PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.     

近交系剑尾鱼的STR遗传检测技术

目的建立近交系实验用鱼DNA检测方法,用于遗传质量控制。方法以RR-B、RW-H、BY-F三个近交系剑尾鱼的基因组DNA为主要研究对象,并以非选育剑尾鱼作对照,采用STR（short tandem repeat）引物扩增方法检测不同品系在DNA上的异同。设计10对STR引物,优化PCR电泳条件,使用相同的实验条件进行重复,以得到可靠的扩增条带,筛选出特异性引物。结果有4对引物可扩增出稳定条带,1对引物（AY204223）的PCR扩增条带在非选育群中表现多态性,在RR-B与BY-F系表型一致,与RW-H表型不同。这些STR位点能够区分剑尾鱼三个近交系品系。结论STR检测技术可望用于近交系剑尾鱼的遗传质量监测。     

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK-1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L-15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.     

DNA polymerase beta mRNA and protein expression in Xiphophorus fish

Herein we report Xiphophorus DNA polymerase beta (XiphPolbeta) mRNA and protein expression levels in brain, liver, gill, and testes tissues from Xiphophorus maculatus, Xiphophorus helleri, and Xiphophorus couchianus parental line fish and two different tumor-bearing Xiphophorus interspecies hybrids. Polymerase beta protein levels in the Xiphophorus tissues were measured by Western blot, and mRNA was measured with a quantitative real time RT-PCR method which employed cRNA construction to produce accurate calibration curves. We found significant differences in both mRNA and protein levels between the tumor-bearing hybrid animals and the three parental species. However, there were no significant differences in either mRNA levels or protein expression observed between the parental species. Thus, interspecies hybridization results in dysregulation of Polbeta expression and this may manifest a modulation in DNA repair capability and susceptibility to latent tumorigenesis.     

TWO TYPES OF MELANOMAS IN A NEW EXPERIMENTAL SYSTEM OF PLATYFISHSWORDTAIL HYBRIDS

A new experimental system for investigating platyfish-swordtail hybrid melanomas was developed using ornamental platyfish ( Xiphophorus maculatus ) and swordtails ( Xiphophorus helleri ). The incidence of melanomas in the hybrids was studied in relation to individual age factors. Based on the age of the onset of the melanomas, two types of the melanomas were discerned and were designated as the fry and the adult melanomas, respectively. The fry melanomas were characterized by early onset, wide-ranged lesions, an invasive nature owing to densely pigmented cells, and a tendency toward regression. The adult melanomas were characterized by occurrence in the later stage of life, a small number of primary sites, and rapid, expanding, and invasive growth due to sparsely pigmented cells.     

Contributions of the Gordon-Kosswig melanoma system to the present concept of neoplasia

Modern cancerology is based on the oncogene concept. This is rather new. The idea of the oncogene, however, is old, and can be traced back to two sources, namely to "cancer families," reported in 1866 by P. Broka, and to "virus induced" neoplasia, detected by P. Rous in 1911. A gene which is--to my knowledge--the first reported oncogene by definition was detected in the little ornamental Mexican fish Xiphophorus by Myron Gordon, Curt Kosswig, and Georg H?ussler in 1928 when they observed the terrible hereditary melanomas that we are now coming to understand and to compare with other kinds of neoplasms in Xiphophorus and in mammals, including humans. Although the Xiphophorus model was always modest in its claims, it has--sometimes too early in its history--contributed many facts to the present concept of neoplasia.     

Melanoma formation in xiphophorus: A model system for the role of receptor tyrosine kinases in tumorigenesis

Cancer is one of the most frequent fatal human diseases. It is a genetic disease, and molecular analysis of the genes involved revealed that they belong to several distinct classes of molecules, one of which is the receptor tyrosine kinases. Neoplastic transformation is regarded as the result of a multistep process and, in most cases, it is hard to evaluate what the initial events in tumor formation are. What makes it difficult to approach this question is the paucity of animal models for tumorigenesis allowing investigation of the mechanisms leading to uncontrolled cell proliferation. Melanoma formation in Xiphophorus is one of these model systems. Here, overexpression and activation of a receptor tyrosine kinase causes neoplastic transformation of pigment cells. Xiphophorus provides all the advantages of a well-characterised genetic system. In addition, some crucial components of the transformation pathway have been identified at the molecular level. As a vertebrate, Xiphophorus might serve as a model system to aid understanding, in more general terms, of the mechanisms of tumorigenesis in human diseases.     

DNA repair in hybrid fish of the genus Xiphophorus

The genus Xiphophorus is an important vertebrate model for investigating the etiology and genetics of both spontaneous and induced cancers. Xiphophorus are comprised of 23 species most of which can be crossed to produce fertile interspecies hybrid progeny. The Xiphophorus gene map is well developed and allows genetic associations to be studied among cohorts of progeny derived from backcrossing interspecies hybrid animals to one of the parental strains. In interspecies cross-progeny from select Xiphophorus backcrosses, ionizing radiation, ultraviolet light (UVB), and exposure to methylnitrosourea (MNU) have all been shown to induce tumors. Induced tumor types represented in various models include melanoma, fibrosarcoma, schwannoma, retinoblastoma, etc. The well-established backcross hybrid genetics make Xiphophorus fish an excellent system to study the contribution of DNA repair capability to induced tumorigenesis. DNA repair pathways represent multigenic traits that must be tightly regulated to insure genome fidelity. Herein we review initial DNA repair studies that assess repair capacities among different Xiphophorus species and interspecies hybrids. Assessment of both base excision repair (BER) and nucleotide excision repair (NER) have yielded consistent results indicating reduced DNA repair function in hybrid fish tissues. These data provide molecular support for potential reduced fitness in hybrid fish under conditions of environmental stress and may present a plausible explanation for absence of interspecies hybridization in sympatric environments. In addition, they support the role of direct DNA damage and its repair in the initiation of tumors in Xiphophorus hybrids.     

A microsatellite genetic linkage map for Xiphophorus

Interspecies hybrids between distinct species of the genus Xiphophorus are often used in varied research investigations to identify genomic regions associated with the inheritance of complex traits. There are 24 described Xiphophorus species and a greater number of pedigreed strains; thus, the number of potential interspecies hybrid cross combinations is quite large. Previously, select Xiphophorus experimental crosses have been shown to exhibit differing characteristics between parental species and among the hybrid fishes derived from crossing them, such as widely differing susceptibilities to chemical or physical agents. For instance, genomic regions harboring tumor suppressor and oncogenes have been identified via linkage association of these loci with a small set of established genetic markers. The power of this experimental strategy is related to the number of genetic markers available in the Xiphophorus interspecies cross of interest. Thus, we have undertaken the task of expanding the suite of easily scored markers by characterization of Xiphophorus microsatellite sequences. Using a cross between Xiphophorus maculatus and X. andersi, we report a linkage map predominantly composed of microsatellite markers. All 24 acrocentric chromosome sets of Xiphophorus are represented in the assembled linkage map with an average intergenomic distance of 7.5 cM. Since both male and female F1 hybrids were used to produce backcross progeny, these recombination rates were compared between "male" and "female" maps. Although several genomic regions exhibit differences in map length, male- and female-derived maps are similar. Thus Xiphophorus, in contrast to zebrafish, Danio rerio, and several other vertebrate species, does not show sex-specific differences in recombination. The microsatellite markers we report can be easily adapted to any Xiphophorus interspecies and some intraspecies crosses, and thus provide a means to directly compare results derived from independent experiments.     

Multi-step genetic regulation of oncogene expression in fish hereditary melanoma

Melanoma occurring spontaneously in Xiphophorus fish hybrids is a model system in which involvement of cellular oncogenes and multi-step regulation of their expression have been identified by classical genetics. The macromelanophore gene in platyfish (Xiphophorus maculatus) is a sex-linked codominant gene which determines the black spot patterns of macromelanophores in the skin. The macromelanophore locus includes a cellular oncogene which potentially induces neoplasms of the pigment cells. Expression of the oncogene is regulated by a multi-step genetic process and brings about a characteristic phenotype associated with pigment cell differentiation at each step. The multi-step genetic regulation of oncogene expression can be recognized by interspecific hybridization of the platyfish with swordtails (Xiphophorus helleri) which have not developed the macromelanophore gene. When platyfish are hybridized with swordtails, the F1 offspring carrying this gene develop a preneoplastic state. When the F1 offspring are back-crossed to swordtails, the backcross offspring develop a heritable form of melanoma with a characteristic inheritance pattern. This heritable form of melanoma occurs at an early age and has a well differentiated character. Thus, the first and second steps of oncogene expression bring about a preneoplastic state in the F1 offspring and a heritable form of melanoma in the backcross offspring, respectively. These steps may be due to progressive substitution of platyfish chromosomes with swordtail chromosomes in germ line cells, resulting in a progressive reduction of the dosage of regulatory genes in the platyfish genome. The third step of oncogene expression brings about a sporadic form of melanoma in the hybrid offspring bearing the preneoplastic state and heritable form of melanoma spontaneously or through induction by carcinogens. This form of melanoma has a poorly differentiated character. The incidence of this form is considerably enhanced by aging in adult life, thus exhibiting age-specific incidence. It is likely that this step is due to mutational events in regulatory genes, which occur in somatic cells following chromosome substitution in germ line cells by hybridization. The albino gene enhances the malignancy of the two forms of melanoma and the incidence of the sporadic form of melanoma, possibly by suppressing the differentiation of transformed pigment cells. These facts and speculations are summarized in Fig. 6. The molecular identification of oncogenes in this melanoma system and their transfer into the swordtail eggs may provide a useful means for studying oncogene expression during development, growth, and aging of animals.     

Pharmacological properties of angiotensin II receptors in cultured rabbit gingival fibroblasts

Certain interspecific hybrids of the fish Xiphophorus spontaneously develop melanoma induced by the derepression of the Xmrk oncogene. Xmrk is a recent duplicate of an orthologue of the mammalian epidermal growth factor receptor gene Egfr. In addition to a specific overexpression in melanoma, amino-acid substitutions in the extracellular domain leading to ligand-independent dimerisation and constitutive autophosphorylation are responsible for the tumorigenic potential of Xmrk. The Xmrk receptor induces several signal transduction pathways mediating cell proliferation and resistance to apoptosis and initiating dedifferentiation. Moreover, Xmrk upregulates the expression of the secreted protein osteopontin, inducing an autocrine loop possibly allowing invasion and survival in the dermis as a first step in malignancy. Hence, Xmrk is able to induce pathways essential for a transformed phenotype. Some of these events are equivalent to those found downstream of the mammalian Egfr, but others have clearly evolved differently or are specific for pigment cells. Xmrk is potentially hazardous, nonessential and located in a very unstable genomic region. Nevertheless, Xmrk has been maintained under purifying selection in divergent Xiphophorus species. Hence, Xmrk has probably a beneficial function under certain conditions. The analysis of this function is a major challenge for future research in the Xiphophorus model.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.     

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.     

Use of immunocytochemical staining of somatostatin for correlative light and electron microscopic investigation of D cells in the pancreatic islet of Xiphophorus helleri H. (Teleostei)

Summary Somatostatin-containing cells have been demonstrated by immunocytochemistry in semithin sections of the pancreatic islet of the teleost fish, Xiphophorus helleri. These cells were shown by correlative light and electron microscopy to be identical with D cells previously defined in this species by the silver impregnation method of Hellman and Hellerström.Supported in part by grants from the British Council and from the Medical Research Council of Great Britain     

Identification of a second egfr gene in Xiphophorus uncovers an expansion of the epidermal growth factor receptor family in fish

The epidermal growth factor receptor (EGFR) gives name to a family of receptors formed by four members in mammals (EGFR, ErbB2, ErbB3, and ErbB4). Members of this family can be activated to become potent oncogenes, and many human and animal tumors express qualitatively or quantitatively altered receptors from this group. We have isolated and characterized a second egfr gene in the melanoma model fish Xiphophorus. Both Xiphophorus egfra and egfrb duplicates are co-orthologs of the mammalian egfr gene. Database analysis showed that not only egfr but also erbB3 and erbB4 are present as duplicates in some fish species. They originated from ancient duplication events that might be consistent with the hypothesis of a fish-specific genome duplication. In Xiphophorus, the egfrb gene underwent a second duplication that generated the melanoma-inducing oncogene Xmrk. The study and comparison of some of the functional characteristics of both Xiphophorus EGF receptors, including expression profile, ligand-binding abilities, and intracellular signal transduction revealed that Xiphophorus Egfra not only shares common features with Egfrb and the human EGFR but also shows significant differences in its functional characteristics. The mechanism of maintenance of these duplicates remains to be clarified.