The impact of age, exposure and genetics on homologous recombination at the engineered repeat sequence in mice

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references.Mitotic homologous recombination is a critical pathway for the repair of DNA double-strand breaks and broken replication forks. Although homologous recombination is generally error-free, recombination between misaligned sequences can lead to deleterious sequence rearrangements, and conditions that stimulate homologous recombination are associated with an increased risk of cancer. To study homologous recombination in vivo, we used Fluorescent Yellow Direct Repeat (FYDR) mice in which a homologous recombination event at a transgene yields a fluorescent cell. To study homologous recombination using FYDR mice, we developed one- and two-photon in situ imaging techniques that reveal both the frequency and the sizes of isolated recombinant cell clusters within intact pancreatic tissue. We then applied these tools to analyze the effects of cancer risk factors such as exposure, genetic predisposition and age on homologous recombination in vivo. To determine the effect of exposure to exogenous carcinogens on homologous recombination, FYDR mice were treated with two different chemotherapeutic agents, cisplatin and mitomycin-C.(cont.) Results show that exposure to these DNA damaging agents causes an induction of recombinant pancreatic cells in vivo, indicating that homologous recombination is an active repair pathway in adult pancreatic cells and that exposure to certain carcinogens stimulates recombinational repair. As a first step towards exploring the effect of genetic predisposition to genomic instability on homologous recombination in vivo, FYDR mice were crossed with mice carrying a defect in p53, a critical tumor suppressor that is mutated in almost 50% of all human tumors. Although loss of p53 is known to promote genomic instability, results show that p53 status does not significantly affect the spontaneous recombinant cell frequency in the pancreas in vivo or the rate of homologous recombination in cultured fibroblasts in vitro. Age is a risk factor for many types of cancers. Here we examined the effect of age on homologous recombination in two tissues of FYDR mice, pancreas and skin. In the pancreas, a dramatic accumulation of recombinant cells is seen with age, resulting from both de novo recombination events and clonal expansion of recombinant cells. In contrast, the skin shows no increase in recombinant cell frequency with age.(cont.) In vitro studies using primary fibroblasts indicate that the ability to undergo homologous recombination in response to endogenous and exogenous DNA damage does not significantly change with age, suggesting that these skin cells are able to undergo de novo homologous recombination events in aged mice. Thus, we propose that tissue-specific differences in the accumulation of recombinant cells with age result from differences in the ability of these cells to persist and clonally expand within the tissue. To further characterize the FYDR mice as a tool for studying homologous recombination, we exploited positive control FYDR-Recombined mice in which all cells carry the full-length coding sequence for enhanced yellow fluorescent protein. Studies show that expression of the FYDR transgene varies among mice, among tissues, and even among cells within a tissue. However, the variation in FYDR expression does not significantly change with age or exposure to exogenous carcinogens. Furthermore, positive control mice reveal that several tissues, in addition to the pancreas and skin, may be amenable for studying homologous recombination in the FYDR mice.(cont.) Thus, our studies demonstrate that FYDR mice combined with in situ imaging technology provide powerful tools to study the effects of cancer risk factors on homologous recombination in vivo. Ultimately, by applying these techniques to study additional cancer risk factors, we may better understand the relationship between DNA damage, homologous recombination and cancer.by Dominika M. Wiktor-Brown.Ph.D

p53 null Fluorescent Yellow Direct Repeat (FYDR) mice have normal levels of homologous recombination

The tumor suppressor p53 is a transcription factor whose function is critical for maintaining genomic stability in mammalian cells. In response to DNA damage, p53 initiates a signaling cascade that results in cell cycle arrest, DNA repair or, if the damage is severe, programmed cell death. In addition, p53 interacts with repair proteins involved in homologous recombination. Mitotic homologous recombination (HR) plays an essential role in the repair of double-strand breaks (DSBs) and broken replication forks. Loss of function of either p53 or HR leads to an increased risk of cancer. Given the importance of both p53 and HR in maintaining genomic integrity, we analyzed the effect of p53 on HR in vivo using Fluorescent Yellow Direct Repeat (FYDR) mice as well as with the sister chromatid exchange (SCE) assay. FYDR mice carry a direct repeat substrate in which an HR event can yield a fluorescent phenotype. Here, we show that p53 status does not significantly affect spontaneous HR in adult pancreatic cells in vivo or in primary fibroblasts in vitro when assessed using the FYDR substrate and SCEs. In addition, primary fibroblasts from p53 null mice do not show increased susceptibility to DNA damage-induced HR when challenged with mitomycin C. Taken together, the FYDR assay and SCE analysis indicate that, for some tissues and cell types, p53 status does not greatly impact HR.National Institute of Environmental Health Sciences (ES02109)National Cancer Institute (U.S.) (R33CA112151)National Cancer Institute (U.S.) (R01CA79827)United States. Dept. of Energy (DE-FG01-04ER04-21)National Institute of Environmental Health Sciences (T32 ES007020, NIEHS Training Grant in Environmental Toxicology)National Science Foundation (U.S.) (Fellowship

Irradiated Esophageal Cells are Protected from Radiation-Induced Recombination by MnSOD Gene Therapy

Radiation-induced DNA damage is a precursor to mutagenesis and cytotoxicity. During radiotherapy, exposure of healthy tissues can lead to severe side effects. We explored the potential of mitochondrial SOD (MnSOD) gene therapy to protect esophageal, pancreatic and bone marrow cells from radiation-induced genomic instability. Specifically, we measured the frequency of homologous recombination (HR) at an integrated transgene in the Fluorescent Yellow Direct Repeat (FYDR) mice, in which an HR event can give rise to a fluorescent signal. Mitochondrial SOD plasmid/liposome complex (MnSOD-PL) was administered to esophageal cells 24 h prior to 29 Gy upper-body irradiation. Single cell suspensions from FYDR, positive control FYDR-REC, and negative control C57BL/6NHsd (wild-type) mouse esophagus, pancreas and bone marrow were evaluated by flow cytometry. Radiation induced a statistically significant increase in HR 7 days after irradiation compared to unirradiated FYDR mice. MnSOD-PL significantly reduced the induction of HR by radiation at day 7 and also reduced the level of HR in the pancreas. Irradiation of the femur and tibial marrow with 8 Gy also induced a significant increase in HR at 7 days. Radioprotection by intraesophageal administration of MnSOD-PL was correlated with a reduced level of radiation-induced HR in esophageal cells. These results demonstrate the efficacy of MnSOD-PL for suppressing radiation-induced HR in vivo.National Institutes of Health (U.S.) (NIH Grant R01-CA83876-8)National Institute of Allergy and Infectious Diseases (U.S.) (NIH grant U19A1068021)National Institutes of Health (U.S.) (Grant T32-ES07020)United States. Dept. of Energy (DOE DE-FG01-04ER04)National Institutes of Health (U.S.) (NIH P01-CA26735

Quantitative morphometric measurements using site selective image cytometry of intact tissue

Site selective two-photon tissue image cytometry has previously been successfully applied to measure the number of rare cells in three-dimensional tissue specimens up to cubic millimetres in size. However, the extension of this approach for high-throughput quantification of cellular morphological states has not been demonstrated. In this paper, we report the use of site-selective tissue image cytometry for the study of homologous recombination (HR) events during cell division in the pancreas of transgenic mice. Since HRs are rare events, recombinant cells distribute sparsely inside the organ. A detailed measurement throughout the whole tissue is thus not practical. Instead, the site selective two-photon tissue cytometer incorporates a low magnification, wide field, one-photon imaging subsystem that rapidly identifies regions of interest containing recombinant cell clusters. Subsequently, high-resolution three-dimensional assays based on two-photon microscopy can be performed only in these regions of interest. We further show that three-dimensional morphology extraction algorithms can be used to analyse the resultant high-resolution two-photon image stacks providing information not only on the frequency and the distribution of these recombinant cell clusters and their constituent cells, but also on their morphology