Browsing by Author "Weil, Michael M., advisor"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Open Access Comparison of radiobiological endpoints in cells from CXB RI mice(Colorado State University. Libraries, 2008) Xiao, Guanxiong, author; Weil, Michael M., advisorRecombinant inbred (RI) mouse strains have been used both for trait cosegregation studies and genetic linkage analysis. They are created by using a breeding scheme that consists of a cross between two inbred mouse strains (progenitor strains) followed by at least 20 generations of brother-sister inbreeding. Thus, RI strains are inbred (homozygous at every locus) and derive roughly half their genome from each of the two progenitor strains. The CXB RI strain set consists of 13 RI strains derived from matings of BALB/c (C) and C57BL/6(B) mice. The CXB progenitor strains, BALB/c and C57BL/6, differ in their susceptibility to radiation-induced mammary tumors with BALB/c being susceptible and C57BL/6 being resistant. In part, the susceptibility difference can be explained by a polymorphism in the Prkdc gene which encodes the catalytic subunit of DNA-dependent protein kinase. However, other, as yet unknown, loci may be involved. The CXB RI strain set provides a useful tool to unravel the events that lead to radiation-induced mammary tumorigenesis and to understand the interrelationships of cellular radiobiological endpoints to one-another. We have generated fibroblast strains from each of the CXB RI strains and from the progenitor strains. The fibroblast strains were assayed for a number of radiobiological endpoints including clonogenic survival following acute and low dose-rate exposures, γ-H2AX focus formation and clearance following acute and low dose-rate exposures, and G2 chromosomal aberrations. In addition, we genotyped the strains for a polymorphism in the gene encoding the catalytic subunit of the DNA-dependent protein kinase, Prkdc. We then determined the correlations of different endpoints between the RI strains. As expected, clonogenic survival at low dose rates and following acute exposures were positively correlated. γ-H2AX focus formation at low dose rate correlated well with survival endpoints, particularly clonogenic survival under low dose rate irradiation and the surviving fraction at 2 Gy acute exposures. These three endpoints are all significantly associated with the Prkdc genotype with radiosensitive strains having the BALB/c genotype. The data we have collected provides a baseline description of cellular radio sensitivity in CXB fibroblasts. The approach used in this dissertation can be used to correlate these cellular radiobiological endpoints with susceptibility to clinically significant adverse outcomes from cancer radiotherapy, such as normal tissue injury and radiation-induced second cancers.Item Open Access Correlation of DNA double strand break repair efficiency and susceptibility to lung tumor development(Colorado State University. Libraries, 2015) Ochola, Donasian Ocan, author; Weil, Michael M., advisor; Bedford, Joel S., advisor; Thamm, Douglas M., committee member; Dow, Steven W., committee member; Kato, Takamitsu A., committee memberIn this dissertation we describe the use of many CcS/Dem recombinant congenic strains (RCS) of mice to determine if there is any correlation between the DNA double strand break (DSB) repair efficiency and susceptibility to lung tumor development. A previous study involving 20 different CcS/Dem RCS of mice all derived from cross of BALB/c x STS progenitors (BALB/c is the recipient strain that is susceptible to tumor development and STS the donor is resistant) showed wide inter-strain variations in susceptibility to radiation-induced lung tumor development. As formalin fixation was used to obtain paraffin embedded tissue sections for immunofluorescence, we first evaluated different methods of euthanasia, perfusion techniques, autofluorescence reduction and antigen retrieval methods to optimize the procedures used so as to obtain reproducible results. The formation of phosphorylated histone H2AX (γ-H2AX) into discrete foci was used as the marker for DSB repair and its co-localization with 53BP1, another component of repair foci, was examined during the optimization. From the optimization phase, CO₂ asphyxiation, right ventricular perfusion, use of sodium borohydride for quenching autofluorescence and the use of sodium citrate for heat-induced epitope retrieval (antigen retrieval) gave very good quality images and were adopted for use in all subsequent experiments. To explore a possible link between heritable differences in DNA DSB repair efficiency and susceptibility to RI lung cancer in a mouse model, we quantified residual γH2AX foci in lungs of 16 different CcS/Dem RCS mice together with their founders after irradiation from a ¹³⁷Cs source of γ-rays at a low-dose rate of 10 cGy/hr for 24 h. We also explored residual γH2AX foci in the peripheral blood leukocytes to compare it with foci in the lungs with the intention of using PBLs as a surrogate to assess DNA repair efficiency in the lungs for possible use in clinical applications to pre-screening patients and assess their suitability as candidates for radiotherapy, especially in fairly young. In the lungs, the results showed a high correlation between mean residual γH2AX foci number per nucleus and radiation-induced lung tumor observed in the previous study (R=0.968, p <0.0001) indicating that γH2AX can be used as a good predictor of the potential to develop RI lung tumor. Though the absolute mean values of foci in PBLs and the lungs were different, the inter-strain differences in DNA repair efficiencies correlated very closely to those in the lungs for all the six strains that were compared (Spearman rank correlation coefficient, R = 0.948), and indication that PBLs are good surrogates for DNA DSB repair efficiency in the lungs of CcS/Dem RCS mice. These results indicate that γH2AX can be used as a good predictor of the potential to develop RI lung tumor, and that, measuring γH2AX foci in PBLs can be used as a surrogate to determine DDR in the lungs of the CcS/Dem RCS mice. Since the product of the Prkdc gene is known to be an important player in the DDR, we tested whether the R2140C substitution has a major influence on DSB repair and a determinant in the foci counts difference in the CcS/Dem strains. We amplified the segment of the Prkdc gene that contains the c>t transition resulting in amino acid substitution that abolishes a BsmBI restriction site. The outcome of these restriction patterns suggests no direct correlation between DNA-PK and DSB repair efficiency and that another gene (or other genes) polymorphic between BALB/c and STS/A may determine the strain differences in DSB repair efficiencies.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.