Date of Graduation

2016

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Laura F Gibson

Committee Co-Chair

John B Barnett

Committee Member

William P Petros

Committee Member

John M Ruppert

Committee Member

James M Sheil

Abstract

The cells comprising the stromal compartment of the bone marrow microenvironment are critical to the maintenance of several homeostatic processes of the body. For example, mesenchymal stem cells (MSCs) and osteoblasts are vital to the regulation of differentiation and quiescence of hematopoietic cells, maintaining the skeletal system, and regulating tumor microenvironments. In addition to contributing to these processes, MSCs display several properties that make them favorable for the use in transplantation therapies. The work described herein summarizes the means by which chemotherapy damages these cells, and potential consequences of such damage with regard to their function.;Previously, our laboratory has investigated chemotherapy induced damage of osteoblast potential to support hematopoiesis, describing an increased presence of tumor growth factor beta (TGF-beta) and interleukin-6 as contributing factors. We have expanded upon these observations to describe damage elicited by etoposide (VP16) and melphalan on the expression of extracellular matrix and hematopoietic support proteins in the murine pre-osteoblast cell lines MC3T3E1 and 7F2. We showed that chemotherapy dysregulates extracellular matrix (ECM), resulting in reduced type I collagen expression in cell lines, as well as altered morphology of the endosteum in VP16 treated mice. In addition, chemotherapy reduced the abundance of hematopoietic support proteins CXCL12 and osteopontin. Chemotherapy exposure also reduced the expression of osteogenic differentiation associated transcription factors, coincident with reduced differentiation potential of pre-osteoblast cells. These observations highlight the vulnerability of osteoblasts to dysregulation of both hematopoietic support and osteogenic functions following chemotherapy exposure.;In the second study, we expanded our evaluation of stromal cell vulnerability to chemotherapy to mesenchymal stem cells (MSCs). MSCs are beginning to be utilized clinically for transplantation therapies, a process which requires in vitro expansion of cells prior to patient administration. To determine whether in vitro expansion affects the susceptibility of MSCs to chemotherapy stress, we evaluated the cellular response to etoposide (VP16) at various passages in vitro. Although passaging did not influence the susceptibility of MSCs to VP16, we found the repair of VP16 induced DNA damage was altered with extended passage. Exposure of MSCs to VP16 reduced homologous recombination (HR) associated transcripts, a phenomenon that was augmented with passage in vitro. Using plasmid based reporter assays, we found that HR mediated repair was reduced in untreated cells, and MSCs were less able to increase non-homologous end joining (NHEJ) following VP16 after extended passage. These results indicated an alteration of the ability of passaged MSCs to perform DNA repair following VP16 stress, indicating that MSCs should not be passaged too extensively prior to utilization for transplantation.;In contrast to the beneficial aspects of MSC function during MSC transplantation, MSCs can display pathogenic interactions with their surrounding environments, such as during cancer progression. In the case of the tumor microenvironment, MSCs have been shown to regulate tumor phenotype through the secretion of various signaling molecules. For example, the Wnt signaling pathway has been shown to regulate the phenotype of certain tumors. We investigated the expression of Wnt signaling molecules by MSCs after chemotherapy exposure and found that Dkk-1, a secreted inhibitor of Wnt signaling, was increased following exposure to VP16, melphalan, and 5-fluorouracil. Through the use of chemical inhibitors and activators of p53, as well as siRNA silencing, we showed that Dkk-1 elevations in MSCs after chemotherapy were mediated by p53, consistent with the published presence of a p53 promoter element within the DKK1 promoter. These results suggested the potential for MSC derived Dkk-1 to elicit negative effects on patients harboring Dkk-1 responsive tumors, and the possible approach of targeting Dkk-1 pharmacologically in these patients.;Collectively, these findings highlight the dynamic nature of bone marrow derived stromal cells, and illustrate that they are responsive to stress in ways that could negatively impact the health of patients receiving chemotherapy treatment. Given the numerous processes that are regulated by these cells, it is important to identify mechanisms by which chemotherapy elicits damage, providing a conceptual framework for developing means of reducing toxicities associated with chemotherapy treatment by maintaining the ability of bone marrow derived stromal cells to perform critical functions.

Download

Share

COinS