Eel WT1 sequence and expression in spontaneous nephroblastomas in Japanese eel.

Nephroblastomas spontaneously developing in Japanese eel reared at farms for 5 to 9months after collection from the wild [Masahito et al., Cancer Res., 52 (1992) 2575-2579] were investigated to cast light on the role of Wilms' tumor 1 gene (WT1) in eel kidney tumorigenesis. Cloning of the WT1 counterpart, EWT1, revealed that conservation of an alternative splice II site, located between the third and fourth zinc fingers, was conserved. The zinc finger domain was highly conserved. The transregulator region, sequences corresponding to exons 4 and 5 in WT1, were lacking in EWT1 cDNA. EWT1 was found to be expressed in kidney, testis and spleen and in situ hybridization revealed dark-stained immature cells in elver kidney to be positive. Although no EWT1 gene mutations were found in 38 eel nephroblastomas, 26 polymorphic nucleic acid changes were observed. Aberrant WT1 expression was noted in epithelial (12 out of 27; 44%) and nephroblastic cell histological types (three out of five; 60%) of eel nephroblastomas. On in situ hybridization the EWT1 expressive cells resembled human blastema cells, similar to those in human Wilms' tumor. These data demonstrated strong signals that the EWT1 protein may function in the development of eel kidney and play a role in genesis of nephroblastomas as in mammals.

Frequent development of pancreatic carcinomas in the Rana nigromaculata group.

In 1979, 2 species of pond frogs (Rana nigromaculata and Rana plancyi plancyi) were imported from China, and hybrids were made between these and Japanese, Korean, and Taiwanese pond frogs (R. nigromaculata, Rana plancyi fukienensis and Rana brevipoda) that had been kept for a number of years in the Laboratory for Amphibian Biology of Hiroshima University. From 1982, development of tumors, especially in the peritoneal cavity, was noticed frequently in the hybrids and also later, although rarely, in the Japanese pond frogs. Such tumors had never previously been observed among pond frogs in the laboratory. Histological and immunohistochemical studies identified the i.p. tumors to be pancreatic carcinomas with occasional production of insulin and/or somatostatin. Ultrastructural investigation revealed both endocrine and exocrine secretion granules together with C-type retrovirus particles in the carcinoma cells. Other tumors included a retroperitoneal rhabdomyosarcoma, liver adenomas, and an unclassifiable mesenchymal tumor of the foot pad.

Age dependence of O6-methylguanine-DNA methyltransferase activity and its depletion after carcinogen treatment in the teleost medaka (Oryzias latipes).

O6-Methylguanine-DNA methyltransferase (O6-MT) is considered to play an important role in the repair of DNA lesions induced by alkylating carcinogens in a wide range of animals. The activity of O6-MT was compared in liver extracts from the teleost medaka (Oryzias latipes) at various ages (3-5 years old) reared under natural conditions. O6-MT activity decreased significantly with advancing age. When medaka were exposed continuously to the alkylating agent methylazoxymethanol (MAM) acetate at levels of 0.1, 0.15 and 0.3 ppm in water, O6-MT activity was markedly reduced from days 1 to 7, with a slight increase thereafter. Furthermore, when fish were exposed to MAM acetate at levels of 1-2 ppm for 1 h and then maintained in normal tap water, O6-MT activity remained suppressed for 2 weeks, followed by a partial recovery.

Appearance of tumorous phenotypes in goldfish erythrophores transfected with ras, src, and myc oncogenes and spontaneous differentiation of the transformants in vitro.

When goldfish erythrophores isolated from the skin by tissue digestion and centrifugation in a Percoll density gradient were transfected in a monolayer-culture with v-Ha-ras or v-src oncogene either singly or in combination with v-myc by means of calcium phosphate-DNA co-precipitation, there appeared a certain number of transformants manifesting a chromatoblast-like profile and tumorous phenotypes as seen in the capability for unlimited growth, and piling-up in a monolayer-culture or colony formation in semi-solid soft agar. After successive growth in vitro for longer than one month which was scarcely observed with the erythrophores, the vast majority of such transformants began to differentiate into erythrophores and ceased proliferation spontaneously. The onset of their differentiation was ascertained by the deposition of marker pteridine pigments. None of the transformants differentiated into melanophores or iridophores or other neural crest derivatives as seen in goldfish erythrophoroma cells. Little difference was observed in their transforming efficiency (0.2-0.3 transformants/micrograms DNA) between the combinations of oncogenes applied but a tendency was noted that cells transfected with ras or src in combination with myc developed the capacity to grow for a longer period and differentiated at a later stage than those transfected solely with ras or src. One cell line (ESM-1) derived from the erythrophores transfected with src/myc grew successively over nine months, indicating its acquisition of immortality. The expression of the transfected oncogenes in this cell line was examined in comparison with the erythrophoroma cells by Western and Northern blot analyses.

Firefly luciferase gene transmission and expression in transgenic medaka (Oryzias latipes).

Plasmids containing the luciferase gene from the firefly (Photinus pyralis) fused to the Chinese hamster metallothioneine I promoter (ChMTI) were microinjected into the pronuclei of medaka (Oryzias latipes) eggs, which were then artificially inseminated. Evidence of integration into the genome was gained from observation of germ-line transmission in a mendelian fashion from the F1 to the F2 generation. However, gene expression (light emission) could not be demonstrated in the established transgenic line. In a separate program, transient expression of gene constructs containing the luciferase gene fused to various promoters was compared in medaka embryos. Plasmids were microinjected into pronuclei, and homogenates from 3-day-old embryos were measured for light emission using a luminometer. Among the various promoters tested (SV40, RSV-LTR, ChMTI, HSP70, and mouse albumin), the highest levels of luciferase gene expression were observed in gene constructs containing ChMTI and HSP70 gene promoters. Expression in these two constructs was significantly increased following administration of ZnSO4 or heat treatment, respectively. Plasmids were also introduced into goldfish fibroblast-like cells in vitro, in which enzymatically active luciferase was transiently expressed. Assaying for expression of luciferase provided a rapid and sensitive method for monitoring promoter activity. The potential usefulness of this fish species for cancer research is discussed based on accumulated information from carcinogenesis studies.

Nephroblastomas in the Japanese eel, Anguilla japonica Temminck and Schlegel.

Nephroblastomas were observed in 50 Japanese eel reared at a farm for 5 to 9 months from 1989 after collection in the wild. The tumors, arising from the trunk kidney near the anus, were noted externally as abdominal swellings and varied in size from 30 to 75 mm in maximum diameter. Most were elastic, solid, and well encapsulated. Histologically, the nephroblastomas were composed of combinations of three main tissue elements. Spindle- or oval-shaped cells resembling human blastema cells were observed in most tumors to some larger or smaller degree. Although variation was evident from tumor to tumor, and even within the same tumor, the most common histological type was epithelial with formation of alveolar nests, the cells sometimes being arranged in tubular structures simulating normal renal tubules. A muscle tissue element with distinct cross-striations was also observed. Liver metastases were found in one case. Histological examination of apparently normal kidneys from 100 eels revealed one early-stage nephroblastoma. The cause of these tumors is unclear, although they have been discovered with increasing incidence after the spread of indoor eel culture with raised water temperatures (26-27 degrees C) in Japan. Environmental factor(s) associated with the new aquaculture method may thus play a role in their genesis.

Immunological detection of in vivo aflatoxin B1-DNA adduct formation in rats, rainbow trout and coho salmon.

Immunological detection of carcinogen-DNA adducts in organs or tissues should prove a particularly useful approach for monitoring carcinogen exposure, for characterization of carcinogen binding to DNA and for investigating DNA repair processes in vivo. In one of a series of experiments aimed at raising antibodies against several carcinogen-modified DNAs, rabbits were immunized in our laboratory with aflatoxin B1 (AFB1)-modified DNA. After the titer and specificity of the antibodies produced were checked against standards by the enzyme-linked immunosorbent assay (ELISA) method, they were used to investigate DNA extracted from livers of rats and rainbow trout (Salmo mykiss) injected with tritium-labeled AFB1 at doses of 1-2 mg/kg (rats) or 0.1-0.5 mg/kg (rainbow trout). Adduct levels were compared using both radioactivity and ELISA methods. Positive DNA binding could be detected in both rats and rainbow trout by the immunological method at similar levels to those estimated with the radioactive analysis. To throw light on possible mechanisms underlying the wide variation in response to aflatoxins among salmonid fish, DNA extracted from the livers of rainbow trout (susceptible species) and coho salmon (Oncorhynchus kisutch) (less susceptible species) were compared following AFB1 treatment. A high rate of DNA binding was observed in rainbow trout, whereas significantly lower values were evident in coho salmon, suggesting a direct relationship between binding levels and susceptibility to mycotoxin carcinogenicity.

Life-span studies on spontaneous tumor development in the medaka (Oryzias latipes).

A total of 961 medaka, separated chronologically from the first to the fifth year of life, were examined for spontaneous tumor development. While no liver tumors were found in either male or female medaka under the age of 1 year and the incidence in 2-year-old fish was relatively low (males 1.9% and females 1.7%), they became more common with advancing age. The incidence was higher in females than in males from 3 to 5 years of age, reaching 7.1% in 5-year-old female stock. These liver tumors included a total of 12 adenomas and 9 hepatocellular carcinomas. The hepatocellular carcinomas were histologically well differentiated and were all observed in female medaka. Spontaneous tumors occurring in organs other than the liver were rare and sporadic. Four squamous cell carcinomas, 5 melanomas and 4 lymphosarcomas were observed with no sexual or pronounced age bias being evident. The squamous cell carcinomas developed in the surface epithelium with local invasion into the dermis. Melanomas occurred in the abdominal cavity and demonstrated systemic invasion into various parts of the body. Three out of the 4 lymphosarcomas arose from the inner part of the operculum suggesting that these tumors were of thymic origin. They also showed extensive invasion. The data indicate a particular susceptibility of older female medaka to liver but not other tumor development.

Pigment cells and pigment cell tumors in fish.

The three basic pigment cell types found in poikilothermic vertebrates, melanocytes (melanin-producing cells), erythrophores (red or yellow pigment cells), and iridophores (iridescence-producing cells), are derived from neural crest. Neoplasms of pigment cells in fish are also of three phenotypes, melanomas (melanophoromas), erythrophoromas, and iridophoromas, showing the phenotypes of their corresponding normal pigment cells. These pigment cell tumors are among the most common types in bony fish and seem to be more common in fish than in mammals, including humans. Moreover, there are no mammalian neoplasms corresponding to erythrophoromas and iridophoromas in fish. The complexities in the nature and classification of pigment cell tumors in fish will be discussed on the basis of a survey of our collection of these tumors at the Cancer Institute. The etiology of pigment cell tumors in fish is obscure. In order to know whether activated oncogene is involved in the genesis of erythrophoromas in goldfish, the ras genes from normal and erythrophoroma cells were cloned and their nucleotide sequences were compared. The goldfish ras gene and human ras genes showed striking homology. However, no point mutation at the 12th codon was observed in ras genes isolated from erythrophoromas. Besides pigment cell tumors in fish, abnormal pigmentation or depigmentation in flounders associated with diseased conditions is also described.

O6-methylguanine DNA methyltransferase activity in liver from various fish species.

O6-Methylguanine DNA methyltransferase (O6-MT) is considered to play an important role in the repair of alkylating carcinogen-induced lesions in a wide range of mammalian species. Fish are used widely in cancer research, one advantage being their high sensitivity to a variety of alkylating agents. To throw light on the mechanisms of DNA repair in the hitherto uninvestigated fish group, O6-MT activity was measured in liver from eight fish species belonging to six classes. Levels of O6-MT activity comparable with mouse values were found in liver of Japanese medaka (Oryzias latipes). Relatively low, but appreciable, levels of O6-MT activity were also observed in the other seven species examined. No adaptive increase in enzyme activity could be established in liver of rainbow trout following chronic dimethylnitrosamine pretreatment.

Nucleotide sequence comparison of the predicted first exonic region of goldfish ras gene between normal and neoplastic tissues.

The first exonic and its flanking regions of a ras-related gene in cultured erythrophoroma cells and normal liver of goldfish (Carassius auratus) were sequenced and their sequences compared. The two samples demonstrated identical sequences for the Alu I fragment of 245 nucleotides, of which 70 nucleotides might correspond to the 5'-upstream segment and 64 to the first intervening sequence, indicating that the predicted exonic region encodes from the 1st to the 37th amino acids from the N-terminal of mammalian ras protein. The length of the predicted first exon of goldfish was the same as that of mammals. There was a 5 nucleotide difference in this exonic region (length 111 nucleotides) from another previously determined ras-related sequence of normal goldfish. This difference was much smaller than that between any two of the three mammalian ras genes. Enhanced expression of ras gene was observed in erythrophoroma cells, although it was uncertain which ras gene was responsible for generation of the observed RNA.

Spontaneous neurinoma in an African lungfish, Protopterus annectens, and DNA repair studies on normal and neoplastic tissues.

Neurinomas developed in an African lungfish (Protopterus annectens), living in an aquarium in Western Japan. The 2 tumors, measuring 7.5 X 9.0 X 6.5 and 13 X 4 X 6 cm, were located on the skin. As shown by light microscopy, tumor cells were composed of spindle-shaped cells with huge pleomorphic nuclei, which were arranged in parallel rows or whorls in interlacing connective tissue. Long-term culture of these tumor cells was achieved in vitro at 25 degrees C with use of conditioned medium over a period of more than 4 months. The nuclear DNA contents of erythrocytes (normal diploids, 2C) and tumor cells dispersed from the fixed tumor tissues were measured by 4',6-diamidino-2-phenylindole hydrochloride-DNA microfluorometry by using mouse cerebellar small granule cells (normal diploids, 2C) as a reference. The 2C value of the lungfish was approximately 28-fold greater than that of the mouse. Furthermore, consistent with the nuclear pleomorphism observed by light microscopy, the nuclear DNA contents of tumor cells showed a wide distribution from hypo-2C to hyper-4C. DNA repair synthesis was measured autoradiographically in organ cultures of the tumor, lung, and skin, exposed to chemical carcinogens or UV radiation. Considerable repair was observed in the tumor and skin cells exposed to 1-methyl-1-nitrosourea (CAS: 684-93-5), N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 70-25-7), or 254-nm or sunlamp UV light. Only traces of repair synthesis were detected in lung exposed to 1-methyl-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. 4-Hydroxyaminoquinoline 1-oxide (CAS: 4637-56-3) did not induce repair in any of the three tissues. The observed values for repair, relative to the amount of DNA, were similar to those in other fishes.

Spontaneous hepatocellular carcinomas in lungfish.

Liver tumors were observed in 2 South American lungfish (Lepidosiren paradoxa) and in 1 African lungfish (Protopterus amphibius) kept in 3 different aquariums in Japan for 2-5 years. In the first case a single, large nodule (70 X 60 X 55 mm) in the liver had been noted externally as a swelling 1 year prior to death. In the second case 2 rounded nodules (25 X 15 X 15 mm and 15 X 10 X 10 mm) were incidentally found at autopsy. In the third case, which also demonstrated abdominal swelling for 1 year preceding death, 2 green nodules (50 X 50 X 40 mm and 17 X 17 X 17 mm) were found in the liver together with multiple metastatic lesions. Histologically, variation was evident from tumor to tumor, even within the same animal. The most common histologic type was typical trabecular hepatomas. Admixtures of glandular or papillary structures were noted in some tumors. A region of poorly differentiated hepatocellular carcinoma with spindle-shaped cells in a sheetlike arrangement was noted in one case. The cause of these tumors is unknown. Since liver tumors were discovered at relatively high incidence (3/14 autopsied cases, including 6 species), lungfish seem to be predisposed to the development of liver tumors.

Extensive sequence homology of the goldfish ras gene to mammalian ras genes.

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurogenic tumors in coho salmon (Oncorhynchus kisutch) reared in well water in Japan.

Neurogenic tumors were found protruding from various parts of the body of 23 coho salmon. The tumor-bearing fish were first- or second-generation fish derived from eggs imported at the eyed stage to Japan from the United States. Twenty-two of the tumors were in young adults and varied from 14 to 80 mm in maximum diameter. Histologically, the tumors were composed of spindle-shaped cells with abundant fibrous stroma. One tumor showed typical nuclear palisading. All tumors in young adults invaded locally muscle and adipose tissues. These tumors were similar in histologic appearance to malignant schwannomas in humans. One tumor found in a fingerling coho salmon was identified as an ependymoblastoma. The Vectstain avidin-biotin-peroxidase complex immunoperoxidase staining procedure for S-100 protein revealed that the S-100 protein existed in an ependymoblastoma and in areas of typical nuclear palisading in a malignant schwannoma in coho salmon. The occurrence of soft tissue tumors in coho salmon was first recorded in Japan. The morphology and etiology of the present cases were compared with those of the tumors in salmon reported from the United States. Judging from the conditions in which the fish were reared, the development of these tumors was not related to halogenated compounds formed during water chlorination, as suggested previously. The environmental factor(s) responsible for their development has not yet been identified, and genetic influences may have been a contributory factor.

Gonadal neoplasms in largemouth bass, Micropterus salmoides and Japanese dace (ugui), Tribolodon hakonensis.

Massive abdominal enlargement was observed at relatively high incidence in two species of fish kept in two public aquariums for several years. Three seminomas, one dysgerminoma and two fibromas were found in 14 largemouth bass, Micropterus salmoides, kept in a public aquarium for over 80 months. Four seminomas and one nephroblastoma were found in 24 Japanese dace (ugui), Tribolodon hakonensis, kept for more than 57 months in two different aquariums. The seminomas in M. salmoides showed rapid growth, and the affected fish died within a few months. At necropsy, a single large tumor, measuring 10.5-12.0 cm, was found in the abdominal cavity. The seminomas in T. hakonensis were single or multiple tumors, measuring 2.0-4.5 cm in diameter. Histologically, these seminomas were composed mainly of a typical germ cells similar to those in human seminomas or embryonal carcinomas. A nephroblastoma in one T. hakonensis showed extensive metastases to various organs. The cause of these tumors is unknown, but the prolonged longevity of fish kept under artificial conditions may have enhanced their development.

Usefulness of the medaka, Oryzias latipes, as a test animal: DNA repair processes in medaka exposed to carcinogens.

Results of previous studies indicate that the medaka, Oryzias latipes, a small aquarium fish found in Japan and neighboring countries, is a suitable animal for use in cancer research. This fish showed a high incidence of liver tumors after a short induction period when kept in water containing diethylnitrosamine. Unscheduled DNA synthesis (UDS) was demonstrated in ganglion cells of the medaka in vivo by autoradiography. For this experiment, part of the bony skull of the medaka was removed surgically, and the living fish was kept in an isotonic solution containing a carcinogen and tritiated thymidine. Its brain was then examined histologically. By this method, UDS was clearly demonstrated over the nuclei of ganglion cells after exposure to a wide variety of chemical carcinogens. The abdominal cavity was opened and treated with carcinogens in the same way as the brain; however, no UDS was detected in liver or intestinal cells. We conducted comparative studies on DNA repair in the brains of 5 species of small fish from widely different families. These 5 species (blond cave fish, Oriental weatherfish, medaka, guppy, and Siamese fighting fish) were exposed to various concentrations of 4-hydroxyaminoquinoline 1-oxide or methyl methanesulfonate. We detected UDS in all 5 species after both treatments, although the extent varied in the different species.

Clonal heterogeneity in physiological properties of melanized cells induced from goldfish erythrophoroma cell lines.

Cells of the uncloned goldfish erythrophoroma lines, GEM-81 and GEM-218, have been induced to melanize by cultivation in autologous serum. The melanized cells continue to proliferate and exhibit clonal heterogeneity in terms of morphology, growth rates, contact behavior and pigment content, and distribution and translocation in response to hormones. Based on these characteristics and those of their normal counterparts, the melanized tumor cells have been categorized as type-I and type-II melanocytomas, and melanophoromas. The melanophoroma cells are capable of pigment translocation in response to epinephrine, melatonin, and/or MSH, whereas melanocytoma cells are not. The distinguishing characteristics of each type are apparent at the first appearance of melanized cells and appear to be stable except in some type-II melanocytoma clones which contain cells capable of differentiating into melanophoroma cells in long-term cultures. It appears that the parent erythrophoroma lines contain stem cells, melanoblastomas, which are capable of melanogenesis. These stem cells may themselves be a heterogeneous population with respect to the characteristics of the melanized cells to which they give rise.

Permanent cell lines from erythrophoromas in goldfish (Carassius auratus).

Attempts to establish permanent cell lines from spontaneous erythrophoromas (tumors derived from red pigment cells or erythrophores) of goldfish were made with the use of biopsy specimens from 12 tumors in 10 fish. Three cell lines were established that grew in vitro in synthetic medium (L-15 or Dulbecco's modified Eagle's minimum essential medium) supplemented with 20% fetal bovine serum for more than 8 months. One of these lines (GEM-81) with high proliferative activity was cultured for over 200 generations without an obvious change in growth rate. From this cell line, clonal cultures were obtained that formed clones with relatively intense yellow pigmentation. Descendants of these cell lines and clones contained low but detectable amounts of pteridine pigments (such as 7-hydroxybiopterin, biopterin, xanthopterin, and isoxanthopterin) and numerous cytoplasmic organelles analogous to pterinosomes. Both these characteristics are phenotypic markers of normal erythrophores and their neoplastic counterparts. After numerous subcultivations, these long-term cultures differed from those of the initial explants in having much lower contents of total pteridines and relatively lowered contents of 7-hydroxybiopterin. As manifestations of their neoplastic origins, all cell lines examined showed disoriented growth with dense focal mounding in monolayer cultures. The population doubling time of the uncloned GEM-81 cell line was 31 hours at 35 degrees C over a feeder cell layer and 3 days at 25 degrees C without feeder cells. Injection of cultured cells (1.5 x 10(7) cells/fish) into normal goldfish that had not been immunosuppressed did not give rise to tumors within 5 months.