The role of leukaemia inhibitory factor and a leukaemic associated inhibitor in the control of the proliferation of haematopoietic stem cells
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Activities associated with, or interacting with, leukaemic cell populations were assayed for the ability to influence in vitro haematopoiesis. The first of these, the glycoprotein leukaemia inhibitory factor (LIF), has a role in aspects of murine, non human primate and human haematopoiesis. It is thought to be particularly important in the development of megakaryocytes and is also known to induce the terminal differentiation of certain leukaemic cell lines. LIF was assayed both for direct and indirect effects on the proliferation of haematopoietic precursor cell populations in vitro. As a direct acting agent in semi-solid agar culture of haematopoietic cell populations derived from normal bone marrow or 15 day foetal liver, LIF was unable to support colony formation. In cultures of cells derived from normal bone marrow stimulated with single, or combinations of, growth factors, the addition of LIF had no statistically significant effect on the level of colony formation. In cultures of cells derived from foetal liver, stimulated with particular growth factor combinations (medium conditioned by the Wehi3B leukaemic cell line + medium conditioned by the lung fibroblast cell line, L929); GM-CSF + M-CSF; IL-la + IL-3 + M-CSF), LIF, was shown to decrease the level of colony formation. LIF did not directly alter the proportion of the population in DNA synthesis in cell populations derived from normal femoral marrow, 15 day foetal liver or y- irradiated femoral marrow. As an indirect acting agent LIF failed to block the synthesis of a stem cell stimulator, or it's action, on a population of high proliferative potential colony forming cells derived from normal femoral marrow, cloned in the presence of Wehicm+L929cm. (HPP-CFC (Wehicm + L929cm)) LIF's actions on clones of a murine myeloid leukaemia (SA2JMB1) were also assessed. LIF had no statistically significant effect on colony formation or the level of DNA synthesis in populations of SA2JMB1 leukaemic cells. A second group of associated activities was produced by the X- irradiation induced murine myeloid leukaemia (SA2JMB1). Medium conditioned by the leukaemic cells was assayed in vitro both for direct and indirect effects on the proliferation of haematopoietic cells derived from femoral marrow. As a direct acting agent in 7 and 14-day semi-solid agar culture of femoral marrow, leukaemic conditioned medium alone stimulated limited colony formation. In 7 and 14 day cultures stimulated with single and combinations of specific colony stimulating factors: (rmGM-CSF, rhM-CSF, rhIL-1a) a significant increase in colony number was noted in all cases when cultures were supplemented with leukaemic conditioned medium. SA2JMBlcm was shown to support the proliferation of an IL-3 dependent cell line (FDCP-A4 cells). The colony enhancing ability of SA2JMBlcm was shown to be blocked by pretreatment with antibodies to IL-3. This suggested that SA2JMB1 conditioned medium contained IL-3 or an IL-3 like activity, as one of its components. The conditioned medium failed to directly alter the level of DNA synthesis in a population of HPP-CFC (Wehicm+L929cm) derived from normal bone marrow or y- irradiated bone marrow. As an indirect acting agent the conditioned medium did block the action of a stem cell proliferation stimulator on normal bone marrow derived HPP-CFC (Wehicm+L929cm). This leukaemia associated activity was shown to be larger than 50KD, sensitive to heat treatment and able to act in a different manner to the stem cell inhibitor MIP-1-a. Thus this novel activity may be important in blocking stimulator action in haematopoietic stem cells and thus contribute to the haematopoietic insufficiency seen in leukaemia.
Thesis, PhD Doctor of Philosophy
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