Mitotic homologous recombination (HR) is critical for the repair of double-strand breaks, and conditions that stimulate HR are associated with an increased risk of deleterious sequence rearrangements that can promote cancer. Because of the difficulty of assessing HR in mammals, little is known about HR activity in mammalian tissues or about the effects of cancer risk factors on HR in vivo. To study HR in vivo, we have used fluorescent yellow direct repeat mice, in which an HR event at a transgene yields a fluorescent phenotype. Results show that HR is an active pathway in the pancreas throughout life, that HR is induced in vivo by exposure to a cancer chemotherapeutic agent, and that recombinant cells accumulate with age in pancreatic tissue. Furthermore, we developed an in situ imaging approach that reveals an increase in both the frequency and the sizes of isolated recombinant cell clusters with age, indicating that both de novo recombination events and clonal expansion contribute to the accumulation of recombinant cells with age. This work demonstrates that aging and exposure to a cancer chemotherapeutic agent increase the frequency of recombinant cells in the pancreas, and it also provides a rapid method for revealing additional factors that modulate HR and clonal expansion in vivo.aging ͉ homologous recombination ͉ mutation ͉ chemotherapy C ells are constantly exposed to endogenous and exogenous DNA-damaging agents that can lead to double-strand breaks, either by causing breaks in both strands of DNA or by causing replication fork breakdown (1). Homologous recombination (HR) is critical for repairing double-strand breaks in mammalian cells. By using homologous DNA sequences present on the sister chromatid or homologous chromosome, damage can be repaired accurately without loss of sequence information (2, 3). Thus, the frequency of HR reflects both the levels of double-strand breaks and the ability of cells to use HR during DNA repair.Although HR is generally error-free, recombination between misaligned sequences can cause insertions, deletions, and translocations. Furthermore, recombination between homologous chromosomes can lead to loss of heterozygosity (4), and HR has been estimated to be the underlying cause of loss of heterozygosity 25-80% of the time in mammalian cells (e.g., see ref. 5). Germ-line mutations in genes that modulate the frequency of HR are associated with an increased risk of cancer. For example, inherited mutations in the HR helicases BLM and WRN lead to increased rates of HR (6, 7) and increase the risk of cancer (8).Whereas too much HR can be problematic, too little HR can also destabilize the genome, possibly as a result of nonhomologous end-joining of DNA ends created at broken replication forks (4, 9). In the pancreas, inherited mutations in BRCA1 (8), BRCA2 (10), and FANCC (11) increase the risk of pancreatic cancer, and loss of function of these genes suppresses HR (12-14), causing an increased frequency of tumorigenic sequence rearrangements (15,16). Although these findings sug...

Background: In the event of a nuclear accident, people are exposed to elevated levels of continuous low dose-rate radiation. Nevertheless, most of the literature describes the biological effects of acute radiation.Objectives: DNA damage and mutations are well established for their carcinogenic effects. We assessed several key markers of DNA damage and DNA damage responses in mice exposed to low dose-rate radiation to reveal potential genotoxic effects associated with low dose-rate radiation.Methods: We studied low dose-rate radiation using a variable low dose-rate irradiator consisting of flood phantoms filled with 125Iodine-containing buffer. Mice were exposed to 0.0002 cGy/min (~ 400-fold background radiation) continuously over 5 weeks. We assessed base lesions, micronuclei, homologous recombination (HR; using fluorescent yellow direct repeat mice), and transcript levels for several radiation-sensitive genes.Results: We did not observe any changes in the levels of the DNA nucleobase damage products hypoxanthine, 8-oxo-7,8-dihydroguanine, 1,N6-ethenoadenine, or 3,N4-ethenocytosine above background levels under low dose-rate conditions. The micronucleus assay revealed no evidence that low dose-rate radiation induced DNA fragmentation, and there was no evidence of double strand break–induced HR. Furthermore, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2. Importantly, the same total dose, when delivered acutely, induced micronuclei and transcriptional responses.Conclusions: These results demonstrate in an in vivo animal model that lowering the dose-rate suppresses the potentially deleterious impact of radiation and calls attention to the need for a deeper understanding of the biological impact of low dose-rate radiation.

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The clonal expansion of mutant cells is hypothesized to be an important first step in cancer formation. To understand the earliest stages of tumorigenesis, a method to identify and analyze clonal expansion is needed. We have previously described transgenic Fluorescent Yellow Direct Repeat (FYDR) mice in which cells that have undergone sequence rearrangements (via homologous recombination events) express a fluorescent protein, enabling fluorescent labeling of phenotypically normal cells. Here, we develop an integrated one-and two-photon imaging platform that spans four orders of magnitude to permit rapid quantification of clonal expansion in the FYDR pancreas in situ. Results show that as mice age there is a significant increase in the number of cells within fluorescent cell clusters, indicating that pancreatic cells can clonally expand with age. Importantly, >90% of fluorescent cells in aged mice result from clonal expansion, rather than de novo sequence rearrangements at the FYDR locus. The spontaneous frequency of sequence rearrangements at the FYDR locus is on par with that of other classes of mutational events. Therefore, we conclude that clonal expansion is one of the most important mechanisms for increasing the burden of mutant cells in the mouse pancreas.aging ͉ cancer ͉ homologous recombination ͉ imaging ͉ pancreas C ancer is caused by the accumulation of mutations within a single cell lineage. This multistep process occurs through successive rounds of clonal expansion and selection of cells that have acquired mutations that confer growth advantages (1-4). Although the clonal expansion of premalignant cells is hypothesized to be an important precursor to the development of cancer (3), no methods have been developed for studying clonal expansion within intact histologically normal tissue.Mitotic homologous recombination (HR) events are an important class of mutations that can promote tumorigenesis (5). During the repair of DNA double strand breaks by HR, homologous DNA is used as a template for repair (for review see ref.6). Although HR is considered to be error-free, recombination between misaligned sequences can occur, resulting in deleterious sequence rearrangements (5). Given the inherent risk of recombinational repair of DNA damage, it is not surprising that genetic and environmental factors that stimulate HR are risk factors for cancer (7)(8)(9)(10)(11)(12)(13)(14)(15).To study HR in vivo, we used transgenic Fluorescent Yellow Direct Repeat (FYDR) mice, in which a HR event at an integrated transgene results in expression of a fluorescent protein (16). Our previous work shows that recombinant cells accumulate in the pancreas of FYDR mice with age as the result of de novo recombination events. In addition, results suggest that clonal expansion of previously existing recombinant cells may also contribute to the accumulation of recombinant cells (17). The extent of clonal expansion had been assessed by using standard wide-field fluorescence microscopy, but because of the inherent limitations of standard...

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm 3 /h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10 5 . This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important. ' International Society for Analytical CytologyKey terms 3D image cytometry; tissue cytometry; rare cell detection; multiphoton microscopy CYTOMETRY is the quantitative measurement of the physical and biochemical states of cell populations. Cytometry provides information of the cell population on a cellby-cell basis rather than the population average. Many cytometric approaches are high-throughput and allow for categorizing large cell populations into subgroups revealing rare subpopulations. Flow cytometry is a widely used technique in which cellular specimens are prepared in fluid suspensions and the properties of individual cells are measured in a narrow fluid stream (1-4). The properties of cells are assayed based on optical characteristics, such as fluorescence, light scattering, and light absorption. Flow cytometry has very high throughput reaching a rate up to 10,000 cells/s (3). In combination with a cell sorting apparatus, precise physical separation of the cellular subpopulations is routinely achieved. Flow cytometry is an indispensa-