Irradiated cells induce chromosomal instability in unirradiated bystander cells in vitro. Although bystander effects are thought to be linked to radiation-induced secondary cancers, almost no studies have evaluated bystander effects in vivo. Furthermore, it has been proposed that epigenetic changes mediate bystander effects, but few studies have evaluated epigenetic factors in bystander tissues in vivo. Here, we describe studies in which mice were unilaterally exposed to X-irradiation and the levels of DNA damage, DNA methylation and protein expression were evaluated in irradiated and bystander cutaneous tissue. The data show that X-ray exposure to one side of the animal body induces DNA strand breaks and causes an increase in the levels of Rad51 in unexposed bystander tissue. In terms of epigenetic changes, unilateral radiation suppresses global methylation in directly irradiated tissue, but not in bystander tissue at given time-points studied. Intriguingly, however, we observed a significant reduction in the levels of the de novo DNA methyltransferases DNMT3a and 3b and a concurrent increase in the levels of the maintenance DNA methyltransferase DNMT1 in bystander tissues. Furthermore, the levels of two methyl-binding proteins known to be involved in transcriptional silencing, MeCP2 and MBD2, were also increased in bystander tissue. Together, these results show that irradiation induces DNA damage in bystander tissue more than a centimeter away from directly irradiated tissues, and suggests that epigenetic transcriptional regulation may be involved in the etiology of radiation-induced bystander effects.

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...

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow directrepeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 ؎ 0.1 per 10 6 cell divisions. Interestingly, the rate is Ϸ10-fold greater in fibroblasts derived from embryonic tissue. We observe an Ϸ15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.H uman cells incur Ϸ10 6 base lesions per day (1), many of which inhibit DNA replication and͞or induce DNA strand breaks. Homology-directed repair provides an important strategy for preventing toxicity caused by such DNA lesions. More specifically, when the replication machinery stalls, recombination between sister chromatids can replace the damaged template with an undamaged copy (2). In addition, should a replication fork collapse to form a double-strand break (e.g., because of an encounter with a single-strand break in the template DNA), the fork can be repaired by homology-directed reinsertion of the broken end (3-5). Thus, the frequency of recombination reflects the levels of certain types of DNA damage.Repair and lesion-avoidance pathways that involve homology searching are integral to DNA replication (3-7). It is estimated that Ϸ10 double-strand breaks are formed each time the mammalian genome is replicated (3), and proteins that are essential for homologous recombination (e.g., Rad51) are also essential for life (8-10). Most of the time, sequences are aligned perfectly, and flanking sequences are not exchanged. However, misalignments may result in deletions, and exchanges between homologous chromosomes may lead to loss of heterozygosity, events that are known to promote cancer (11)(12)(13)(14).A useful approach to studying recombination is to engineer two mutant expression cassett...

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