An androgen-dependent mouse mammary carcinoma cell line (SC-3) requires androgen for growth stimulation. We have shown previously that androgen acts on SC-3 cells to induce secretion of a fibroblast growth factor (FGF)-like growth factor, which in turn stimulates growth of the cells in an autocrine manner. In this study, the androgen-induced growth factor (AIGF) was purified from a conditioned medium of SC-3 cells stimulated with testosterone. cDNA cloning of AIGF by use of its partial amino acid sequence data revealed that AIGF is a distinctive FGF-like growth factor. An AIGF cDNA (pSC17) encodes a 215-amino acid protein with a putative signal peptide, which shares 30-40% homology with known members of the FGF family. The AIGF mRNA was markedly induced by 10 nM testosterone in Northern blot analysis. Expression of AIGF cDNA in mammalian cells clearly showed remarkable stimulatory effects of AIGF on growth of SC-3 cells in the absence of androgen. Thus, it is clear that the androgen-induced growth of SC-3 cells is mediated in an autocrine manner by AIGF, which is secreted by the tumor cells themselves in response to hormonal stimuli.

We have used mouse mammary tumor virus (MMTV) infection of Wnt-1 transgenic mice to accelerate mammary tumorigenesis and to molecularly tag insertionally activated proto-oncogenes that cooperate oncogenically with Wnt-1 (G. M. Shackleford, C. A. MacArthur, H. C. Kwan, and H. E. Varmus, Proc. Natl. Acad. Sci. USA 90:740-744, 1993). Here we report the identification and characterization of a 31-kb genomic locus that contains clonal MMTV integrations in 8 of 80 mammary tumors from MMTV-infected Wnt-1 transgenic mice. Two genes were identified within this locus, one of which was transcriptionally activated by MMTV insertions. This activated gene is identical to androgen-induced growth factor (AIGF/Fgf-8) (A. Tanaka, K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto, Proc. Natl. Acad. Sci. USA 89:8928-8932, 1992), the eighth member of the fibroblast growth factor (FGF) family. Transcriptional activation of Fgf-8 was found in all tumors with MMTV insertions in this locus. Fgf-8 mRNA was absent in normal mammary glands and was detected only in adult testis and ovary and in midgestational embryos. The sequences of Fgf-8 genomic and cDNA clones revealed five coding exons, in contrast to the three coding exons found in other FGF genes. cDNAs encoding three isoforms of the FGF-8 protein were isolated. The three corresponding mRNAs resulted from the alternative use of two 5' splice sites and two 3' splice sites for the second and third exons, respectively. These results implicate Fgf-8 as the third FGF gene found to cooperate with Wnt-1 in MMTV-induced murine mammary tumorigenesis, suggesting that FGFs and Wnts are strong collaborators in this process.

Enamel-renal syndrome (ERS) is an autosomal recessive disorder characterized by severe enamel hypoplasia, failed tooth eruption, intrapulpal calcifications, enlarged gingiva, and nephrocalcinosis. Recently, mutations in FAM20A were reported to cause amelogenesis imperfecta and gingival fibromatosis syndrome (AIGFS), which closely resembles ERS except for the renal calcifications. We characterized three families with AIGFS and identified, in each case, recessive FAM20A mutations: family 1 (c.992G>A; g.63853G>A; p.Gly331Asp), family 2 (c.720-2A>G; g.62232A>G; p.Gln241_Arg271del), and family 3 (c.406C>T; g.50213C>T; p.Arg136* and c.1432C>T; g.68284C>T; p.Arg478*). Significantly, a kidney ultrasound of the family 2 proband revealed nephrocalcinosis, revising the diagnosis from AIGFS to ERS. By characterizing teeth extracted from the family 3 proband, we demonstrated that FAM20A(-/-) molars lacked true enamel, showed extensive crown and root resorption, hypercementosis, and partial replacement of resorbed mineral with bone or coalesced mineral spheres. Supported by the observation of severe ectopic calcifications in the kidneys of Fam20a null mice, we conclude that FAM20A, which has a kinase homology domain and localizes to the Golgi, is a putative Golgi kinase that plays a significant role in the regulation of biomineralization processes, and that mutations in FAM20A cause both AIGFS and ERS.

Androgen-induced growth factor (AIGF/FGF-8) has purified from a mouse mammary carcinoma cell line and its cDNA cloning revealed that AIGF is the eighth member of the fibroblast growth factor family. To clarify a biological role of FGF-8, Northern blot analysis of various adult tissues was carried out, but no expression was found. However, whole mount in situ hybridization of 7.5- to 14.5-day gestation (E7.5-14.5) embryos revealed six predominant expression domains: [1]primitive streak region (E7.5-9.75); [2]midbrain-hindbrain border (E8.0-10.5); [3]rostral forebrain (E8.0-10.5); [4]limb ectoderm and apical ectodermal ridge (E9.25-13.5); [5]nasal placode and epithelium (E9.5-12.5); and [6]branchial arch ectoderm (E8.5-11.5). The embryonic expression pattern suggests a unique role of FGF-8 in mouse development, especially in gastrulation, brain development, and limb and facial morphogenesis.

Amelogenesis imperfecta (AI) is a genetically and clinically heterogeneous group of inherited dental enamel defects. Commonly described as an isolated trait, it may be observed concomitantly with other orodental and/or systemic features such as nephrocalcinosis in Enamel Renal Syndrome (ERS, MIM#204690), or gingival hyperplasia in Amelogenesis Imperfecta and Gingival Fibromatosis Syndrome (AIGFS, MIM#614253). Patients affected by ERS/AIGFS present a distinctive orodental phenotype consisting of generalized hypoplastic AI affecting both the primary and permanent dentition, delayed tooth eruption, pulp stones, hyperplastic dental follicles, and gingival hyperplasia with variable severity and calcified nodules. Renal exam reveals a nephrocalcinosis which is asymptomatic in children affected by ERS. FAM20A recessive mutations are responsible for both syndromes. We suggest that AIGFS and ERS are in fact descriptions of the same syndrome, but that the kidney phenotype has not always been investigated fully in AIGFS. The aim of this review is to highlight the distinctive and specific orodental features of patients with recessive mutations in FAM20A. We propose ERS to be the preferred term for all the phenotypes arising from recessive FAM20A mutations. A differential diagnosis has to be made with other forms of AI, isolated or syndromic, where only a subset of the clinical signs may be shared. When ERS is suspected, the patient should be assessed by a dentist, nephrologist and clinical geneticist. Confirmed cases require long-term follow-up. Management of the orodental aspects can be extremely challenging and requires the input of multi-disciplinary specialized dental team, especially when there are multiple unerupted teeth.

A mouse testis cDNA library in lambda pCEV27 eukaryotic expression vector was transfected in NIH3T3 fibroblasts and several transformed foci were identified. A plasmid with high-titered focus forming activity was rescued from one of these transformants. Structural analysis of this cDNA predicted a protein identical to androgen induced growth factor (AIGF), recently identified as the eighth member of the fibroblast growth factor (FGF) family. A 1.6 kilobasepair transcript of the FGF-8 gene was detected in testis but not in other adult tissues analysed. During development, expression of FGF-8 was restricted to embryonic days 9 through 13 suggesting that the growth factor plays a role during a discrete stage of mouse embryogenesis. An exon-containing genomic clone of human FGF-8 was isolated and structural comparisons indicated that the gene structure of this region is highly conserved among the FGF genes. Using a panel of human-rodent somatic cell hybrids, the FGF-8 gene was localized to human chromosome 10.

BACKGROUND

Using array analysis for screening RNA from BRCA1-mutated and sporadic breast tumors, we observed that AIGF/FGF-8 expression was lost in BRCA1-mutated breast tumors. Since this growth factor is induced by androgens, we studied the androgen receptor (AR) expression in BRCA-mutated tumors and in matched sporadic breast tumors.

METHODS

Paraffin embedded breast tumors of carriers of a BRCA1 mutation (n=41, median age of patients at time of surgery was 41 years [range 28-59 years]) or a BRCA2 mutation (n=14, median age 41 years [range 31-85 years]) were analyzed for the presence of ER-alpha, PR, P53 and AR using standard immunohistochemical techniques. All statistical tests used, Pearson chi2 and Fisher exact, were two-sided.

RESULTS

The AR was only present in 12% of BRCA1-mutated tumors, with mutations located at the C-terminal half of the BRCA1-gene. The AR expression was significantly more prevalent, however, in a series of 61 sporadic breast tumors (80%) and in BRCA2-mutated tumors (50%). In contrast to an increased percentage of p53 positive cells, in 66% of the BRCA1-mutated tumors, the ER-alpha expression was observed only in 25% and the PR in 13% of these specimens. The three steroid hormone receptors were expressed in about half of the BRCA2-mutated specimens studied.

CONCLUSIONS

Our data add to the emerging evidence that the biological phenotype of BRCA1-associated tumors may be different from BRCA2 and non-hereditary cases. The loss of the AR expression, as shown by immunohistochemistry, together with the observed loss of other steroid hormone receptors in BRCA1-mutated tumors may lead to a hormone-independent growth or to anti-hormone resistant growth of these tumors.

Androgen-induced growth factor (AIGF or FGF-8) was originally isolated from the conditioned medium of an androgen-dependent Shionogi carcinoma, SC-3, cell line. It shares structural similarity with other members of the FGF family. The temporal and spatial expression patterns of the FGF-8 gene suggest its involvement in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor. In the adult, expression of FGF-8 is restricted to gonads including testes and ovaries. Since FGF-8 is identified as a corroborating gene in MMTV-induced mammary tumors in Wnt-1 transgenic mice and because FGF-8 manifested its autocrine mitogenic activity in SC-3 cells, it is possible that aberrant expression of FGF-8 may be present in human cancers which are hormone dependent. However, very little is known about human FGF-8. To determine whether FGF-8 plays a role in human breast cancer, we have isolated the full-length cDNA from SK-BR-3 breast cancer cells. We have also isolated the corresponding genomic DNA in a P1 cloning vector. The FGF-8 gene has been mapped to chromosome 1Oq24 using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. Finally, we show that FGF-8 gene expression in a human breast cancer cell line, MDA-MB-231, is inducible by androgen. The findings presented here will facilitate our understanding of the molecular mechanism underlying hormone-responsive breast and prostate cancers.

Our previous study demonstrated that androgen-dependent growth of mouse mammary carcinoma cells (SC-3) was mediated through an induction of heparin-binding growth factor, termed as androgen-induced growth factor (AIGF). Here, we report that NIH3T3 cells stably transfected with AIGF expression vector exhibit the abilities of tumor formation in nude mice, focus formation in monolayer culture and colony formation in soft agar. Thus, this newly cloned growth factor can be categorized as an oncogene. In addition, androgen-induced enhancement of DNA synthesis in SC-3 cells can be blocked by simultaneous incubation with AIGF antisense oligonucleotides. The possibility is also addressed that AIGF exerts its biological activity through an interaction with fibroblast growth factor (FGF) receptor. Transfection of expression vector encoding a variant form of FGF receptor-1 cloned from SC-3 cells into FGF receptor-negative L6 cells results in AIGF-dependent inhibition of differentiation. These results demonstrate that the ability of androgen to elicit transformed phenotype of SC-3 cells is mediated through AIGF induction and its interaction with FGF receptor-1.

Fibroblast growth factor 8 (FGF8) is an androgen-induced growth factor (AIGF) that is crucial for embryonic development. This study was developed to investigate the role of FGF8 in developmental abnormalities of the genital tubercle (GT) in hypospadiac male rats when prenatally exposed to di-n-butyl phthalate (DBP). DBP was administered to timed-pregnant rats to establish the hypospadiac rat model where the incidence of hypospadias in male offspring was 43.6%. On postnatal day (PND) 7, decreased mRNA and protein expression levels for androgen receptor (AR) and FGF8 were observed in the GT of hypospadiac rats. Decreased serum testosterone (T) levels were observed in groups displaying hypospadias, which was confirmed using histological analysis. Further anatomical examination using digital photography helped to reveal visualized expression of dysplasia in organs strongly associated with hypospadias. In addition, changes in body weight (BW) and anogenital distance (AGD) were recorded, showing definitive decreases. Collectively, these data clearly demonstrate an interaction between androgen and FGF8, which might play an important role in the occurrence of hypospadias and abnormal organ development induced by DBP.

Androgen-induced growth factor (AIGF) has hormone-regulated properties in the mouse Shionogi carcinoma cell line. To investigate whether or not it is involved in growth of human hormone-responsive cancers, we isolated the human AIGF gene from a placental genomic library. Genomic analyses suggested that the AIGF gene was about 6.5 kilobases in length containing five exons. The deduced amino acid sequence of human AIGF was completely identical with that of the mouse. RT-PCR analyses showed that prostate and breast cancer cell lines, LNCaP, PC-3, and MCF-7, slightly expressed the AIGF gene. Recombinant AIGF enhanced the growth of the human prostate cancer LNCaP cells, and it also markedly stimulated the growth of fibroblasts. These in vitro findings suggest that AIGF might be a possible autocrine or paracrine factor in hormone-responsive cancers.

Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

Androgens are essential for normal prostatic and testicular function. However, paracrine and/or autocrine actions of a number of growth factors have been implicated in the function of these tissues. A recent addition to the fibroblast growth factor family, the so called androgen-induced growth factor (AIGF) or fibroblast growth factor-8 (FGF-8), has been proposed to be under strict androgen regulation and induction in the mouse mammary carcinoma cell line SC3. FGF-8, therefore, may have a local role in the prostate, which is known to be an androgen-responsive organ. This study reports, for the first time, the presence of FGF-8 mRNA in normal adult rat tissues (heart, brain, lung, kidney, testis, prostate and ovary), using an optimised reverse transcription and nested polymerase chain reaction (RT-PCR) procedure, although androgen-dependent FGF-8 expression was not demonstrated in these adult tissues. Consistent with the oncogenic characteristics of FGF-8, the corresponding mRNA was detected in the human prostate tumour cell lines LNCaP and DU145. Because the DU145 cell line is known to be androgen-independent, and the expression of FGF-8 mRNA in cultured LNCaP cells also occurred in the absence of exogenous androgens, it can be concluded that the expression of FGF-8 mRNA in these human cell lines, in the rat prostate and in other rat tissues is not under the regulation of androgens as hitherto proposed.

Androgen-induced growth factor (AIGF) is essential for the androgen-induced autocrine growth of a mouse mammary Shionogi carcinoma cell line (SC-3 cells). Because glucocorticoid and estrogen have been observed to weakly stimulate DNA synthesis in SC-3 cells, the expression of AIGF mRNA after stimulation with various concentrations of androgen, glucocorticoid, or estrogen was examined by Northern blot analysis. Testosterone, dexamethasone, and estradiol-17 beta (E2) induced AIGF mRNA expression, although the maximum AIGF mRNA expression levels induced by dexamethasone or E2 were lower than that by testosterone. Yet, diethylstilbestrol showed no induction, suggesting that the effect of E2 could be mediated through the androgen receptor. The induction levels of AIGF mRNA by each steroid hormone were correlated positively with hormone-induced DNA synthesis. In addition, the DNA synthesis induced by each steroid hormone was almost completely inhibited by AIGF antisense oligonucleotides, indicating that AIGF is an obligatory component in not only the androgen- but also the glucocorticoid-inducible autocrine loop in SC-3 cells.

Shionogi carcinoma 115 (SC 115) has been extensively used to analyze the mechanism of androgen-dependent cancer growth. This tumor exhibits marked androgen-dependent growth in vivo and one cell line whose growth is markedly stimulated by androgen in serum-free culture condition is isolated from SC 115 tumor. This androgen-dependent growth is mediated through an induction of heparin binding growth factor termed as androgen-induced growth factor (AIGF). In addition, fibroblast growth factor receptor 1 (FGFR 1) is identified as a receptor for AIGF. The expression of FGFR 1 mRNA is up-regulated by androgen in SC 115 cells, indicating that this androgen-inducible autocrine loop is potentiated at two sites by androgen. An androgen-independent cell line is also established from this androgen-dependent SC 115 tumor. The growth of these androgen-independent cells is stimulated by AIGF, indicating that AIGF acts not only as an autocrine growth factor to androgen-dependent cells but also as a paracrine growth factor to androgen-independent cells. In addition, transfection of AIGF expression vector into androgen-dependent cells results in a facilitation of conversion from androgen-dependent to -independent phenotype. Thus, AIGF might play a role from tumor progression. These results indicate that a blockade of AIGF activity is an important therapeutic target. Actually, some compounds such as heparin and suramin are found to inhibit this androgen-induced autocrine loop. These basic observations will be discussed in relation to their possible clinical application.

Methylcobalamin (MeCbl) is an important enzyme cofactor required for methionine synthase activity. It also inhibits, in a dose-dependent manner, the proliferation of an androgen-dependent cell line, SC-3, derived from an androgen-dependent mouse mammary tumor (Shionogi carcinoma 115). In SC-3 cells, androgen induces the production of androgen-induced growth factor (AIGF), an autocrine growth factor increasing the proliferation of SC-3 cells. MeCbl treatment suppressed the androgen-induced, AIGF-mediated growth of SC-3 cells, as well as the androgen-induced increase of AIGF mRNA. In SC-3 cells, androgen receptors linked with androgen form complexes that tightly bind DNA and act as transcription factors in the nucleus to regulate the expression of specific genes such as AIGF. The number and dissociation constants of androgen receptors in control and MeCbl-treated SC-3 cells were the same. Similarly, the extent of binding of normal androgen receptors in nuclei from control and MeCbl-treated cells was virtually identical. The androgen receptors from control and MeCbl-treated cells showed similar capacities for conversion to a form that tightly binds to DNA on heat activation. These results suggest that the reduction of AIGF mRNA, subsequent to the nuclear binding of androgen receptors, may be a partial cause of the growth-inhibitory activity of MeCbl in SC-3 cells.

Recent studies have suggested that the growth of sex steroid-dependent cancer is mediated through sex steroid-induced growth factors in an autocrine manner. In order to prove the hypothesis, estrogen-responsive human breast cancer cell lines such as MCF-7 have been used by many investigations. However, these studies have been unable to prove the hypothesis, since estrogen-dependent growth system in serum-free medium could not be established. We established in 1987 an androgen-dependent growth system in a serum-free medium using androgen-dependent mouse mammary cancer cells (SC-3). By use of this androgen-dependent growth system in serum-free medium, we could demonstrate that androgen-dependent growth of SC-3 cells is mediated via an androgen-induced new growth factor in fibroblast growth factor (FGF) family (AIGF; 8th member of FGF family) in an autocrine mechanism.

The growth of the mouse mammary Shionogi carcinoma 115 (SC 115)-derived cell line (SC-3) is markedly stimulated by androgen through induction of a heparin-binding growth factor termed androgen-induced growth factor (AIGF). This androgen-induced growth is partly blocked by thyroid hormone(s) such as triiodothyronine (T3). Transforming growth factor beta 1 (TGF beta 1) also inhibits the growth of SC-3 cells. Thus, we have investigated the possibility that T3 exerts its inhibitory effects on SC-3 cell growth through an alteration in AIGF or TGF beta 1 mRNA expression. Unexpectedly, T3 failed to modulate the expression of AIGF mRNA. T3 was also unable to significantly affect TGF beta 1 mRNA levels in androgen-stimulated SC-3 cells. More importantly, a neutralizing antibody against TGF beta 1 could not block T3-dependent inhibition of androgen-induced SC-3 cell growth. However, the inhibitory ability of T3 was potentiated by TGF beta 1. In addition, T3 treatment resulted in a significant inhibition of AIGF-induced DNA synthesis. Thus, T3 treatment renders SC-3 cells somehow refractory to AIGF. The signal pathway for T3 to reduce AIGF responsiveness may be shared by TGF beta 1.

SC-3 cells derived from mouse mammary carcinoma (Shionogi carcinoma 115) exhibit remarkable growth enhancement and cell morphology change in response to androgen stimuli. These events are mediated through an androgen-induced growth factor (AIGF). Amino acid sequence deduced from cDNA reveals that AIGF has 215 amino acids with a signal peptide and scattered regions homologous to fibroblast growth factor (FGF) family proteins. The biological ability of AIGF to stimulate SC-3 cell growth is inhibited by heparin or suramin. More importantly, antisense oligodeoxynucleotide of AIGF can block androgen-induced growth of SC-3 cells. Upon synthesis under the control of androgen, AIGF is immediately secreted into the extracellular space without intracellular accumulation. At the early phase (18-24 h) of androgen stimulation, however, AIGF is mainly associated with the glycosaminoglycan on the cell surface or extracellular matrix. In addition, treatment of SC-3 cells with sulfation blocker (chlorate) or heparitinase results in the abolishment of their ability to respond to androgen or AIGF, indicating that heparan sulfate has important roles for condensing AIGF on or near the cell surface as well as potentiating the biological activity of AIGF. Then, AIGF can bind to the FGF receptor. Northern blot analysis and cDNA cloning indicate that SC-3 cells predominantly express the FGF receptor 1 with some altered amino acid sequences. Transfection of expression vectors of AIGF and this variant form of FGF receptor 1 into FGF receptor-negative myoblast cells (L 6 cells) confirms that a variant form of FGF receptor 1 is a receptor of AIGF. These results clearly demonstrate that an autocrine mechanism is operating in androgen-induced growth of SC-3 cells.

IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms.

Many insect species have several genes coding for insulin-related peptides (IRPs), but so far only a single IRP gene has been identified in migratory locusts. Here, we report and characterize two other genes coding for peptides that are related to insulin, namely gonadulin and arthropod insulin-like growth factor (aIGF); peptides postulated to be orthologs of Drosophila melanogaster insulin-like peptides 8 and 6 respectively. In Locusta migratoria the aIGF transcript is expressed in multiple tissues as was previously reported for IRP in both L. migratoria and Schistocerca gregaria, but there are significant differences in expression patterns between the two species. The gonadulin transcript, however, seems specific to the ovary, whereas its putative receptor transcript is expressed most abundantly in the ovary, fat body and the central nervous system. Since the central nervous system-fat body-ovary axis is essential for successful reproduction, we studied the influence of gonadulin on vitellogenesis and oocyte growth. A reduction in the gonadulin transcript (via RNA interference) led to a significant reduction in vitellogenin mRNA levels in the fat body and a strong oocyte growth inhibition, thus suggesting an important role for gonadulin in reproduction in this species.

Keywords: IGF; IRP; fat body; locust; oocytes; ovary.

SC-3 cells, derived from androgen-dependent mouse breast cancer Shionogi carcinoma 115, have shown not only androgen-dependent growth stimulation but also morphological changes in serum-free culture. We have demonstrated that this androgen-dependent growth is mediated through an induction of heparin-binding growth factor (termed as androgen-induced growth factor: AIGF). We studied the molecular mechanism of androgen-dependent growth. NIH3T3 cells transfected to express AIGF showed focus-forming activity, anchorage-independent growth and tumor formation in nude mice indicating AIGF is an oncogene. The findings that AIGF inhibited binding of [125I]basic fibroblast growth factor (bFGF) and only FGF receptor (FGFR)-1 mRNA expressed in SC-3 cells suggested that AIGF acts through FGFR-1. Therefore, we cloned FGFR-1 from cDNA library of SC-3 cells, which was found to have three mutations. When this variant FGFR-1 was expressed in FGFR-negative myoblast L6 cells, AIGF-dependent inhibition of the differentiation to myocytes was observed suggesting that variant FGFR-1 is receptor of AIGF. Since expressions of c-myc mRNA as well as AIGF mRNA were induced by testosterone treatment, we studied the role of c-myc in androgen-dependent growth in SC-3 cells using phosphorothioate derived antisense oligonucleotides (antisense) of c-myc or AIGF. Not only AIGF antisense but also c-myc antisense inhibited androgen-dependent growth. c-myc antisense did not affect androgen-induced AIGF mRNA expression. c-myc antisense inhibited AIGF-, but not bFGF-induced DNA synthesis. These results suggest that c-myc has a critical role of AIGF-, but not bFGF-induced growth.

Androgen-induced growth factor (AIGF) is an autocrine growth factor for androgen-dependent SC-3 cells, which is induced by androgen stimuli. To elucidate the mechanism of the progression from hormone-dependent to -independent tumor, we transfected an expression vector of cDNA encoding AIGF into SC-3 cells and established a stable transfectant (A1) expressing AIGF. A1 cells showed enhanced DNA synthesis. This enhanced DNA synthesis was blocked by exposing the cells to AIGF antisense oligonucleotides, heparin, or suramin, indicating that enforced AIGF expression is responsible for the increase in DNA synthesis. However, A1 cells did not grow in serum-free medium unless stimulated with androgen. Recloning from A1 cells in semi-solid agar supplemented with fetal calf serum but without androgen quickly generated an autonomous subline that was able to grow rapidly in the serum-free medium irrespective of androgen stimulus. Mock-transfected SC-3 cells failed to form any colony under identical conditions. These results suggest that stable expression of AIGF alone is not sufficient for, but facilitates the conversion of SC-3 cells from androgen-dependent to -independent phenotype.

Fibroblast growth factor 8 (FGF-8), also known as androgen-induced growth factor (AIGF), is presumed to be a potent mitogenic cytokine that plays important roles in early embryonic development, brain formation and limb development. In the bone environment, FGF-8 produced or received by chondrocyte precursor cells binds to fibroblast growth factor receptor (FGFR), causing different levels of activation of downstream signalling pathways, such as phospholipase C gamma (PLCγ)/Ca²⁺ , RAS/mitogen-activated protein kinase-extracellular regulated protein kinases (RAS/MAPK-MEK-ERK), and Wnt-β-catenin-Axin2 signalling, and ultimately controlling chondrocyte proliferation, differentiation, cell survival and migration. However, the molecular mechanism of FGF-8 in normal or pathological cartilage remains unclear, and thus, FGF-8 represents a novel exploratory target for studies of chondrocyte development and cartilage disease progression. In this review, studies assessing the relationship between FGF-8 and chondrocytes that have been published in the past 5 years are systematically summarized to determine the probable mechanism and physiological effect of FGF-8 on chondrocytes. Based on the existing research results, a therapeutic regimen targeting FGF-8 is proposed to explore the possibility of treating chondrocyte-related diseases.

Keywords: FGF-8; cartilage; chondrocyte; osteoarthritis; skeletal system.