Correlation of DNA double strand break repair efficiency and susceptibility to lung tumor development
Date
2015
Authors
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 member
Journal Title
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Volume Title
Abstract
In 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.
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Subject
double strand break
lung tumor
ionizing radiation
DNA repair