Semester

Fall

Date of Graduation

2010

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Yon Rojanasakul

Abstract

The bone marrow microenvironment is characterized as the anatomical site including specialized niches that support stem cells. In addition, these niches also provide both soluble and physical cues leading to the differentiation of stem cells into all the cells of the blood. The studies in this dissertation focus on two supportive niches in the bone marrow microenvironment, osteoblasts (HOB) and bone marrow stromal cells (BMSC), in the setting of high dose chemotherapy and the potential damage that chemotherapy treatment causes to the cells of the bone marrow microenvironment.;In the first study, we investigated the effects of melphalan and Etoposide (VP-16) treatment on osteoblasts. Previous studies from our lab had shown that chemotherapy treatment increased the amount of active TGF-beta secreted from BMSC, leading to decreases in the ability to support pro-B cells. Here we describe the novel observation that osteoblasts pre-treated with chemotherapy have increased active TGF-beta and a decreased capacity to support human embryonic stem cells (hESC), CD34+ bone marrow derived cells and pro-B cells. We also evaluated the effects of adding recombinant TGF-beta (rTGF-beta) to osteoblasts to mimic the autocrine and paracrine TGF-beta in the microenvironment during chemotherapy treatment. rTGF- beta treatment of osteoblasts increased TGF-beta secretion and also led to a decreased ability to support hESC, CD34+ bone marrow derived cells and pro-B cells. Microarray analysis of the cells treated directly with chemotherapy or rTGF-beta or conditioned media from BMSC that were treated with chemotherapy suggested that many genes are changing in response to all of these treatment groups, indicating that osteoblasts are a vulnerable cell population that can be affected by high dose chemotherapy, potentially resulting in decreased hematopoietic support.;We also investigated Interleukin-6 (IL-6), a known hematopoietic factor important in both myeloid and lymphoid differentiation, acute and inflammatory immune responses and bone metabolism. Neuroendocrine modulation of the bone marrow microenvironment is thought to be important in both hematopoiesis and immune regulation. We investigated the roles of neurotrophins in the bone marrow and their effects on BMSC. We show that BMSC express functional neurotrophin receptors and that treatment of BMSC with two neurotrophins, NGF or BDNF, led to an increase in IL-6 expression. Increased IL-6 is associated with a number of inflammatory diseases and our data support the idea that increased neurotrophins in the bone marrow microenvironment could lead to dysregulated hematopoiesis.;Additionally, we also evaluated the effects of chemotherapy treatment of BMSC and HOB, focusing on IL-6. Previous data has suggested IL-6 to be involved in graft versus host disease and we investigated the effects of melphalan on IL-6 expression in BMSC and HOB. Interestingly, we determined that melphalan treatment led to a decrease in IL-6 mRNA and protein, and compared to other chemotherapeutic agents used in our studies, melphalan had the most pronounced effect. We also evaluated the effects of recombinant IL-6 (rIL-6) in combination with melphalan and determined that the addition of rIL-6 restored both IL-6 mRNA and protein expression, suggesting that pathways associated with IL-6 expression may be disrupted. The decrease in IL-6 could potentially affect hematopoiesis and further studies in vivo need to be completed. Additionally, melphalan is used as first-line therapy in the treatment of multiple myeloma (MM). IL-6 is a proliferative factor in MM, allowing for disease progression. The melphalan-induced decrease in IL-6 observed in our studies may, in part, contribute eradicating the tumor population by decreasing the potent proliferative factor, IL-6.;Collectively, these data contribute to our understanding of alterations to the bone marrow microenvironment that occur during high dose chemotherapy and emphasize the importance of understanding the mechanisms that underlie the potential damage leading to altered ability to support normal hematopoiesis.

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