DISSECTING THE HEMATOPOIETIC GROWTH FACTOR NETWORK

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Hematopoietic regulation involves an elaborate network of interacting cells and cytokines in which hierarchies of gene expression are complicated by autocrine and paracrine amplification mechanisms. Each cytokine exhibits a variety of biological activities, some of which are synergistic with those of other cytokines. In a system of such complexity, an experimental technique which inhibits the expression of specific genes would greatly facilitate the analysis of questions of function, linkage, and causality. To this end, we have demonstrated that antisense oligodeoxynucleotides complementary to GM-CSF or G-CSF MRNA inhibit expression of the corresponding growth factor in IL-1-induced endothelial cells or fibroblasts in a sequence-specific fashion. In this proposal, we will examine the determinants of the efficiency of inhibition of gene expression by antisense oligonucleotides and the cellular and molecular mechanisms which mediate this inhibition. This approach would be particularly useful in two experimental settings: (1) long-term bone marrow culture (in which it is difficult to deliver antibodies that interrupt juxtacrine mechanisms) and (2) studies on mechanisms of IL-1 signal transduction (these are entirely intracellular and not amenable to manipulation with antibodies). Accordingly, we will employ antisense oligonucleotides to analyze directly the role of GM- CSF, G-CSF, IL-6, and stem cell factor (kit ligand) in the initiation and maintenance of hematopoiesis in long-term bone marrow culture. We will clarify the role of c-fos, c-jun, and c-raf-1 in IL-1-induced hematopoietic growth factor expression using antisense oligonucleotides to specifically inhibit their expression.