Extensive neuronal localization and neurotrophic function of fibroblast growth factor 8 in the nervous system.

Fibroblast growth factor (FGF) 8 has been well established to play a critical role in the early development of the central nervous system (CNS). We report here extensive neuronal localization and neurotrophic function of FGF8 in the nervous system. In sections of mouse embryos at E10.5, FGF8 was immunohistochemically found in neurons at the marginal zones of the CNS and in the dorsal root ganglia (DRG). Neuronal localization of FGF8 was marked at later embryonic stages and in adults, involving most of the central and peripheral neurons, including intermuscular enteric neurons, DRGs, and paraaortic sympathetic ganglia. Functionally, FGF8 promoted neurite outgrowth in human neuroblastoma SK-N-MC cells as well as in rat pheochromocytoma PC12 cells, suggesting that FGF8 acts as a neurotrophic factor. FGF8 also supported neuronal survival and differentiation in cultured human neural progenitor cells. In a cell growth assay, treatment with 50 ng/ml FGF8 on human cultured neuroblastoma SK-N-MC and IMR32 cells attenuated the growth of both. In accordance with these in vitro findings, the immunohistochemical analysis on human neurological diseases showed that FGF8 expression is evident in differentiating histological types of neuroblastoma and ganglioneuroblastoma, and that the levels of FGF8 immunoreactivity in the substantia nigra from Parkinson's disease are significantly lower than those in age-matched controls. Taken together, the present findings strongly suggest that FGF8 acts as a more generalized neurotrophic factor than previously reported.

Purification of activins from androgen-independent Shionogi carcinoma cells demonstrates enhanced expression of activin betaB-subunit under androgen-depleted cell conditions in vitro and in vivo.

Here, we report characterization of growth factors secreted from androgen-independent mouse mammary Shionogi carcinoma cells. Previous isolation of fibroblast growth factor 8 (FGF8) from androgen-dependent Shionogi carcinoma SC-3 cells prompted us to characterize growth factors secreted from the androgen-independent cells. After several purification procedures, mitogens for NIH3T3 cells from the androgen-independent cells were identified as activins on the grounds that activin betaA- and betaB-subunits are detected in the active fractions by Western blotting and that the growth-promoting effects by the active fractions are specifically inhibited in the presence of follistatin. In addition, exogenous activins, but not inhibin, stimulated the growth of NIH3T3 cells in a dose-dependent manner. Interestingly, transcripts of activin betaB-subunit were predominantly found in the androgen-independent cells while its betaA-subunit was universally expressed in both androgen-dependent and -independent Shionogi carcinoma cells. In concordant with this in vitro finding, transcripts of activin betaB-subunit were enhanced in murine prostates after castration. Therefore, expression of activin betaB-subunit, but not its betaA-subunit, is likely to be related with androgen-depleted cell conditions in prostates, and possibly in androgen-related cancers.

Isolation of an androgen-inducible novel lipocalin gene, Arg1, from androgen-dependent mouse mammary Shionogi carcinoma cells.

Here we report isolation of an androgen-regulated novel gene from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line. Using a polymerase chain reaction-based subtraction method and Northern blotting analysis, we isolated four androgen-inducible genes from SC-3 cells. Nucleotide sequencings identified three of the genes as cyclin D1, beta-catenin, and fatty acid synthase, respectively, but the fourth, a gene tentatively named as Arg1 (androgen-regulated gene 1), remained undefined. The cloned 2.0-kb sized Arg1 cDNA encoded 414 amino acid sequences. The deduced amino acid sequences, sharing about 30% homology with cathepsin family members at a protein level, had relatively conserved residues around the three proteinase active sites reported earlier. In Northern blotting, Arg1 mRNA was found in kidney, heart, lung, and to a lesser degree, in spleen and liver. Its transcripts were also detected in male reproductive organs on RT-PCR. In addition, its expression levels in prostate were markedly reduced after castration. Unexpectedly, Arg1-expressing COS1 cells showed no significant proteinase activity to various synthesized substrates under neutral or acidic conditions in this study. This might have been due to the replacement of the cysteinyl active site for proteinase to serine residue in the Arg1 amino acid sequences. Given that Arg1 also contains a lipocaline signature known as a binding motif for small hydrophobic molecules at the center of its amino acid sequences, Arg1 is a lipocalin family gene regulated by androgens in prostate and Shionogi carcinoma cells.

Predominant expression of fibroblast growth factor (FGF) 8, FGF4, and FGF receptor 1 in nonseminomatous and highly proliferative components of testicular germ cell tumors.

Nonseminomatous components within testicular germ cell tumors affect patient prognosis to varying degrees. These components are well known to mimic early embryonic totipotential tissues. Prompted by the recent observation that fibroblast growth factor (FGF) 8, FGF4, and FGF receptor (FGFR) 1 are required for the growth of early postimplantational embryonic tissues, we investigated the expressions of FGF8, FGF4, and FGFRI in surgically resected specimens of primary testicular germ cell tumors using an immunohistochemical method. All cases of embryonal carcinoma (14 cases), yolk sac tumor (3 cases), and choriocarcinoma (3 cases) showed positive immunostaining for FGF8, FGF4, and FGFR1. In contrast, out of 13 cases of seminoma, immunostaining was negative for FGF8, FGF4, and FGFR1 in 8 cases (61.5%), 6 cases (46.1%), and 7 cases (53.8%), respectively. In 7 cases of mature and immature teratoma, most areas showed negative immunostaining. In addition, the Ki-67 labeling index showed extremely high mitogenic activity in embryonal carcinoma, yolk sac tumor, and choriocarcinoma, which are precisely the carcinomas with the highest expressions of FGF8, FGF4, and FGFR1. It is in keeping with the immunohistochemical result that murine teratocarcinoma P19 cells were shown to express FGF8, FGF4, and FGFRI only under undifferentiated growth conditions. Taken together, these findings confirm the involvement of FGF8, FGF4, and FGFR1 in highly proliferative conditions of nonseminomatous germ cell tumors.

Integration of proviral sequences, but not at the common integration sites of the FGF8 locus, in an androgen-dependent mouse mammary Shionogi carcinoma.

A retroviral insertional mutation, especially by mouse mammary tumor virus (MMTV), is a major cause of murine mammary tumorigenesis. Prompted by our previous finding that FGF8, an insertionally activated cellular oncogene, is highly expressed in androgen-dependent mouse mammary Shionogi carcinoma cells, we here investigated retroviral integration adjacent to the fgf8 locus in Shionogi carcinoma. In the genomic Southern blots for fgf8 and its 5'-upstream gene npm3, the hybridized fragments were identical to the host DD/Sio mice, the original Shionogi carcinoma 115 tumor, and a pair of cultured Shionogi carcinoma cell lines of SC-3 and SC-4, suggesting that no retroviral integration occurred around either loci. The genomic cloning for the fgf8 locus from SC-3 cells also confirmed no MMTV integration. In addition, npm3, which is usually coactivated with fgf8 by MMTV insertion,was not up-regulated by androgens in SC-3 cells. All these findings led us to conclude that no retroviral insertion was present at the common integration sites adjacent to the fgf8 locus in Shionogi carcinoma although we demonstrated in this study that multiple proviral sequences of MMTV, Moloney murine sarcoma virus and FBJ-murine sarcoma virus are integrated into SC-3 cells in association with their distinct promoter activity in SC-3 cells.

High frequency of fibroblast growth factor (FGF) 8 expression in clinical prostate cancers and breast tissues, immunohistochemically demonstrated by a newly established neutralizing monoclonal antibody against FGF 8.

Fibroblast growth factor (FGF) 8, also known as androgen-induced growth factor, was originally isolated from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line, in which it was shown to have androgen-regulated properties. We previously demonstrated that Fgf 8 transcripts were detected in several human prostate and breast cancer cell lines and that recombinant FGF 8 was mitogenic to an androgen-sensitive prostate cancer LNCaP cell line. In this study, to characterize the roles of FGF 8 in clinical hormone-responsive cancers, we established a monoclonal antibody against FGF 8. In Western blots, this antibody specifically interacted with a FGF 8b isoform that was identical between mouse and human but was not identical to other murine 8a and 8c isoforms. In a cell growth assay using SC-3 cells, the newly established anti-FGF 8 antibody blocked androgen- and FGF 8-stimulated growth but not basic FGF-stimulated growth. Immunohistochemical analyses by use of the established anti-FGF 8 antibody demonstrated that FGF 8 was frequently expressed in human prostate cancers, appearing in 40 of 43 cases (93%), whereas both prostatic hyperplasia specimens and normal prostate tissues included in biopsy specimens were negative for FGF 8 expression. On the other hand, FGF 8 was detected in normal ductal and lobular epithelial cells in breast tissues. FGF 8 was also frequently expressed in various breast diseases, including fibroadenomas (5 of 5 cases, 100%), intraductal papillomas (3 of 3 cases, 100%), ductal hyperplasias (3 of 6 cases, 50%), and breast cancers (8 of 12 cases, 67%). Androgen receptors were also immunohistochemically detected in FGF 8-positive prostate cancers (40 of 40 cases, 100%) and FGF 8-positive breast diseases (17 of 19 cases, 89%). These findings strongly suggest that FGF 8 is involved in hormone-related tumorigenesis of the prostate and breast.