UV Radiation Induces Delayed Hyperrecombination Associated with Hypermutation in Human Cells

Abstract

Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an ϳ5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m 2 ), revealed as mixed GFP ؉/؊ colonies. UV-B did not induce DHR at an equitoxic (75 J/m 2 ) dose or a higher dose (150 J/m 2 ). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were ϳ5-fold higher in strains derived from GFP ؉/؊ (DHR) colonies than in strains in which recombination was directly induced by UV (GFP ؉ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP ؉ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP ؉/؊ strains were small (1-to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression.UV radiation elicits many cellular stress responses attributed to its induction of reactive oxygen species (ROS) and its ability to directly damage DNA, predominantly forming cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone 6-4 photoproducts (12,38,68). UV also indirectly produces single-strand breaks and double-strand breaks (DSBs) during DNA replication (42). Because UV can produce strand breaks and other DNA lesions in several ways, it is a powerful mutagen and carcinogen. Solar UV-A (320 to 400 nm) and the higher-energy and shorter wavelength UV-B (290 to 320 nm) penetrate the Earth's atmosphere, and chronic exposures have been linked to melanoma, the most fatal form of skin cancer (56). UV-C (100 to 290 nm) has the highest energy but is blocked by the ozone layer. However, UV-C exposures are possible from artificial sources, including germicidal lamps, arc welding equipment, and mercury arc lamps in older tanning beds. A significant fraction of UV damage is repaired by the nucleotide excision repair system, and patients with nucleotide excision repair defects show marked susceptibility to skin cancer (1, 27, 56). Human nonmelanoma skin cancer correlates with sun exposure (1) and is often associated with p53 mutations at dipyrimidines...

Low- and High-LET Ionizing Radiation Induces Delayed Homologous Recombination that Persists for Two Weeks before Resolving

“…In prior studies DHR was detected as mixed GFP +/− colonies arising 7–10 days, after derivatives of human RKO or hamster–human hybrid GM10115 cells were exposed to low-to-moderate doses of ionizing radiation or ultraviolet (UV) radiation (22–24). In the current study, we modified the assay to detect DHR over a longer time period and to expand the range of detectable outcomes.…”

“…Ionizing and nonionizing radiation also induce late effects seen many cell generations after exposure, including mutations, chromosome translocations, chromosome aberrations, micronuclei, microsatellite instability, giant cells and cell death (17–21). We defined delayed hyperrecombination (DHR) as a novel delayed event induced by ionizing and nonionizing radiation (22–24). Although both delayed chromosomal instability (DCI) and DHR are induced by low to moderate radiation doses at high frequencies (indicative of nontargeted effects), DCI and DHR arise through distinct mechanisms, since delayed death is associated with DCI, but not DHR (24).…”

Targeted and Nontargeted Effects of Low-Dose Ionizing Radiation on Delayed Genomic Instability in Human Cells

“…Genomic instability was evaluated in RKO36 cells by analysis of homologous recombination and mutation/deletion at the GFP direct repeat substrate and measured as the number of GFP ). Genomic instability, as measured by this GFP-based assay, is defined by colonies with both GFP + and GFP À cells, with >10 cells of each type in a colony (23,26). The frequency of induced instability was calculated as the number of GFP +/À colonies per total surviving colonies scored.…”

Genomic landscape of intracranial meningiomas

“…Such instability could be innate to an oncogenic driver within the incipient tumor or induced by external factors. 3,4,29 Despite the recognition of recurrent chromosomal changes in malignant progression of meningiomas, underlying gene targets that may be driving such changes largely remain elusive. It may be that gains and losses of specific chromosomal arms reflect a more pervasive underlying genomic instability, resulting from the convergence of several aberrant signaling pathways.…”