Nucleotide excision repair proteins and their importance for radiation-enhanced transfection.

PURPOSE: Irradiated cells transfect more efficiently than unirradiated cells because of a radiation-induced increase in plasmid integration. However, the molecular mechanism is unclear. Because of recent observations that nucleotide excision repair (NER) proteins can be involved in certain types of recombination in yeast, it was hypothesized that NER proteins might play a role in this radiation-enhanced integration. MATERIALS AND METHODS: Hamster and human cells with inactivating mutations in NER genes were irradiated at doses from 0 to 6 Gy and then immediately transfected with a linearized selectable marker plasmid. Transfection-enhancement ratios (TERs) were calculated as the ratio of the number of drug-resistant colonies in unirradiated cells to the number of transfectants in irradiated cells, corrected for cytotoxicity from radiation. RESULTS: Transfection into unirradiated rodent cells was unaffected by NER mutation status. Transfection into unirradiated human cells, however, was increased by NER mutation. The TERs were 5 and 100 for CHO and primary human fibroblasts, respectively, after exposure of the cells to 6 Gy. Mutations in ERCC1, XPA, XPB, XPC, XPF, XPG and CSB dramatically reduced TER. Mutations in ERCC1, XPC, XPF, XPG and CSB suppressed transfection so that the TER was significantly below 1. CONCLUSIONS: The mechanism of radiation-enhanced plasmid integration was distinct from that of plasmid integration in unirradiated cells, and NER gene products were critical for enhanced integration to occur.

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.

Characterization of the CDKN2A and ARF genes in UV-induced melanocytic hyperplasias and melanomas of an opossum (Monodelphis domestica).

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

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

Xiphophorus interspecies hybrids provide genetically controlled models of tumor formation. Spontaneous melanomas form in first-generation backcross (BC(1)) hybrids produced from backcrossing F(1) 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 BC(1) 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 BC(1) 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 BC(1) hybrids from some crosses, but not others. Homozygosity for X. helleri and X. couchianus CDKN2X alleles in BC(1) 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 BC(1) 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.

Genetic analysis of neoplasia induced by N-nitroso-N-methylurea in Xiphophorus hybrid fish.

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 BC(1)-hybrid CDKN2X genotypes and UV-induced melanoma formation exist in a backcross produced from 2 inbred parental lines. However, when BC(1) 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 BC(1) hybrid fish.

A proposed classification scheme for Xiphophorus melanomas based on histopathologic analyses.

We studied the histopathologic characteristics of melanomas induced in the Xiphophorus model. This fish model has been used for several decades to study the molecular and genetic mechanisms underlying its susceptibility to melanoma induction. Numerous distinct interspecific hybrid crosses currently are being used in research on carcinogenesis. We previously reported that tumors were induced in such hybrid crosses after treatment with N-methyl-N-nitrosourea or UV radiation. In this report, we describe the histopathologic features of Xiphophorus melanomas and propose a new classification system. We suggest that melanomas in these fishes can be classified as follows: melanocytic melanomas; melanophorous-macromelanophorous polymorphic melanomas; spindle cell type melanomas; epithelioid cell melanomas; and amelanotic melanomas. The new classification of Xiphophorus melanomas should allow correlations between histopathologic characteristics and carcinogen treatment, and between histopathologic characteristics and the genetic background of the hybrid fish.

Xiphophorus genetic linkage map: beginnings of comparative gene mapping in fishes.

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.

Aquaria fish models of human disease: reports and recommendations from the working groups.

On September 21-24, 2000, the National Cancer Institute, Southwest Texas State University, and the Roy and Joan Mitte Foundation sponsored an international conference entitled "Aquaria Fish Models of Human Disease" at Southwest Texas State University (SWT), San Marcos, Texas, USA. Over 100 scientists, representing various fish model systems, participated in four roundtable working groups. We considered the first step in promoting the exciting research with fish models was to unify the efforts within this scientific community towards accomplishing specific goals. With this objective in mind, the following four working groups were convened: (1) fish cancer models: sustenance and enhancement; (2) fish genomics and transgenics: resources and technology; (3) fish pathology: standards for tumor pathology classification; and, (4) resources underpinning aquaria fish research. Each working group was charged with preparing a report of their discussions with recommendations on how researchers and funding agencies might best direct and strengthen research support to ensure a healthy future for such work. Included are the final reports from these working groups, together with a brief summary of the discussions held during the sessions and the consensus recommendations from each group.

Role of ERCC1 in removal of long non-homologous tails during targeted homologous recombination.

The XpF/Ercc1 structure-specific endonuclease performs the 5' incision in nucleotide excision repair and is the apparent mammalian counterpart of the Rad1/Rad10 endonuclease from Saccharomyces cerevisiae. In yeast, Rad1/Rad10 endonuclease also functions in mitotic recombination. To determine whether XpF/Ercc1 endonuclease has a similar role in mitotic recombination, we targeted the APRT locus in Chinese hamster ovary ERCC1(+) and ERCC1(-) cell lines with insertion vectors having long or short terminal non-homologies flanking each side of a double-strand break. No substantial differences were evident in overall recombination frequencies, in contrast to results from targeting experiments in yeast. However, profound differences were observed in types of APRT(+) recombinants recovered from ERCC1(-) cells using targeting vectors with long terminal non-homologies-almost complete ablation of gap repair and single-reciprocal exchange events, and generation of a new class of aberrant insertion/deletion recombinants absent in ERCC1(+) cells. These results represent the first demonstration of a requirement for ERCC1 in targeted homologous recombination in mammalian cells, specifically in removal of long non-homologous tails from invading homologous strands.

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells.

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.

Overexpression of a fish CDKN2 gene in a hereditary melanoma model.

The fish genus Xiphophorus provides a vertebrate model useful in etiological studies of cancer. Hybrid fish can spontaneously develop melanomas deriving from the inheritance of melanistic pigment patterns and the simultaneous absence of proper genetic regulation. A cyclin-dependent kinase inhibitor gene, termed CDKN2X, was mapped to a genomic region that is implicated in fish melanoma tumor suppression. The related human tumor suppressor locus CDKN2A (P16, INK4A, MTS1) is deleted, mutated or transcriptionally repressed through methylation of cytosine bases within the 5' CpG island in a variety of neoplasms, including melanoma. The fish CDKN2X locus harbors a CpG island within its promoter and first exon, analogous in location to CpG islands in human CDKN2A and CDKN2B loci. The methylation state of individual CpG dinucleotides was investigated in genomic DNA derived from control tissues and melanomas within the CDKN2X 5' CpG island. The studied genomic area was found to be virtually unmethylated in all tested tissues including melanomas. In addition, RNA expression studies of the fish CDKN2X locus revealed that it is significantly overexpressed in melanoma, in contrast to what has been reported for the human CDKN2A locus in melanoma. Such overexpression may be a consequence of the pronounced upregulation of the Xmrk-2 receptor tyrosine kinase oncogene reported in several Xiphophorus melanoma models.

Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model.

We have cloned, sequenced, and characterized the RNA expression properties of a fish CDKN2 gene from Xiphophorus helleri and X. maculatus. This gene, termed CDKN2X, shows a high degree of amino acid sequence similarity to members of the mammalian CDKN2 gene family, which includes the tumor suppressor loci CDKN2A (P16) and CDKN2B (P15). Comparative sequence analysis suggests that fish CDKN2X is similarly related to all four mammalian gene family members, and may represent a descendant of an ancestral prototypic CDKN2 gene. CDKN2X was mapped to a region on autosomal Xiphophorus linkage group V (LG V) known to contain the DIFF gene that acts as a tumor suppressor of melanoma formation in X. helleri/X. maculatus backcross hybrids. Thus, CDKN2X may be a candidate for the tumor suppressor DIFF gene. Here we have sequenced CDKN2X in both Xiphophorus species and have characterized its expression in normal and melanotic tissues within control and backcross hybrid fish. A simultaneous expressional analysis of the Xmrk-2 tyrosine kinase receptor gene, which is strongly implicated in melanomagenesis in this system, was also performed. RT - PCR analyses revealed that both genes were highly expressed in melanomas. For CDKN2X, this result contrasts numerous findings in human tumors including human melanoma in which either CDKN2A (P16) deactivation or LOH was observed.

Mapping of tyrosine kinase gene family members in a Xiphophorus melanoma model.

Xiphophorus fish have been the subject of intensive genetic research for more than 60 yr, primarily because of the availability of a number of interspecific hybrids that are malignant melanoma models with apparently simple oncogene and tumor suppressor gene determinants. The gene map of Xiphophorus is one of the most extensive among nonhuman vertebrates, with about 100 genes assigned to at least 20 independently assorting linkage groups (LGs), as well as more than 250 anonymous DNA sequence markers, providing coverage for most of the genome for genetic mapping studies. This characteristic has resulted in the mapping of a tumor suppressor locus, DIFF, which is one of two genetic determinants of melanoma formation in the best-studied hybrid melanoma, the Gordon-Kosswig melanoma model. The other gene responsible for melanoma formation in this model is a sex-linked tyrosine kinase gene related to EGFR and called Xiphophorus melanoma receptor kinase (Xmrk). The cellular oncogene homologues of the non-receptor tyrosine kinase family orthologous toyes and fyn have also been found to be overexpressed in malignant melanomas of Xiphophorus and may be involved in tumor progression. We report here the map location of a Xiphophorus yes gene, YES1, in LG VI, closest to the EGFR gene and the assignment of a fyn gene homologue to newly designated LG XV, linked to the gene for cytosolic alpha-galactosidase. We also confirmed that an EGFR-related sequence (EGFRL1) that we previously assigned to Xiphophorus LG VI by cross-hybridization to a viral erbB probe was the EGFR orthologue. Our results suggest that the presence of expressed duplicates of members of the tyrosine kinase gene family in teleost fishes may increase the potential number of targets in oncogenic cascades in fish tumor models.

Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes.

We have isolated two major histocompatibility complex (MHC) class II B genes from the inbred fish strain Xiphophorus maculatus Jp 163 A. We mapped one of these genes, designated here as DXB, to linkage group III, linked to a malic enzyme locus, also syntenic with human and mouse MHC. Comparison of genomic and cDNA clones shows the gene consists of six exons and five introns. The encoded beta1 domain has three amino acids deleted and a cytoplasmic tail nine amino acids longer than in other teleost class II beta chains, more similar to HLA-DRB, clawed frog Xela-F3, and nurse shark Gici-B. Key residues for disulfide bonds, glycosylation, and interaction with alpha chains are conserved. These same features are also present in a swordtail (Xiphophorus helleri) genomic DXB PCR clone. A second type of class II B clone was amplified by PCR from X. maculatus and found to be orthologous to class II genes identified in other fishes. This DAB-like gene is 63% identical to the X. maculatus DXB sequence in the conserved beta2-encoding exon and was mapped to new unassigned linkage group LG U24. The DXB gene, then, represents an unlinked duplicated locus not previously identified in teleosts.

Characterization and analysis of Chinese hamster ovary cell ERCC1 mutant alleles.

We isolated and characterized the ERCC1 coding sequence from three Chinese hamster ovary (CHO) parental (CHO-AA8, CHO-AT3-2 and CHO-9) and 10 ERCC1 mutant cell lines. Two general classes of mutations were observed: two mutant cell lines exhibited nucleotide additions or deletions to produce frameshift mutations and seven mutant cell lines exhibited point mutations that resulted in transitions or transversions, including nonsense mutations and mutations that generated intron/exon splicing errors. One mutant (UV201) which had been provisionally assigned to ERCC1 complementation group 1 (CG1) had no detectable mutation in its coding sequence. Of the nine ERCC1 mutant alleles characterized two mutations were identified in the XpA binding region of the Ercc1 protein; no mutations were found in the N-terminal portion of the Ercc1 protein. Results of Northern hybridization analysis showed that the relative levels of ERCC1 mRNA differed significantly both among the parental cell lines and among the mutant cell lines derived from each parental cell line. Western analysis with a CHO Ercc1-specific antibody detected Ercc1 protein in each of the parental cell lines and also in UV201. The marked reduction in Ercc1 protein levels observed in all the other mutants examined supports the hypothesis that ERCC1 mutations may destabilize this polypeptide.

Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor.

The Xiphophorus hybrid melanoma model represents one of the earliest reported cases of genetically regulated tumor susceptibility. Melanoma formation in Xiphophorus hybrids may be explained by the inheritance of two genes: a sex-linked oncogene, Xmrk, and a putative tumor suppressor locus, termed DIFF, located in Linkage Group V (LG V). Several genetic mapping procedures were used to produce a new Xiphophorus LG V map with 20 loci. All markers, particularly a recently cloned Xiphophorus CDKN2 gene family member, called CDKN2X, were tested for associations of genotype with degree of macromelanophore pigment pattern modification and susceptibility to melanoma formation in backcross hybrids of seven genetic types, involving 1,110 fish and three pigment patterns. Highly significant associations of CDKN2X genotypes with such phenotypic effects suggests that this gene is a strong candidate for the classically defined DIFF tumor suppressor gene. Because published results have documented the involvement of the CDKN2A (p16, MTS1, and INK4A) tumor suppressor gene in human melanoma formation, the possibility of CDKN2 genes acting as tumor suppressors in both man and Xiphophorus is likely.

Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models.

We have cloned and sequenced the p53-encoding cDNA of green swordtail (X. helleri) and southern platyfish (X. maculatus). These two fish species are often used to produce hybrids that develop melanomas after genetic crossing. Computer translation of derived cDNA sequences revealed that p53 polypeptides from these two species are virtually identical, exhibiting only two conservative amino acid substitutions. TP53 mRNA expression was detected in virtually all tissues tested. Comparison of these fish p53 polypeptide sequences with those of other vertebrates, including other fishes, amphibians, and mammals, revealed that conservation is especially high in several previously defined protein domains. In addition, sequencing of the 3' TP53 genomic region of X. maculatus reveals similarity to the human TP53 locus in overall organization. Knowledge of the Xiphophorus TP53 sequences will allow assessment of mutational alterations within tumors generated from numerous fish genetic crosses.

Targeted recombination at the Chinese hamster APRT locus using insertion versus replacement vectors.

In this study, we have examined the effects of targeting vector configuration and site of vector linearization on the frequency of targeted recombination at the endogenous CHO APRT locus, and have analyzed the types and class distributions of APRT+ recombinants obtained in APRT targeting experiments employing uncut circular, insertion-type (ends-in), and replacement-type (ends-out) configurations of the same pAG7 targeting vector, including configurations produced by introduction of a double-strand break (DSB) at sites either within, or at the 5' or 3' boundaries of APRT targeting homology. Our results suggest that: 1) plasmid-chromosome targeted recombination in mammalian cells may not be stimulated to the same degree by a DSB in the targeting vector as by a DSB in the chromosomal target; 2) recombinant class distributions are highly dependent upon targeting vector configuration; and 3) one-sided invasion mechanisms may play a significant role in homologous recombination in mammalian cells.