The significance of LMO2 expression in the progression of prostate cancer.

LIM domain only 2 (LMO2) proteins are important regulators in determining cell fate and controlling cell growth and differentiation. This study has investigated LMO2 expression in human prostatic tissue specimens, prostate cancer cell lines, and xenografts; and has assessed the possible role and mechanism of LMO2 in prostate carcinogenesis. Immunohistochemical analysis on a tissue microarray consisting of 91 human prostate specimens, including normal, prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and invasive carcinoma, revealed that overexpression of LMO2 was significantly associated with advanced tumour stage, as measured by Gleason score (p = 0.012), as well as with the development of distant metastasis (p = 0.018). These data were supported by quantitative real-time PCR experiments, where LMO2 mRNA levels were found to be significantly higher in prostate tumour specimen than in normal epithelium (p = 0.037). The expression of LMO2 in cell lines and xenografts representing androgen-dependent (AD) and androgen-independent (AI) prostate cancer stages was further studied. Consistent with the in vivo data, LMO2 mRNA and protein were found to be overexpressed in the more aggressive AI cells (PC3, DU145, and AI CWR22 xenografts) compared with less aggressive AD cells (LNCaP and AD CWR22 xenografts). Furthermore, stable introduction of LMO2 into LNCaP cells conferred enhanced cell motility and invasiveness in vitro, accompanied by down-regulation of E-cadherin expression. Taken together, these findings provide the first evidence to support the hypothesis that LMO2 may play an important role in prostate cancer progression, possibly via repression of E-cadherin expression.

Allele-specific marker generation and linkage mapping on the Xiphophorus sex chromosomes.

There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.

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.

The melanoma-specific XMEL antigen is expressed in dysplastic naevi.

We have previously shown that a novel monoclonal antibody, XMEL, exhibited reactivity with deep primary melanomas while showing no reactivity with other tumours and normal tissue. XMEL was raised against a part of the extracellular domain of Xmrk, a growth factor receptor presumed to mediate melanoma formation in the Xiphophorus fish model. Here we investigate the range of XMEL immunohistochemical reactivity in paraffin sections from human common acquired and dysplastic naevi of both junctional and compound type. The strongest reactivity was observed with the compound dysplastic naevi. We conclude that the antigen recognized by XMEL acts early in the cascade of genetic alterations underlying progression into malignant melanoma. Our results also support the notion that the dysplastic naevus may play a role in progression of human malignant melanoma and may indeed represent the precursor stage.

The Xiphophorus-derived antibody, XMEL, shows sensitivity and specificity for human cutaneous melanoma but is not a prognostic marker.

XMEL is a monoclonal antibody raised against part of the extracellular domain of the putative tyrosine kinase receptor protein implicated in the pathogenesis of melanoma formation in the Xiphophorus fish melanoma model. Our objective in this study was to determine the diagnostic and prognostic utility of XMEL for human melanoma. Formalin-fixed tissue from 82 melanomas, 42 carcinomas, 23 neural tumors, 12 lymphomas and 12 sarcomas were immunostained with XMEL and compared with a widely used melanoma antibody, HMB-45. The sensitivity of HMB-45 (83.1%) was similar to that of XMEL (79.8%). XMEL detected 7 melanomas that were HMB-45 negative. Specificity for detection of melanoma was greater with HMB-45 (95.5%) as compared to XMEL (80.9%). Of interest, all 4 prostatic adenocarcinomas were XMEL positive. These data suggest that XMEL is as sensitive but not as specific as HMB-45 in the detection of cutaneous melanoma but may serve as an ancillary antibody to improve diagnostic yield. The consistent positivity of XMEL in melanoma lends support to the hypothesis that the detected protein plays a role in melanoma pathogenesis. XMEL reactivity is not an independent prognosticator of death from melanoma in 37 melanomas from patients with at least 10 years' follow-up. These data and the fact that XMEL shows variable reactivity with metastatic melanomas but almost 100% reactivity with the primary melanomas suggest that the antigen recognized by the XMEL antibody may be important in the early stages of melanoma progression. This is supported by our earlier observation that XMEL is reactive with dysplastic nevi, a precursor of malignant melanoma.

Generation of poly- and mono-clonal antibodies against trout fibronectin (FN) and their use in FN immunodetection in teleost fishes.

Immunization of rats with gelatin-affinity column purified fibronectin (FN) from rainbow trout (Oncorhynchus mykiss) plasma produced a polyclonal antiserum that reacts specifically with FN in immunoblotted protein extracts and cultured cells, not only from trout but also from swordtails (Xiphophorus helleri). Most importantly, this antiserum specifically stains FN-containing structures in sections from embryos, as well as skin and dorsal fin of swordtails and platyfish (Xiphophorus maculatus), allowing, e.g., correlation of the distribution of FN with neural crest cell development in Xiphophorus. The antiserum also cross-reacts with FN in sections from embryos of the Japanese medaka (Oryzias latipes) and coho salmon (Oncorhynchus kisutch). In addition to the polyclonal antibodies, monoclonal anti-trout FN antibodies were produced in rats. These did not exhibit reactivity on sections, but stained the cultured fish cells and FN in immunoblots. Both types of antibodies may be of interest to the fish industry for marking the level of FN as an indicator, not only for infectious diseases, but also for certain developmental stages such as smoltification and spawning.

Differences in transcription and promoters of Xmrk-1 and Xmrk-2 genes suggest a role for Xmrk-2 in pigment pattern development in the platyfish, Xiphophorus maculatus.

Pigment (macromelanophore) patterns in the platyfish Xiphophorus maculatus are due to a complex pigmentary locus; for example, the spotted-dorsal (Sd) fin pattern is due to the Sd locus. In interspecific backcross hybrids with the swordtail X. helleri, the Sd pattern changes into benign or malignant dorsal fin melanoma as a result of hemi- or homozygous loss of a platyfish regulatory (R) gene, the tumor suppressor gene Diff, that appears to play a role in the final differentiation of macromelanophores. Closely linked to the pigmentary locus is an epidermal growth factor receptor-like gene, Xmrk-2, that has arisen by duplication from the linked Xmrk-1. The transcriptional expression of the Xmrk genes was determined in various tissues including Sd pigment patterns and melanomas of various growth potential using reverse transcription-polymerase chain reaction. While Xmrk-1 expression was found in all tissues examined, Xmrk-2 expression correlated with pigment cell growth. Xmrk-2 was highly expressed in the dorsal fin exhibiting the Sd pattern but drastically reduced in a platyfish mutant which has lost the capacity to form these pigment cells in the dorsal fin. Most interestingly, Xmrk-2 expression increased with the proliferative capacity of the melanomas but declined once melanoma growth ceased. We conclude that Xmrk-2 plays a role in the formation of the pigment pattern cell type, perhaps in proliferation of precursor cells, which, in melanoma, are kept in a proliferative state due to loss of Diff.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between variant forms of estrogen receptor RNA and an apoptosis-related RNA, TRPM-2, with survival in patients with breast cancer.

BACKGROUND: Although smaller variant forms of estrogen receptor (ER) messenger RNA (mRNA) have been detected in breast tumors, neither their prevalence nor their prognostic significance have been evaluated. Similarly, TRPM-2 mRNA, the product of a gene induced principally during the onset of apoptosis, is present in mouse and human breast cancer cell lines, but whether it also occurs in primary breast tumors and is related to disease outcome is unknown. METHODS: The relative expression and transcript size of ER mRNA and TRPM-2 mRNA in 126 primary breast tumors were measured by Northern analysis and compared with tumor grade, hormone receptor status, extent of tumor necrosis, and survival. RESULTS: In ER-positive tumors, 64% of the tumors had only the normal 6.5 kb ER mRNA, an additional 9% had the normal plus smaller ER mRNA, and 2% had variant forms. Only 8% of ER-negative tumors had ER mRNA transcripts. There were significant relationships between the occurrence of ER mRNA and low tumor grade, ER-positive receptor status, and better survival. In contrast, TRPM-2 mRNA was found in only 17% of breast tumors, none of which could be grouped with respect to grade, hormone receptor status, or survival. CONCLUSIONS: The presence of smaller variant forms of ER mRNA either alone or in association with the normal ER transcript is not indicative of an unfavorable prognosis, whereas TRPM-2 mRNA occurs in many primary breast tumors, but has no apparent relationship to survival.

A putative marker for human melanoma. A monoclonal antibody derived from the melanoma gene in the Xiphophorus melanoma model.

A MoAb was raised against a peptide corresponding to an exposed domain of the putative tyrosine kinase receptor protein encoded by Xmrk, a gene involved in melanoma formation and/or progression in the Xiphophorus fish melanoma model. The antibody reacts specifically with cells from human melanocytic lesions, ie, common acquired nevi, primary and metastatic melanoma biopsies. No reactivity with other cells, including normal melanocytes, was observed in the biopsies or with cells in biopsies from normal tissue (skin, liver, lung, spleen) and from other malignancies including those of neuroectodermal origin. The reactivity was very weak and variable in metastatic melanomas but very strong and characteristic of a receptor-type antigen in primary melanomas, a stage in melanoma progression in which cells have acquired metastasizing potential. It is suggested that the antigen recognized may be involved in growth promotion and represents the human equivalent of the fish melanoma gene product.

Functional analysis and temporal expression of promoter regions from fish antifreeze protein genes in transgenic Japanese medaka embryos.

Several series of sequences that are upstream of the transcriptional start site of different types of fish AFP genes were fused to the bacterial CAT gene, and their transcriptional role was examined in a transient expression assay after microinjection into Japanese medaka (Oryzias latipes) embryos at the 1-4 cell stage. Our studies demonstrated that the AFP genes have functional promoter regions containing positive as well as negative regulatory regions, indicating that these genes could be regulated at multiple sites. We also observed a promoter-specific pattern of temporal expression. Typically, the CAT expression was low in the first 4 days of embryonic development or before the stage of body pigmentation, followed by a sharp increase. The high level was maintained until hatching (11-13 days after fertilization), by which time the activity decreased to a very low level.

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes).

Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.

Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.

To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.

Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes.

Neural crest cells obtained from explanted neural tubes take up, express, and retain exogenous DNA applied by the CaPO4 co-precipitation method during their differentiation into melanocytes. High efficiencies of gene transfer were obtained with both supercoiled DNA and intact phage particles; linear DNA or DNA from the phage yielded very low efficiencies. There is some evidence that transferred gene expression is differentiation dependent. The system should be useful for studies concerned with the analysis of cell developmental genes and their regulatory elements.

Expression and fate of CAT reporter gene microinjected into fertilized medaka (Oryzias latipes) eggs in the form of plasmid DNA, recombinant phage particles and its DNA.

Fertilized medaka (Oryzias latipes) eggs were cytoplasmically injected with the chloramphenicol acetyltransferase (CAT) gene encompassed in supercoiled and linear plasmid DNA, as well as in intact recombinant phage particles and DNA isolated from the phage. Expression for the CAT plasmid DNA was highest at the gastrula/neurula stage, while for the DNA of the phage, it peaked in the 1-week old embryo; then expression declined but was still detectable in early adulthood (4 weeks post injection). Following the fate of exogenous DNA, an extensive replication was observed in early embryogenesis, and DNA was still found 4 weeks after injection, suggesting a possibility of integration. The system is useful as a transient expression system for the analysis of early developmental genes in particular, but also as a test system for the analysis of cloned genes of interest for the farming of economically important fish species. The fact that DNA transferred in intact phage particles or its DNA is functionally active opens the possibility to introduce larger DNA pieces (20 kb), e.g., for the functional test of larger and more distant control regions.

Culture of embryos and cells from platyfish and swordtails for carcinogenicity testing and studies on melanoma formation.

We introduce inbred strains of platyfish and swordtails as well as their hybrids as laboratory animals for carcinogenicity testing. We especially propose to take advantage of the platyfish-swordtail melanoma system, which permits the breeding of animals with high susceptibility to carcinogen-induced melanoma. Based on our present view that, in melanoma formation, such fundamental processes as the determination and differentiation of a specific cell type are affected, we suggest the use of embryos and embryo-derived cell cultures. In both systems, all stages of pigment cell differentiation can be followed and screened for abnormal development. The in vitro culture of the embryos of the viviparous fish and the establishment of cell cultures are described.

The induction of a specific pigment cell type by total genomic DNA injected into the neural crest region of fish embryos of the genus Xiphophorus.

We report genetic transformation in an intact higher organism, i.e., in xiphophorine fish. The gene to be transferred (Tu) is responsible for the formation of T-melanophores in the platyfish and is involved in the formation of melanomas in platyfish-swordtail hybrids. After injection of Tu-donor DNA into the neural crest region of embryos from Tu-free fish, some of the recipients developed T-melanophores. In a few cases, one or two single T-melanophores were formed during late embryo-genesis. In most cases, many T-melanophores developed in young fish and were arranged in several colonies or in a pattern. DNase-degraded Tu-donor DNA, Tu-free fish DNA, as well as DNA from E. coli and adenovirus-2, did not induce T-melanophores. When using DNA from different strains of Tu-donor fish which differed in a regulating gene linked to Tu, the percentages of fish showing T-melanophores paralleled the degree of phenotypic expression of the Tu gene in the DNA donor. The results suggest that the Tu gene has been successfully transferred together with the linked regulating gene.

Suppression of melanoma development and regression of melanoma in xiphophorine fish after treatment with immune RNA.

In xiphophorine fish suffering from a genetically caused melanoma, both suppression of melanoma development and regression of the existing melanoma were observed after treatment of the fish with an "anti-melanoma immune RNA." This RNA was extracted from the lymphoid organs of guinea pigs immunized with fish melanoma. RNA from guinea pigs immunized with fish skin or liver and RNA from nonimmunized guinea pigs were ineffective. RNase treatment of the anti-melanoma immune RNA diminished its activity, although Pronase treatment did not. Analysis of antisera obtained from guinea pigs and rabbits immunized with either melanoma or normal skin of xiphophorine fish revealed differences in the immune responses induced by these tissues. The anti-melanoma sera recognized antigens in melanoma extracts, which were not present in skin extracts. These antigens were not recognized by the anti-skin sera. The results suggest specificity of the anti-melanoma immune RNA.