Browsing by Author "Ullrich, Robert L., advisor"
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Item Open Access Characterizing in vitro propagation and radiation response of murine mammary stem cells(Colorado State University. Libraries, 2009) Magers, Tonya Sirisalee, author; Ullrich, Robert L., advisorStem cells in breast tissue may be sensitive to known carcinogens (i.e. ionizing radiation), which impact their susceptibility to transformation. The involvement of mammary stem cells in tumorigenesis could explain the heterogeneity and molecular complexity of breast cancer. However, the involvement and the underlying mechanisms of such targets have yet to be fully elucidated. This study was designed to investigate mammary stem cells as plausible targets of radiation-induced damage in radiation-induced mammary carcinogenesis. We utilized an in vitro system (mammospheres) that was developed for the detection of mammary stem cells. We expanded the applicability of this in vitro assay through the development of a methodology and novel size criteria to address specific radiation biology endpoints. We applied the methodology and size criteria to analyze the effects of ionizing radiation (IR) on the survival of mammary stem cells derived from mice carrying one mutated copy of Atm. Our results demonstrated that mammary stem cells derived from Atm-ΔSRI heterozygous mice (Atm(+/ΔSRI)) do not exhibit increased radiation sensitivity compared to their wildtype littermates (Atm(+/+)). In fact, mammary stem cells derived from Atm-ΔSRI heterozygous mice exhibited increased radioresistance. To our knowledge, this is the first study to examine the radiation response of mammary stem cells as mammospheres using Atm heterozygous mice carrying a known missense mutation found in human A-T. These studies demonstrated the proof of principle for this model development and the utility of this methodology. Our improved methodology has expanded the feasibility and the applicability of this model to examine numerous functional in vitro endpoints. We believe the methodology described here will facilitate investigating the radiation response of mammary stem cells and their progeny, and key components involved in early events of the carcinogenic process in murine model systems.Item Open Access Examination of molecular genetic factors involved in sensitivity to breast cancer following radiation exposure(Colorado State University. Libraries, 2008) Williams, Abby J., author; Ullrich, Robert L., advisor; Bailey, Susan M., advisorUnderstanding DNA repair is not only an important aspect of cell biology, but also has important implications for the field of carcinogenesis since cancer most likely occurs from genetic damage that occurs over one's lifetime. DNA repair needs to be accurate and efficient in order for a cell to maintain genomic stability, and defects in repair systems can result in radiosensitivity. Because radiation exposure, DNA repair deficiency and telomere malfunction are associated with cancer risk, we investigated Lymphoblastoid Cell Lines (LCLs) from breast cancer patients and controls for chromosomal radiosensitivity, relative telomere length, and gene expression changes. The importance of studying peripheral blood lymphocytes from cancer patients lies in the fact that minimally invasive techniques are lacking for the detection of individuals with high risk for cancer, and that telomere length has been proposed to be useful in this regard. Identification of radiosensitivity markers would be a valuable contribution for clinicians in hopes of avoiding excessive radiation or chemotherapy treatment given to patients. Failure to adequately repair DNA damage can result in cell suicide or halting of cell cycle progression in an attempt to allow repair mechanisms to operate. If damage persists, a cell can be pushed toward transformation and the pathway of carcinogenesis. A second aspect of the current work was to study the Homologous Recombination double-strand break repair protein, Rad51D. The emerging interrelations between DNA repair and telomere maintenance also prompted us to evaluate Rad51D's role in telomere function. The final aspect of this research involved examination of how DNA repair related proteins are linked to the indirect effect of ionizing radiation exposure known as the bystander effect (BSE). We are the first to demonstrate that DNA-PKcs and ATM are required to generate, but not receive, a bystander signal. We also show that mouse embryonic fibroblasts do not generate bystander signals to neighboring cells, while their adult cell counterparts do. Taken together, this work makes important contributions to our appreciation of the many and varied roles DNA repair related proteins play in maintenance of chromosomal integrity, proper telomere function, inhibition of carcinogenesis and now, regulation of the BSE.Item Open Access Identification of premalignant target cells using a mouse model of radiation-induced acute myeloid leukemia(Colorado State University. Libraries, 2010) Liu, Xiangfei, author; Ullrich, Robert L., advisor; Weil, Michael M., committee member; Ray, F. Andrew, committee member; Bamburg, James, committee member; Avery, Anne C., committee memberAcute myeloid leukemia (AML) is believed to arise from the clonal expansion of a malignantly transformed blast cell. Whether spontaneous AML arises from a hematopoietic stem cell (HSC) or a more restricted progenitor of myeloid lineage is an area of active investigation. Much less is known about which cell types give rise to radiation-induced or radiotherapy-related AML. We are exploring this question using a mouse model of radiation-induced AML. Several mouse strains including CBA/CaJ are susceptible to radiation-induced AML whereas other strains are resistant. In murine radiation-induced AML the PU.l (Sfpil) gene behaves as a classic tumor suppressor gene. In most murine myeloid leukemias, there is a deletion in chromosome 2 encompassing the PU.l gene. The second ‘hit’ is generally a point mutation in the remaining PU.l allele. We used the PU.l deletion as an early marker for potential radiation-induced AML, and performed immunophenotyping combined with fluorescent in situ hybridization (immunoFISH) to study the persistence of this AML initiating lesion in myeloid and lymphoid lineages. Bone marrow cells were harvested from CBA/CaJ mice irradiated with 3 Gy of 137Cs y-rays 1, 3 and 6 months post-radiation and assayed by immunoFISH for PU.l deletions and cell differentiation markers. We demonstrated that the frequency PU.l deletions were similar in different cell types 1 month after irradiation but increased in the myeloid lineage and decreased in the lymphoid lineage at 3 and 6 months post-irradiation. PU.l deletions were only found in myeloid but not in lymphoid lineages in mice with radiation-induced AML. These results indicate that radiation-induced AML is likely to originate from the more restricted progenitor of the myeloid lineage. Immunostaining the mouse bone marrow cells with a panel of cell differentiation markers, we were able to isolate phenotypically defined hematopoietic stem cells and progenitor subpopulations. In future experiments, studying cytogenetic events in those particular bone marrow subpopulations will help achieve a better understanding on pathogenesis of radiation-induced AML. Identifying a small fraction of bone marrow cells bearing point mutations in the remaining PU.l gene remained as a technical challenge to our study of radiation-induced AML. We assessed a novel in situ hybridization approach that employs padlock probes as a potential tool to detect a PU.l point mutation and found it would only be useful if modifications were made to increase the fidelity of this technology.Item Open Access In-vivo investigation of resveratrol as a preventive for radiation-induced acute myeloid leukemia(Colorado State University. Libraries, 2008) Carsten, Ronald E., author; Ullrich, Robert L., advisorResveratrol has been shown to have cancer preventive properties. It modulates a wide range of molecular targets including those involved with induction of apoptosis and cell cycle arrest in a concentration dependent fashion. This study was designed to investigate resveratrol's ability to reduce radiation-induced chromosome aberrations and PU.1 gene loss in mouse bone marrow cells. Loss of 1 PU.1 gene and missense mutation of the remaining allele leads to acute myeloid leukemia in CBA mice.Item Open Access Of mice, genes, and radiation: the genetics of non-hereditary breast cancer explored using the common laboratory BALB/c mouse(Colorado State University. Libraries, 2008) Ramaiah, Lila, author; Ullrich, Robert L., advisor; Benjamin, Stephen A., advisorIn this dissertation we describe the generation and characterization of two novel strains of mice carrying alternate genetic variants of the DNA repair gene Prkdc (DNA-PKcs). Strains congenic for the common (PrkdcB6) and variant (Prkdc BALB) alleles of Prkdc are developed, genotypically validated, and used to examine the functional consequences of Prkdc BALB and its linkage with radiation susceptibility. DNA-PKcs protein expression, post-irradiation double strand break repair, post-irradiation cell survival, breeding depression, and constitutive and radiation-induced gene expression are examined. By western blot we demonstrate that PrkdcBALB is required and sufficient to decrease DNA-PKcs protein expression. Using three different DSB repair quantification methods we show that PrkdcBALB is required for reduced radiation-induced DSB rejoining in BALB/c. We also show that Prkdc BALB is both sufficient and required for decreased cell survival after exposure to ionizing radiation. Thus we demonstrate that Prkdc BALB modulates and even diminishes the ability of cells to maintain genomic homeostasis. Using our newly developed congenic mice, we present the first evidence that PrkdcBALB has a significant effect on gene expression in unirradiated as well as irradiated mice. Microarray analysis of gene expression reveals that PrkdcBALB may have a greater impact on overall gene expression than does radiation, and that Prkdc may play a role in constitutive and DNA damage-induced apoptotic and transcriptional responses. The results presented within this dissertation support the hypothesis that the main role of PrkdcBALB in radiation-induced breast cancer is the initiation of mammary epithelial cells. Our data show that PrkdcBALB is strongly associated with diminished DNA-PKcs expression and function, diminished survival, and altered transcriptional regulation. The congenic strains developed and characterized in this proposal will be instrumental in ongoing studies aiming to clarify the role of Prkdc and genomic instability in radiation-induced mammary carcinogenesis in the BALB/c mouse. Future studies should endeavor to quantify DNA-PKcs specific kinase activity and protein metabolism and to evaluate cytogenetic instability, with particular emphasis on telomeres. The congenic strains developed and characterized in this work serve as compelling rodent models of sporadic and radiation-induced human breast cancer, and provide proof of principle for the role of genetic polymorphisms and genomic instability in breast cancer susceptibility.Item Open Access Prkdc polymorphisms and radiation effects(Colorado State University. Libraries, 2008) Askin, Kristin Fabre, author; Ullrich, Robert L., advisor; Weil, Michael M., advisorThe Prkdc gene encodes DNA-PKcs which is involved in the immune system, DNA repair and chromosomal integrity. In humans, deficiencies in DNA-PKcs are linked to cancer predisposition. This connection can also be observed in mice models using ionizing radiation as the carcinogenic inducer. In particular, the BALB/c mouse strain is susceptible to mammary cancer after radiation exposure. Subsequent studies have shown that DNA repair is deficient in BALB/c which is attributed to a hypomorphic variant of DNA-PKcs. DNA-PKcs has also been linked to apoptosis because BALB/c is more resistance to ionizing-induced apoptosis in intestinal crypt cells compared to other mouse strains. Furthermore, it has been demonstrated that DNA-PKcs is involved in telomere maintenance. Additional studies have revealed two polymorphisms in Prkdc BALB/c that may be responsible for its DNA-PKcs variant.