Semester

Fall

Date of Graduation

2002

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Kenneth S. Landreth.

Abstract

B lymphocytes are continually produced in bone marrow from pluripotential hematopoietic stem cells. Lymphopoiesis is characterized by a series of highly regulated genotypic and phenotypic changes resulting in immunocompetent effector cells which express cell surface immunoglobulin. Our laboratory has focused on defining extracellular signals regulating lymphoid progenitor cell survival, proliferation, and differentiation. These studies have demonstrated that pro-B cell survival, proliferation, and differentiation are regulated by interactions with fibroblastic stromal cells in the hematopoietic microenvironment. However, specific molecular mechanisms by which stromal cells regulate B lymphoid development are largely unknown. In an attempt to better understand molecular mechanisms regulating maturation in this lineage, we developed a panel of pro-B cell clones from 14-day murine fetal liver. These pro-B cell clones remain dependent on stromal cells for survival, do not form tumors, and reconstitute B lymphocytes in severe combined immunodeficient (SCID) mice. In vitro, pro-B cell clones continuously proliferate and do not differentiate. We noted that pro B cell lines were characterized by expression of high levels of the oncogene c-myb. Although several laboratories have proposed a role for c-myb in regulation of hematopoiesis, virtually nothing is known about the function of c-myb in normal B lineage cells. To investigate the role of c-myb in the survival, proliferation, and differentiation of B lineage cells, we utilized a stromal cell dependent pro-B cell line that expresses mRNA and protein for c-myb. Experiments utilizing RT-PCR and Western blot analysis reveal that c-myb is regulated in pro-B cells by stromal cells, specifically by stromal cell adhesion contacts. Both DMSO and antisense oligonucleotides were used to downregulate c-myb protein to determine the role this intracellular regulator plays in B lymphocyte development. Our investigations revealed that downregulation of c-myb did not affect pro-B cell survival but did interrupt both pro-B cell proliferation and differentiation. In vivo investigations in mice carrying homozygous mutations of the c-myb gene indicate that lymphopoiesis is severely diminished in embryonic knockout animals. These data suggest a central role for c-myb in proliferation and differentiation of developing B lymphocytes.

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