Oncogene database

2249

FGF4

fibroblast growth factor 4;FGF4;fibroblast growth factor 4

FGF-4|HSTF-1|fibroblast growth factor 4 splice isoform|heparin secretory transforming protein 1|heparin secretory-transforming protein 1|heparin-binding growth factor 4|human stomach cancer, transforming factor from FGF-related oncogene|kaposi sarcoma onc

11q13.3

protein-coding

A plasmid containing the K-fgf proto-oncogene linked to the dihydrofolate reductase gene has been constructed, and used in transfection experiments to investigate the effects of K-fgf expression on the tumorigenic and metastatic properties of NIH-3T3 fibroblasts. Analysis of cells transfected with K-fgf revealed that expression of the K-fgf proto-oncogene can, in a single step, induce both tumorigenic and metastatic characteristics, as determined in soft agar cloning experiments, and in tumorigenicity and experimental lung metastasis assays with BALB/c nu/nu mice. Selection for resistance to increasing concentrations of methotrexate lead to the isolation of a series of cell lines containing amplifications of both the dihydrofolate reductase gene and the linked K-fgf gene, which synthesized elevated levels of growth factor message and protein. The most highly resistant and gene amplified cell lines exhibited lower than expected levels of K-fgf mRNA, and also appeared to have down-regulated cell surface growth factor receptors. Further support for the concept that altered K-fgf expression can induce fully malignant and metastatic cells was obtained in experimental metastasis assays, where K-fgf transfected and gene amplified cell lines were highly aggressive.

expression of the K-fgf/hst proto-oncogene appears to be restricted to cells in the early stages of development, such as embryonal carcinoma (EC) cells. When EC cells are induced to differentiate, K-fgf expression is drastically repressed. To identify cis-acting DNA elements responsible for this type of regulation, we constructed a plasmid in which cat gene expression was driven by about 1 kilobase of upstream K-fgf human DNA sequences, including the putative promoter, and transfected it into undifferentiated F9 EC cells or HeLa cells as prototypes of cells which express or do not express, respectively, the K-fgf proto-oncogene. This plasmid was essentially inactive in both cell types, and the addition of more than 8 kilobases of DNA sequences upstream of the K-fgf promoter did not lead to any increase in chloramphenicol acetyltransferase (CAT) expression. On the other hand, when we inserted in this plasmid DNA sequences which are 3 of the human K-fgf coding sequences, we could detect a significant stimulation of CAT activity. Analysis of these sequences led to the identification of enhancerlike DNA elements which are part of the 3 noncoding region of K-fgf exon 3 and promote CAT expression only in undifferentiated mouse F9 or human NT2/D1 EC cells, but not in HeLa, 3T3, or differentiated F9 cells, therefore mimicking the physiological expression of the K-fgf proto-oncogene. Similar elements are also present in the 3 region of the murine K-fgf proto-oncogene, in a region showing high homology to the human K-fgf sequences. These regulatory elements can promote CAT expression from heterologous promoters in an EC-specific manner, suggesting that they interact with a specific cellular transacting protein(s) whose expression is developmentally regulated.

The nucleotide sequence of the 5 -flanking region of the mouse k-FGF oncogene has been determined. This sequence extends 2.1 kb upstream from the transcriptional start point (tsp) and includes two Sp1 and two AP-2 consensus binding sequences immediately 5 of the TATA box. In addition, the sequence contains an alternating purine:pyrimidine motif that lies approx. 1 kb upstream from the tsp.

Embryonal carcinoma (EC) cells provide an effective model system for studying growth factor production and regulation during mammalian embryogenesis. Our earlier data indicated that the mouse EC cell lines F9 and PC-13 and the human EC cell line NT2/D1 produce a factor with properties similar to those ascribed to members of the fibroblast growth factor (FGF) family and that production of this FGF-related factor is suppressed when ALL three EC cell lines are induced to differentiate. Subsequent studies suggested that NT2/D1 EC cells express transcripts for basic FGF (bFGF). The current study confirms and extends these findings using a combination of reverse transcription and polymerase chain reaction (RT-PCR). In this study, the expression of bFGF and other members of the FGF family have been examined in F9 and PC-13 cells in addition to NT2/D1 EC cells. In contrast to NT2/D1 EC cells, bFGF expression could not be detected in F9 and PC-13 EC cells. Additionally, expression of four other members of the FGF family (acidic FGF, int-2, FGF-5, and FGF-6) were not detected in NT2/D1, F9, or PC-13 EC cells. However, expression of another member of the FGF family, the k-FGF oncogene, was detected in NT2/D1, F9, and PC-13 EC cells. Moreover, the expression of this transcript is reduced dramatically when each of the three EC cell lines is induced to differentiate. Taken together, our findings argue that expression of the k-FGF oncogene is predominantly responsible for the FGF-related activity detected in EC cells and that differentiation of these EC cells results in suppression of this oncogene.

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.

The immunohistochemical localization of ras p21 oncogene product was examined in human bladder cancer. These bladder cancers consist of 52 superficial bladder tumors and 10 cases of invasive tumor. Five (9.6%) of 52 superficial tumors were demonstrated to be positive for ras p21 product. Two (20%) of 10 cases which showed deep tumor infiltration were demonstrated to be positive for ras p21 oncogene product. When we analyzed the incidence of positive staining for ras p21 with regard to histological grade of bladder cancer, 2 of 30 (7%) patients with G1, 4 of 26 (15%) with G2, and 1 of 6 (17%) with G3 had positive staining for the ras p21 oncogene product. One of five patients who showed positive staining on tumors had tumor recurrence 3 months later. The remaining 4 patients positive for the ras p21 oncogene product had no tendency for recurrence in 11-19 months follow-up. Therefore, the incidence of detection of ras p21 oncogene product on superficial and deep infiltrating bladder tumor was similar and there was no significant correlation between the incidence of positive ras p21 staining and depth of invasion of bladder tumors or histological grade.

We recently reported the cloning of a rearranged human oncogene following transfection of DNA from Kaposi s sarcoma into NIH 3T3 cells. To identify the protein(s) encoded in two novel mRNAs of 3.5 and 1.2 kb expressed in NIH 3T3 transformants, we constructed a cDNA library. One of the cDNA clones isolated (KS3) corresponded to the 1.2 kb mRNA and transformed NIH 3T3 cell when inserted into a mammalian expression vector. The 1152 nucleotide KS3 cDNA encodes a protein of 206 amino acids with significant homology to the growth factors basic FGF and acidic FGF. expression of the KS3 product as a bacterial fusion protein or in COS cells allowed us to determine that both proteins had significant growth-promoting activity and that the COS cell protein was glycosylated. Thus one of the mRNAs transcribed from the KS oncogene encodes a growth factor that could transform cells by an autocrine mechanism and appears to represent a new member of the FGF family.

The protein encoded in a novel human oncogene isolated by transfection of Kaposi s sarcoma DNA is a growth factor with significant homology to basic and acidic FGFs. The genomic structure of this oncogene (designated K-FGF), as originally isolated, carried DNA rearrangements upstream and downstream of the coding region. The normally discontinuous sequence upstream of the K-FGF coding region derived from the 3 end of the c-fms gene and thus originated from human chromosome 5. In order to determine the normal chromosomal location of the K-FGF gene and of the DNA sequences adjacent to its 3 end, we have correlated the presence of these sequences with retention of specific human chromosome regions in rodent-human somatic cell hybrids. These experiments mapped the K-FGF gene to human chromosome region 11q13----11q23, and in situ hybridization localized it more precisely to region 11q13 near int-2, which also belongs to the FGF family. The sequence downstream of the gene in transfectants and discontinuous with K-FGF in normal human DNA derives from chromosome region 12p12----12q13, possibly near the int-1 locus.

In this report we described the linkage between two oncogenes of the fibroblast growth factor family. Using in situ hybridization to human metaphase chromosomes we mapped the hst gene to chromosome 11 at band q13. This is also the location of the int.2 gene. Furthermore, the two genes are co-amplified in a human melanoma, raising the possibility that amplification in human tumors may be a mechanism of activation of genes of the FGF family.

Transfection of rat thyroid differentiated epithelial cells, PC clone 3, with the K-fgf/hst oncogene results in alleviation of thyrotropin dependency for growth and suppression of the differentiated phenotype without the acquisition of the fully transformed phenotype. An autocrine mechanism is responsible for these effects, since the proliferation of PC clone 3 cells, induced by K-FGF-conditioned medium, is blocked by anti-K-FGF-specific antibodies. Moreover, addition of K-FGF-conditioned medium inhibits the thyroid differentiated functions. Also, such other members of the fibroblast growth factor family as basic and acidic fibroblast growth factor are able to induce thyroid cell growth and to block expression of the differentiated functions.

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained ALL of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.