David Trono

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.

Genetic analysis of susceptibility to spontaneous and UV-induced carcinogenesis in Xiphophorus hybrid fish.

Abstract:Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC1) hybrids produced from backcrossing F1 hybrids derived from the platyfish X. maculatus Jp 163 A and the swordtail X. helleri to the X. helleri parental strain (the Gordon-Kosswig hybrid cross). Nodular melanomas originate in the dorsal fin from cells constituting the spotted dorsal (Sd) pigment pattern. A parallel genetic cross, with X. maculatus Jp 163 B, exhibits the spotted side (Sp) pigment pattern instead of Sd, and produces BC1 hybrids exhibiting a much lower frequency of spontaneous melanoma formation. These hybrids are susceptible to melanoma development if irradiated with UV light as fry. Other hybrids involving these two strains of X. maculatus and different swordtail and platyfish backcross parents also have been investigated as potential tumor models, and show differing susceptibilities to UV-induced and spontaneous melanomas. Genotyping of individual BC1 hybrids from several Xiphophorus crosses has implicated a locus, CDKN2X (a Xiphophorus homologue of the mammalian CDKN2 gene family, residing on Xiphophorus linkage group V), in enhancing pigmentation and the susceptibility to spontaneous and UV-induced melanoma formation in BC1 hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianusCDKN2X alleles in BC1 hybrids can predispose individuals to melanoma, but this susceptibility is modified in other crosses depending both on the contributing sex-linked pigment pattern locus from X. maculatus (Sd or Sp), and the genetic constitution of the backcross parent. Xiphophorus BC1 hybrids constitute unique genetic models offering the potential to analyze the contributions of specific genes to spontaneous and induced tumor formation in different, but comparable genetic backgrounds.

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.

Genetic Analysis of Neoplasia Induced by N-Nitroso-N-methylurea in Xiphophorus Hybrid Fish

Abstract: Interspecific crosses within the genus Xiphophorus have historically been used to study the genetic aspects of melanoma formation. Melanomas typically occur as a result of deregulation of polymorphic, naturally occurring macromelanophore pigment patterns. Hybrid crosses also have been used to study the inducibility of melanoma by physical sources (such as UV light) and chemicals (such as N-methyl-N-nitrosourea, MNU). We previously defined a genomic region that is implicated in fish melanomagenesis and identified a candidate tumor suppressor gene (CDKN2X) within this genomic area. Highly significant associations between BC1-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC1 hybrids are exposed to MNU as the tumor induction agent, a significant association between inheritance of CDKN2X alleles and tumor development is not observed. These data suggest there is mechanistic and genetic heterogeneity in melanomas derived from different etiologies within BC1 hybrid fish.

MNU Induction of Neoplasia in a Platyfish Model

Interspecific hybrid crosses between members of the fish genus Xiphophorus have been used for over 70 years to study the genetic aspects of melanoma formation. In the well-established “Gordon-Kosswig” cross, the platyfish X. maculatus is outcrossed to the swordtail X. helleri, and the resulting backcross segregants spontaneously develop melanoma. We recently produced a distinct cross between X. maculatus and another platyfish species, X. couchianus. X. maculatus strain Jp 163 A is homozygous for several X-linked pigment pattern genes, including the Spotted dorsal (Sd), Dorsal red (Dr), and Anal fin spot (Af). Af is a sex-limited trait, coding exclusively for melanophores distributed on the modified anal fin or “gonopodium” in the adult male fish. Within F1 and BC1 hybrids (to X. couchianus), the Sd pigment pattern is phenotypically suppressed, whereas Dr and Af are enhanced. We exposed BC1 hybrids to the direct-acting carcinogen N-methyl-N-nitrosourea (MNU). Treatment led to the development of schwannomas, fibrosarcomas, and retinoblastomas. In addition, numerous MNU-treated males that inherited Af developed a pronounced melanotic phenotype, with melanin-containing cells oftentimes totally covering the gonopodium and extending further to grow within the ventral regions of the fish. Genetic linkage analysis of the BC1 hybrids revealed a significant (p < 0.01) association between CDKN2X genotype and the phenotypic degree of melanization. Such an association is consistent with a locus within linkage group V playing a role in the development of melanosis and delineates three genetic preconditions and a carcinogenic scheme resulting in melanosis of the ventral regions of hybrid fish. The overall study further alludes to the potential of using Xiphophorus fish to study carcinogenic mechanisms for tumors other than melanoma (schwannoma, fibrosarcoma, and retinoblastoma) and should enable extensive pathologic and molecular genetic studies of derived neoplastic abnormalities.

The Genetic Map of Xiphophorus Fishes Represented by 24 Multipoint Linkage Groups

Hybrids between distinct Xiphophorus species have been utilized for over 70 years to study melanoma and other neoplasms that can develop spontaneously in hybrid offspring. Genetic linkage mapping has proven to be important in delineating genomic areas that harbor oncogenes and tumor suppressors. Within this report, two parallel backcrosses have been utilized to generate a genetic linkage map for Xiphophorus fishes. Isozyme/allozyme, RFLP and PCR-based mapping techniques, including AP-PCR/RAPDs and microsatellite loci were utilized. The derived linkage map provides a total of 403 mapped polymorphisms distributed among 24 linkage groups, representative of 24 acro- and telocentric chromosome pairs. Genomic coverage is approximately one marker per 5.8 cM. Detailed genotypic analysis of the utilized hybrids revealed two areas of the genome that show significant segregation distortion. Loci within the linkage group harboring the sex determining locus (LG 24) and an autosomal linkage group (LG 21) show highly significant deviations from Mendelian expectations. This phenomenon is not present in a hybrid cross that utilizes a different backcross hybrid progenitor species. The derived map with sequence-tagged markers provides a framework for physical map generation, large-scale genomic sequencing and will further enable cross-genome comparisons of vertebrate genomes.

Xiphophorus Genetic Linkage Map: Beginnings of Comparative Gene Mapping in Fishes

Abstract: The explosive expansion of gene maps of mouse and man has provided strong support for hypotheses first advanced from comparing fish and mammalian genomes that the vertebrate genome was derived from multiple ancestral tetraploidizations with subsequent preferential translocations among paralogous chromosomes. At least two genome duplication events have become widely accepted in lineages leading to vertebrates, and a third has been proposed either before, or after, divergence of fishes and tetrapods. Cytogenetic and comparative gene mapping studies suggest that teleost gene maps have diverged more slowly from gene arrangements in the vertebrate ancestor than have those of mammals. The recent assembly of extensive maps of >100 genes in three fish species, medaka (Beloniformes), Xiphophorus swordtails and platyfishes (Cyprinodontiformes), and zebrafish (Cypriniformes) and the development of less extensive maps in several other fish orders provides the first salient opportunity to assess homology of most or all chromosomes among fishes.

Melanoma susceptibility and cell cycle genes in Xiphophorus hybrids

Xiphophorus interspecies hybrids provide genetically defined models of both spontaneous and inducible melanomagenesis. In both models, backcrossing F1 hybrids of different strains of X. maculatus and X. helleri to a X. helleri parental fish results in segregation of melanoma susceptibility, fitting a Mendelian two‐gene inheritance model. The sex‐linked Xmrk oncogene is required for melanoma development in both crosses. The Xiphophorus CDKN2A/B gene, which is homologous to mammalian CDKN2A/B cyclin‐dependent kinase inhibitors (p16 and p15), is a candidate melanoma susceptibility gene. In this model, tumor susceptibility segregates with homozgyosity for CDKN2A/B from the recurrent X. helleri parent in backcross hybrids. We found that both CDKN2A/B mRNA and protein are highly overexpressed in melanoma. Because the p13 protein product of CDKN2A/B is a putative regulator of the G1 checkpoint, we investigated expression of other components of Xiphophorus G1 checkpoint control. By real‐time PCR analysis, retinoblastoma gene (RB) is consistently expressed twofold higher in both tumors and melanized skin than in normal tissue, indicating that RB is not downregulated by the overexpression of CDKN2A/B in Xiphophorus melanoma. We also found a significant correlation between the quantitative level of CDKN2A/B and Xmrk RNA in tumors, suggesting a functional relationship between Xmrk and CDKN2A/B expression. Although X. helleri CDKN2A/B protein contains a non‐conservative substitution, the biochemical function appears to show little overt defect. These studies indicate that in Xiphophorus melanoma, CDKN2A/B is functionally insufficient to mediate cell‐cycle arrest in the presence of Xmrk.

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.

Etiology of MNU-induced melanomas in Xiphophorus hybrids☆

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.