Age-Dependent Alterations in Meiotic Recombination Cause Chromosome Segregation Errors in Spermatocytes.

Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.

Genetic dissection of crossover mutants defines discrete intermediates in mouse meiosis.

Crossovers (COs), the exchange of homolog arms, are required for accurate chromosome segregation during meiosis. Studies in yeast have described the single-end invasion (SEI) intermediate: a stabilized 3' end annealed with the homolog as the first detectible CO precursor. SEIs are thought to differentiate into double Holliday junctions (dHJs) that are resolved by MutLgamma (MLH1/MLH3) into COs. Currently, we lack knowledge of early steps of mammalian CO recombination or how intermediates are differentiated in any organism. Using comprehensive analysis of recombination in thirteen different genetic conditions with varying levels of compromised CO resolution, we infer CO precursors include asymmetric SEI-like intermediates and dHJs in mouse. In contrast to yeast, MLH3 is structurally required to differentiate CO precursors into dHJs. We verify conservation of aspects of meiotic recombination and show unique features in mouse, providing mechanistic insight into CO formation.

Ultraviolet A does not induce melanomas in a Xiphophorus hybrid fish model.

We examined the wavelength dependence of ultraviolet (UV) ra-diation (UVR)-induced melanoma in a Xiphophorus backcross hybrid model previously reported to be susceptible to melanoma induction by ultraviolet A (UVA) and visible light. Whereas ultraviolet B (UVB) irradiation of neonates yielded high frequencies of melanomas in pigmented fish, UVA irradiation resulted in melanoma frequencies that were not significantly different from unirradiated fish. Spontaneous and UV-induced melanoma frequencies correlated with the degree of pigmentation as expected from previous studies, and the histopathology phenotypes of the melanomas were not found in significantly different proportions in UV-treated and -untreated tumor-bearing fish. Our results support the conclusion that a brief early-life exposure to UVB radiation causes melanoma formation in this animal model. These data are consistent with an essential role for direct DNA damage, including cyclobutane dimers and (6-4) photoproducts, in the etiology of melanoma.

Molecular response of nasal mucosa to therapeutic exposure to broad-band ultraviolet radiation.

Ultraviolet radiation (UVR) phototherapy is a promising new treatment for inflammatory airway diseases. However, the potential carcinogenic risks associated with this treatment are not well understood. UV-specific DNA photoproducts were used as biomarkers to address this issue. Radioimmunoassay was used to quantify cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA purified from two milieus: nasal mucosa samples from subjects exposed to intranasal phototherapy and human airway (EpiAirway) and human skin (EpiDerm) tissue models. Immunohistochemistry was used to detect CPD formation and persistence in human nasal biopsies and human tissue models. In subjects exposed to broadband ultraviolet radiation, DNA damage frequencies were determined prior to as well as immediately after treatment and at increasing times post-treatment. We observed significant levels of DNA damage immediately after treatment and efficient removal of the damage within a few days. No residual damage was observed in human subjects exposed to multiple UVB treatments several weeks after the last treatment. To better understand the molecular response of the nasal epithelium to DNA damage, parallel experiments were conducted in EpiAirway and EpiDerm model systems. Repair rates in these two tissues were very similar and comparable to that observed in human skin. The data suggest that the UV-induced DNA damage response of respiratory epithelia is very similar to that of the human epidermis and that nasal mucosa is able to efficiently repair UVB induced DNA damage.

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments.

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate approximately 100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be approximately 400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.

Interspecific variation in the repair of UV damaged DNA in the genus Xiphophorus as a factor in the decline of the Rio Grande Platyfish.

The fish genus Xiphophorus consists of 26 species distributed along the eastern slopes of mountain ranges extending from northern Mexico to Belize and Nicaragua. We analyzed light-dependent repair of UV-induced DNA damage in at least two species from each of the four monophyletic Xiphophorus groups. We found that the northern platyfish had significantly reduced photoenzymatic repair compared to the other three groups, including the northern swordtails, southern platyfish and southern swordtails. All of the species of the northern platyfish, including the Marbled (meyeri), Northern (gordoni) and Monterrey Platyfish (couchianus) are the northernmost species in the genus and are the only three species in the genus that are currently found on the IUCN Red List of Threatened Species. Satellite data from the past 30 years (1979-2008) correlate greater increases in shorter wavelength UVB with higher latitudes within the Xiphophorus range. We suggest that, combined with other consequences of human population growth, anthropogenic deozonation resulting in a disproportionate increase in UVB in temperate latitudes may be a contributing factor in the decline and extirpation of the northern platyfish.

Acute exposure to ultraviolet-B radiation modulates sex steroid hormones and receptor expression in the skin and may contribute to the sex bias of melanoma in a fish model.

Using the Xiphophorus fish melanoma model, we show a strong male bias for sunlight-induced malignant melanoma, consistent with that seen in the human population. To examine underlying factors, we exposed adult X. couchianus fish to a single, sublethal dose of UVB and measured circulating sex steroid hormones and expression of associated hormone receptor genes over a 24-h period. We found that a single exposure had profound effects on circulating levels of steroid hormones with significant decreases for all free sex steroids at 6 and 24 h and increases in conjugated 2-estradiol and 11-ketotestosterone at 6 and 24 h, respectively. Whereas ARα expression increased in male and female skin, neither ARß nor either of the ERs showed significant responses to UVB in either sex. The rapid response of male androgens and their receptors in the skin after UVB irradiation implicates hormones in the male bias of skin cancer and suggests that the photoendocrine response immediately after UV exposure may be relevant to melanomagenesis.

Nucleotide excision repair is reduced in oral epithelial tissues compared with skin.

Ultraviolet radiation (UVR) exposure to internal tissues for diagnostic, therapeutic and cosmetic procedures has increased dramatically over the past decade. The greatest increase in UVR exposure of internal tissues occurs in the cosmetic industry where it is combined with oxidizing agents for teeth whitening, often in conjunction with indoor tanning. To address potential carcinogenic risks of these procedures, we analyzed the formation and repair of the DNA photoproducts associated with the signature mutations of UVR. Radioimmunoassay was used to quantify the induction and repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts in DNA purified from three reconstructed tissues, EpiDerm(TM) , EpiGingival(TM) and EpiOral(TM) . We observed comparable levels of DNA damage in all tissues immediately after UVR exposure. In contrast, repair was significantly reduced in both oral tissues compared with EpiDerm(TM) . Our data suggest that UVR exposure of oral tissues can result in accumulation of DNA damage and increase the risk for carcinoma and melanoma of the mouth. Because NER is a broad-spectrum defense against DNA damage caused by a variety of agents in addition to UVR, our data suggest that the relatively low NER efficiency observed in oral tissues may have wide-ranging consequences in this highly exposed environment.

Recent advances in sunlight-induced carcinogenesis using the Xiphophorus melanoma model.

Unlike breast and prostate cancers, the nature and sequence of critical genetic and epigenetic events involved in the initiation and progression of melanoma are not well understood. A contributing factor to this dilemma, especially given our current understanding of the importance of UV light in melanoma etiology, is the lack of quality UV-inducible melanoma animal models. In this study we elaborate on the capability of UV light to induce cutaneous malignant melanomas (CMM) in Xiphophorus fishes, which were previously found to develop melanomas after acute neonatal UVB irradiation. In two separate tumorigenesis experiments, we exposed adult Xiphophorus hybrids to either acute UVB irradiations (5 consecutive daily treatments) or chronic solar irradiations (continuous UVA/UVB treatment for 9 months). Acute adult UVB irradiation resulted in the significant induction of melanomas, and moreover, this induction rate is equivalent to that of animals exposed to acute neonatal UVB irradiation. This study represents the first evidence that acute adult UVB irradiation, in the absence of any early life exposures, induces CMM. Similar to the findings conducted on other divergent melanoma models, including HGF/SF transgenic mice and Monodelphis domestica, prolonged chronic solar UV was not a factor in melanomagenesis.

An experimental population study of nucleotide excision repair as a risk factor for UVB-induced melanoma.

Nucleotide excision repair (NER) is the primary defense against the DNA damage implicit in skin cancer formation and is negatively affected by chronic exposure to UVB radiation. However, in situ and in vitro studies consistently yield equivocal results when addressing individual DNA repair capacity and melanoma susceptibility. The primary objective of this study was to determine if individual global NER capacity is a risk factor for melanoma formation in a prominent UVB-inducible melanoma model, hybrid Xiphophorus fishes. After neonatal UVB irradiation, adult tumor-bearing and tumor-free fish were given a challenge UVB dose and (6-4) photoproduct repair was quantified in individual fish at 24 h using radioimmunoassay. Despite considerable inter-individual variation in repair capacity, ranging from 13% to 91%, we found no difference in mean NER capacity between fish with and without melanomas, thus detaching global NER from melanomagenesis. Furthermore, despite epidemiological data indicating that sex and age are important risk factors underlying melanoma susceptibility, we found no difference in mean NER rates among the sexes or as a function of age. We conclude with a discussion of the apparent paradox of how inter-individual variation in NER is not a risk factor given the clear evidence that DNA damage underlies melanoma susceptibility.

DNA damage, repair and photoadaptation in a Xiphophorus fish hybrid.

Exposure to sunlight is responsible for most cutaneous malignant melanomas in the human population. It is very likely that DNA damage is an initial event in melanomagenesis, however, the role played by this damage is an open question. To this end, we used a hemipigmented F(1) hybrid of the fish genus Xiphophorus and HPLC tandem mass spectrometry to examine the effects of melanin on the induction and repair of the predominant UV-induced photoproducts formed in skin cell DNA. We found that heavily pigmented skin cells had about half the damage of nonpigmented cells and the relative induction of the major photoproducts was independent of the degree of pigmentation. The efficiency of photoenzymatic repair was the same in nonpigmented and pigmented areas of the fish. We found no evidence of residual damage at 10 days after the last exposure. Most striking was that repeated exposure to multiple doses of UVB caused a very significant photoadaptive response. Rather than an accumulation of damage after five doses of UVB we saw a significant reduction in the amount of damage induced after the final dose compared with the initial dose. The relevance of these observations is discussed in the context of melanoma susceptibility and UVB thresholds.

The etiology of sunlight-induced melanoma in Xiphophorus hybrid fish.

In contrast to sunlight-induced squamous cell carcinoma the etiology of cutaneous malignant melanoma (CMM) is not well understood. In particular, the role that sunlight exposure and DNA damage play in the initiation of this deadly form of cancer is an open question. Early UV carcinogenesis studies in the Xiphophorus backcross hybrid fish model by Richard Setlow indicated that direct DNA damage caused by exposure to the UVB component of sunlight is necessary and sufficient for melanoma formation. Subsequent studies by Setlow suggested that monochromatic UVA radiation that is not directly absorbed by DNA was also sufficient for melanoma induction in Xiphophorus and was, indeed, primarily responsible for initiating human melanoma. These results had significant public health consequences, suggesting that although sunscreens may inhibit UVB-induced erythema they may actually increase exposure to the UVA wavelengths that cause cancer. An intensive worldwide public debate on sunscreen use and "abuse" ensued. Our data do not support a major role of free radical chemistry in melanoma induction. We find evidence that the direct damage caused by the absorption of UVB wavelengths by DNA (e.g., the cyclobutane pyrimidine dimer or CPD) is required for CMM formation and that the ability to repair these lesions plays a significant role in tumor susceptibility. Using the Xiphophorus backcross hybrid fish we are currently in the process of re-evaluating the wavelength- and DNA damage-dependence of UV-induced melanoma and the role nucleotide excision repair and the genes controlling DNA repair and the UV response play in melanoma resistance. From these studies we hope to define the effective solar wavelength boundaries of melanoma, identify the class of critical DNA damage and elucidate the role of DNA repair in tumor suppression.