The overall purpose of this project is to examine the development and regulation superoxide-generating oxidase in phagocytic leukocytes. Studies performed in the first grant period (1983-86) delineated the cell physiology and enzymology of superoxide production during induced differentiation of the HL-60 human promelocytic leukemia cell line and in various forms of chronic granulomatous disease (CGD). During the next five years, the proposed studies will extend to the molecular level of regulation of the oxidase. The major tool for these experiments is complementary DNA (cDNA) from the recently cloned gene (termed """"""""X-CGD"""""""") for X-linked CGD.
The specific aims of the project are: I. Examination of X-CGD gene expression during induced differentiation of HL-60 cells. This section will focus on the steady state levels and rates of synthesis and degradation of X-CGD transcripts (using techniques of blot and solution hybridization, nuclear transcriptional run-off, and 32p pulse-chase labelling). Other studies in HL-60 will explore the role of the cell cycle in X-CGD expression and the effects of introduction of the full-length cDNA linked to differentiation-independent promoters. II. Examination of X-CGD gene expression in differentiating myeloid precursors derived from normal human bone marrow. These studies, utilizing myeloid cells in culture and mixed bone marrow cells in situ, will parallel those in HL-60 in order to verify the findings in non-leukemic cells. III. Examination of X-CGD gene expression in granulocytes and macrophages treated with cytokines that modulate superoxide production. These studies will determine the effects of human recombinant interferon gamma, tumor necrosis factor, and granulocytes-macrophage colony- stimulating factor on X-CGD transcript levels and kinetics. IV. Application of these studies to CGD. These studies will extend preliminary results on the effects of IFN-gamma on CGD phagocytes, develop and refine methods for the prenatal diagnosis of CGD, and determine the molecular nature of genetic defects that cause the disease. V. Probes for other oxidase components will be obtained or cloned from HL-60 libraries to complement the studies with X-CGD. As these probes become available, they will be used to similarly study the regulation of other genes contributing to oxidase activity. Overall, this project is intended both to increase our understanding and to indicate means of therapeutic manipulation of a vital arm of host defense.
