Year of Publication

2006

Document Type

Dissertation

College

Medicine

Department

Physiology

First Advisor

Gary Van Zant

Second Advisor

Brian Jackson

Abstract

Cellular proliferation is a key characteristic of hematopoietic stem and progenitor cells (HSC/HPCs) that allows for the production of all blood cell lineages during an individuals lifetime. While this feature of stem cells is strictly regulated during steadystate and stress hematopoiesis, it also contributes to the development of myeloproliferative disorders, such as chronic myelogenous leukemia, essential thrombocythemia, and polycythemia vera. It should come as no surprise then, that common treatments for these diseases often target the proliferative nature of the dysfunctional HSC/HPCs. Thus, the identification of molecular determinants of cell cycle regulation associated with these disorders could serve as targets for novel therapies. Using the hematopoietic system of the inbred mouse strains, C57BL/6J (B6) and DBA/2J (D2), it was found that the HSC/HPCs of the long-lived B6 mouse strain were less susceptible to the cytostatic agent hydroxyurea (HU) than the short-lived D2 mouse strain. A quantitative trait locus (QTL) analysis revealed a region of proximal chromosome 7 that regulates this response to HU. Congenic mouse strains were generated and phenotypic analysis confirmed that the B6 and D2 loci confer a low and high sensitivity of the HSC/HPCs to HU, respectively. We then showed that while this response of the HSC/HPCs to HU is independent of their cell cycle status, the B6 allele of this QTL confers a proliferative advantage to bone marrow cells after bone marrow transplantation. Having shown that proximal chromosome 7 regulates the response of HSC/HPCs to HU, we found it necessary to characterize the gene and protein expression profiles in order to identify the responsible candidate genes. We first analyzed mRNA expression profiles of HPCs from the parental and congenic mouse strains using gene microarrays and found that four genes within the congenic interval were differentially expressed. Real-time PCR confirmed that the expression profile of only one gene, Ndufa3, is significantly different in HPCs of B6 and D2 mice. Concurrently, we assessed the protein expression profiles of HPC-enriched mononuclear cells. Significant differences were found between the cytoplasmic and nuclear fractions of both strains, with a skewing of protein expression towards the D2 congenic strain.

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