The aim of this research is to characterize the interactions between myeloid progenitors and marrow stromal components. The capacity of CD34+ myeloid progenitors and myeloid leukemic cell lines to bind to stromal layers will be correlated with subsequent proliferative potential and phenotypic maturation. This will be accomplished using clonogenic assays, flow cytometric techniques to assess surface antibody phenotype, and bromodeoxyuridine uptake to determine cell cycle status of the progenitor populations. In addition, antisense RNA cytokine probes to be utilized in phase II studies will be prepared in phase I of this proposal. These will include probes for interleukin-1 alpha (IL01) and interleukin-6 (IL-6), which have a role in promoting early stem cell self-renewal; and transforming growth factor-beta, which is inhibitory to primitive progenitors. In phase iI, the giant cell tumor (GCT) cell line, a mesenchymal cell line which stimulating factor (GM-CSF), granulocyte CSF, and macrophage CSF will be used as a model of marrow stromal cells. The regulation of cytokine messenger RNA expression in GCT monolayers exposed to dexamethasone, tumor necrosis factor alpha, phorbol esters, or hematopoietic progenitor cells will be examined. RNA extraction, NOrthern blotting, and nuclear run-on assays will be used for these measurements. Also, the effect of cycloheximide in this system will be examined to delineate post transcirptional regulatory events. In situ hybridization techniques will be applied to GCT and primary marrow stromal cell monolayers to search for localized asymmetric cytokine (e.g. GM-CSF, IL-6) expression in these cultures after exposing them to fixed CD34+ myeloid progenitors or KG1-a cells. Expression of these cytokines in stromal cells at steady state and after exposure to progenitor cells will also be assessed utilizing RNA polymerase chain reaction techniques. These studies will help to further understanding of stromal cell-hematopoietic progenitor interactions. They will therefore serve to aid in elucidating the role of he marrow microenvironment in stem cell homing, survival, and engraftment after transplantation and in understanding alterations in hematopoiesis which occur in inflammatory and infectious disease states.
