Semester

Spring

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Scott A. Weed.

Abstract

The bone marrow microenvironment serves as the primary site for post-natal hematopoiesis allowing for HSC development and maturation. More specifically, B lineage cells rely on key signals from both the physical and soluble components of the marrow for their survival and maturation. In addition to normal hematopoiesis the bone marrow has been shown to be a sanctuary for leukemic cells, as well as other tumors, that can interact with cues from the microenvironment allowing for their protection during chemotherapy. The studies described in this dissertation focus on two supportive components of the bone marrow microenvironment, osteoblasts and bone marrow stromal cells (BMSC), in the settings of post high dose chemotherapy and survival of leukemic cells during chemotherapy, respectively.;In the first study we investigated the effects of high dose chemotherapy on human osteoblasts (HOB). Previous studies from our laboratory have shown that high dose chemotherapy causes increases in the amount of active TGF-beta released from BMSC and subsequently leads to diminished ability to support pro-B cells. Here we describe the novel observation that HOB treated with chemotherapy have increased levels of active TGF-beta as well as a diminished capacity to interact with, and support, human embryonic stem cells and pro-B cells. Additionally, we determined that HOB treatment with chemotherapy or rTGF-beta led to increased levels of active TGF-beta, as well as decreased CXCL12 mRNA and protein leading to a decreased adherence of pro-B cells to HOB. Chemotherapy or rTGF-beta treatment also lead to the diminished ability of HOB to support pluripotent OCT-4 positive stem cell colonies and microarray analysis of HOB treated with Melphlan, rTGF-beta or Conditioned Media from BMSC treated with Melphlan led to dramatic changes in the gene expression profiles of the HOB. This study elucidates the importance of osteoblasts in the marrow following ablative therapy and indicates how different components of the marrow are altered by chemotherapy.;Our laboratory and others have previously shown that BMSC are able to protect tumor cells from chemotherapy however the mechanisms by which the bone marrow microenvironment regulates tumor cell survival are diverse. This study describes the novel observation that in addition to Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cell lines, primary patient cells also express Vascular Endothelial Cadherin (VE-cadherin) which is regulated by Abl kinase and has been associated with aggressive phenotype and poor prognosis in other models. Targeted knockdown of VE-cadherin rendered ALL cells more susceptible to chemotherapy, even in the presence of BMSC derived survival cues. Pre-treatment of Ph+ ALL cells with ADH100191, a VE-cadherin antagonist, resulted in increased apoptosis during in vitro chemotherapy exposure. Consistent with a role for VE-cadherin in modulation of leukemia cell viability, lentiviral-mediated expression of VE-cadherin in Ph- ALL cells resulted in increased resistance to treatment-induced apoptosis. Collectively, these data contribute to our understanding of the alterations that occur to the different supportive components of the bone marrow microenvironment during chemotherapy and the mechanisms that alter their ability to support both normal and neoplastic cells.

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