The overall objective of this research is to elucidate the signal transduction pathway leading to differentiation as indicated by activation of the kappa light chain gene in a pre-B like lymphocyte cell line, 70Z/3. It has been shown previously that lymphokine-induced differentiation of 70Z/3 is preceded by a number of membrane events which include alterations of ion fluxes. The immediate goals of this project are to examine the relation of ion fluxes to activation of the kappa gene in 70Z/3 cells. Wild type and mutant 70Z/3 cells will be used. Differentiation will be assessed by measuring the levels of transcription factors using gel retardation, kappa mRNA levels by cytoplasmic dot hybridization, and surface immunoglobulin expression by flow cytometry. Ion fluxes and intracellular ion concentrations will be assessed by atomic absorption spectrophotometry, radiolabeled tracers, and fluorescent dyes. It is expected that some ions may serve as transducers while others may serve as modulators of the 70Z/3 differentiation response by interacting, directly or indirectly, with the DNA binding proteins that regulate kappa gene expression. The proposed studies should provide important information about the relationship between lymphokine-triggered ion fluxes, kappa gene specific DNA binding proteins, and kappa light chain gene expression in this cell model for B lymphocyte development. The results obtained in this system should also shed light on signal transduction pathways in other cells in which specific extracellular signal molecules initiate a chain of events leading to changes in gene expression.
