo normal B-lymphocytes derive growth factors through interactions with T-cells and monocytes. In culture however, survival and growth of activated B-cells depends on the availability of external factors such as interleukins. B-CLL cells populations are unable to survive in culture long enough to respond to the addition of growth factors. Such factors are important for the proliferation and survival of many cell types and in the absence of cytokines, these cells die as a result of apoptosis. Chronic lymphocytic leukaemia cells are influenced in vitro by a number of exogenously added cytokines that include IFN- α, IFN-γ, IL-2, IL-4, IL-10, IL-13, IL-15, TGF- β and TNF- α. The aim of this study was to investigate the effect of cytokines e.g., IFN, IL-2, IL-6, IL7 and IL-10 on the proliferation and survival of B-CLL cells and furthermore to compare the induction and reduction of spontaneous and induced apoptosis in vitro. Patients with B-CLL were recruited from three centres. Thirty blood samples were collected, separated using Ficoll Hypaque Gradient and purified by rosetting with AET treated SRBC. The proliferation and survival of B-CLL cells were studied in vitro in response to GM-CSF, IFN, IL-2, IL-6, IL7 and IL-10,. The survival and apoptosis of B-CLL cells in cultures with or without interleukins and other growth factors were studied under microscopic examinations and DNA agarose gel electrophoresis. It was observed in B-CLL cells cultures that IFN and IL-2 enhanced proliferation significantly. IL6, IL-7 and GM-CSF also enhanced proliferation of B-CLL cells but not to the greater extent than IL2 and IFN. IL-10 inhibited proliferation of B-CLL cells when compared to controls. In a long-term (5-day) culture, survival of B-CLL cells was greatly enhanced by IFN and followed by IL-2. Therefore it appeared that IFN and IL-2 are the two most potent growth factors tested in this study to promote B-CLL cells proliferation and survival. The combination of these mitogens did not further enhanced proliferation. IL-6 and GM-CSF enhanced proliferation and survival of B-CLL cells. IL-7 promoted proliferation but had no effect on survival of B-CLL cells in-vitro. IL-10 enhanced apoptosis and did not promote survival of B-CLL cells in-vitro. IFN and IL2 are survival and promoting growth factors for B-CLL cells in culture. In contrast, IL-10 has demonstrated to induce apoptotic cell death of B-CLL cells. In conclusion B-CLL cells proliferated equally well with IFN and IL-2. IL-6, IL-7 and GM-CSF had a much lower proliferation and survival effect with noticeable antiapototic activity when compared to IFN and IL-2. IL-7 was found not to promote survival of B-CLL cells and IL-10 enhanced cell death by apoptosis.Item The effect of differentiation on the expression of phosphoprotein phosphatase in the human promyelocytic leukaemic cell line HL-60(2006-11-16T07:30:27Z) Bhoola, RajeshDynamic cellular activity is fundamental to all life. Virtually all life processes, are modulated by the reversible phosphorylation of proteins, mediated by protein kinases and phosphoprotein phosphatases, respectively. This thesis focuses on three enzymes, namely: phosphoprotein phosphatase 1, phosphoprotein phosphatase 2A and protein tyrosine phosphatase-1B. Temporal variations in the expression of the enzyme proteins were examined in the human acute promyelocytic leukaemic cell line, HL-60. The cells were induced to differentiate along the macrophage pathway using phorbol-12-myristate- 13-acetate and along the granulocytic pathway using dimethyl sulfoxide, all-trans retinoic acid and 9-cis retinoic acid. Modulation of the rhythmic patterns of protein and messenger RNA was monitored in the absence and presence of inducing agents. Expression of protein in cell extracts prepared at various time intervals was determined by western immunoblotting, while mRNA expression was assessed by northern blotting and RT-PCR. The probe used for northern blotting was generated during the RT-PCR procedure. In addition, PTP-1B mRNA was cloned into an expression vector to produce recombinant protein. Results indicate that the expression of phosphoprotein phosphatase 1, phosphoprotein phosphatase 2A and protein tyrosine phosphatase-1B protein is dynamically regulated in proliferating HL-60 cells and modulated after being induced to differentiate along either the macrophage or granulocytic pathway. Similar changes were also noted with PTP-1B mRNA when using northern blot analysis. Using molecular cloning techniques, PTP-1B mRNA was successfully cloned into pGex-4T-1 expression vector to produce recombinant PTP-1B protein, which was checked by sequence and western blot analysis.