KEYNOTE SPEAKER

Dr. Daniel B. Rifkin

Activities of High Molecular Weight Forms of FGF-2

The FGF-2 mRNA codes for four species of FGF-2. Translation of one of these forms is initiated at a AUG codon and encodes an 18 kD protein. The three other forms of 22, 22.5, and 24 kD initiate at three CUG codons that are 5' to the AUG codon. Thus, each high molecular weight (HMW) form contains all of the sequences that are present in the smaller forms. The HMW FGF-2s have been shown to localize preferentially to the nucleus, whereas the 18 kD form is primarily cytosolic. The nuclear localization of the HMW forms of FGF-2 is determined by the additional amino-terminal sequences compared to the 18 kD form. This has been demonstrated by showing that chimereic proteins containing the amino-terminal sequence of HMW FGF-2 fused to cytosolic proteins localize to the nucleus. HMW FGF-2 has previously been shown to contain methylarginine (MR) residues. We have analyzed the three HMW species of FGF-2 to localize the individual MR residues. With one exception, all arginines within the context gly-arg-gly are either mono- or di-methylated.

Previously, we demonstrated that cells over-expressing all three HMW forms of FGF-2 were transformed and grew in l% serum, whereas cells transformed by over-expression of 18 kD FGF-2 did not grow under these conditions. To assess whether the capacity to grow in low serum was associated specifically with expression of a unique form of HMW FGF-2, we transfected cells with DNA constructs that encoded single HMW forms of FGF-2 and characterized the cellular phenotype. The results showed that all expression of any HMW FGF-2 form endowed cells with the ability to grow in low serum.

As each of the HMW forms of FGF-2 contains all of the sequence within the 18 kD form, we next asked whether the transforming ability of HMW FGF-2 was a property of the amino-terminal extension or of the 18 kD sequence. Constructs were prepared that encoded the 18 kD sequence fused to either the SV40 T antigen NLS or the cMyc NLS. The properties of cells over-expressing these proteins were examined. In each case, the ability to grow in low serum was confirmed by the appearance of increased nuclear FGF-2. Therefore, expression of chimeric proteins of 18 kD FGF-2 plus any NLS produced cells that grew in low serum indicating that growth in low serum is a consequence of enhanced concentrations of nuclear FGF-2.

These results suggested that the HMW FGF-2 forms must interact with additional proteins within cells. To test for this, we used the yeast two hybrid assay to screen for proteins that bound to HMW FGF-2. One binding partner was characterized as the ribosomal protein L6/TAXREB107. This molecule binds to both 18 kD and HMW FGF-2. Details of the binding properties as well as potential biological significance of this interaction will be discussed.

Structural and Functional Diversity of the FGF Family

The prototypic fibroblast growth factors, FGF-1 (aFGF) and FGF-2 (bFGF), were originally isolated from the brain and pituitary as mitogens for fibroblasts. FGF-1 and FGF-2 are widely expressed in developing and adult tissues, and are polypeptides with multiple biological activities including pattern formation, angiogenesis, mitogenesis, cellular differentiation and repair of tissue injury. The FGF family consisted of nine members, FGF-1 to FGF-9. FGF-3 was identified to be a common target for activation by the mammary tumor virus. FGF-4 and FGF-6 were identified as oncogene products, and FGF-7 to FGF-9 as mitogens for culture cells. These FGFs have a conserved 120-amino acid residue core with 30 to 60% amino acid identity of the FGF family. They also appear to play important roles in developing and adult tissues. FGFs mediate their biological activities by binding to and activating specific cell surface receptors, FGF receptors. Four FGF receptor genes (FGFR-1 to FGFR-4) have been identified. At least 7 FGF receptors with distinct ligand specificities are generated by alternative splicing of these genes.

Although the FGF family consisted of 9 members, additional members have been proposed. We used a T7 phage display method and a homology-based PCR method to identify novel members of the FGF family. A recombinant extracellular ligand binding domain of the FGF receptor was produced by the baculovirus expression system. A T7 phage display rat embryo cDNA library was constructed, and screened with the extracellular ligand binding domain. The recombinant phage clones which bound to the extracellular domain were isolated and analyzed. However, we identified no novel member of the FGF family with the T7 phage display method. Using a homology-based PCR method with various sets of degenerate primers corresponding to the amino acid sequences of the conserved core of the FGF family, we have identified 4 novel members (FGF-10, FGF-16, FGF-17 and FGF-18) of the FGF family. Among known FGF family members, FGF-10 and FGF-16 are most similar to FGF-7 and FGF-9, respectively. FGF-17 and FGF-18 are similar to FGF-8. In addition, 5 novel members (FGF-11 to FGF-15) have been identified by other groups. Currently, the FGF family consists of at least 18 members (1).

Although FGF-10, FGF-17 and FGF-18 have typical signal sequences at their amino termini, FGF-16 has not the signal sequence. However, FGF-16 is efficiently secreted as well as FGF-10, FGF-17 and FGF-18. FGF-10 is expressed in both developing and adult tissues. During embryonic morphogenesis, FGF-10 was shown to participate in limb outgrowth and branching morphogenesis of the lung. These results indicate that FGF-10 is a mesenchymal factor affecting epithelial cells during pattern formation. Among adult tissues, FGF-10 is most abundantly expressed in white adipose tissues. FGF-10 is expressed in preadipocytes but not mature adipocytes of white adipose tissues. FGF-10 has mitogenic activity for preadipocytes, and is transiently expressed in preadipocytes during adipocyte differentiation. Therefore, FGF-10 is expected to participate in energy homeostasis. FGF-16 is abundantly expressed in embryonic brown adipose tissues but not perinatal or postnatal brown adipose tissues, and has mitogenic activity for brown adipose cells. These results indicate that FGF-16 participates in the development of embryonic brown adipose tissues. FGF-17 is preferentially expressed in the embryonic brain, indicating that FGF-17 plays a role in the induction and pattern formation of the brain. FGF-18 is expressed in the adult lung and several discrete regions of the embryo.

The FGF family is a large family consisting of at least 18 members. Each member of the FGF family has a distinct expression profile and physiological roles. The structural and functional diversity of the FGF family is largely unknown. However, members of the FGF family likely play important roles in intercellular signalling with multiple biological activities in developing and adult tissues.

1. Ohbayashi et al., J. Biol. Chem. 272, 18161-18164 (1998)

Roles of FGFs during Limb Development

Many investigations have been done to elucidate cellular and molecular events in the initial phase of limb development. From the experimental results, it has been speculated that a certain factor present in the prospective limb mesoderm acts in initiation of limb bud formation. Recently, it was found that members of the FGF family can induce an additional limb (Dasoku) in the chick embryonic flank by implantation of FGF-beads or Fgf-expressing cells. It was reported by 1996 that such FGF members were FGF1, FGF2, FGF4, and FGF8. However, at that time, none of FGF members except for FGF8 were likely to function as endogenous signaling factors for limb bud induction, because their expression domains are not restricted to the prospective limb territories in chick and mouse embryos. Therefore, one might supposed that ectopically-applied FGFs merely mimic a function of the endogenous limb inducing factor. In the case of FGF8, Crossley et al. (1996) demonstrated that it is expressed in the intermediate mesoderm (IM), but not in the lateral plate mesoderm (LPM), and plays key roles in induction and initiation of chick limb development. They also suggested that FGF8 in the IM may induce expression of Fgf8 in the prospective apical ectoderm indirectly through the LPM. Thus, it is reasonable to consider that there exists an unidentified endogenous factor in the LPM, which induces expression of Fgf8 in the AER. To explore the endogenous limb inducing factor, a new FGF member was tested whether it can induce the Dasoku. In 1996, FGF10 was discovered from rat embryos by a homology-based PCR method by ItohÕs group of Kyoto University. Then, implantation of Fgf10-expressing cells was demonstrated to gives rise to a Dasoku in the competent embryonic flank through induction of Fgf8 expression in the ectoderm. Since the expression domain of Fgf10 is restricted to the prospective limb mesoderm, and finally the definite limb mesenchyme, FGF10 is considered to be an endogenous mesenchymal factor involving in the initial limb budding and continuous limb bud outgrowth of vertebrates. To determine whether the additional limb is formation by FGF10 by the same mechanism as found in authentic limb formation, expression patterns of Fgf8, Fgf10, and Shh were examined in the FGF10-induced ectopic limb bud. The results indicated that ectopically-applied FGF10 induces ectopic Fgf8 expression in the flank ectoderm at first, and then the induced Fgf8 in the flank ectoderm reciprocally induces Fgf10 expression in the underlying flank mesoderm. In the case of FGF8-induced ectopic limb buds, in contrast, exogenous FGF induces Fgf10 expression in the LPM and probably this endogenous FGF10 then induces Fgf8 expression in the overlying flank ectoderm. These results implicated the presence of FGF10-FGF8 positive feedback loop to initiate the outgrowth of normal limb buds. From the observation of Shh expression, it was confirmed that the FGF10-induced limb has a reversed polarity along the anteroposterior axis, similar to other Dasokus. From the gene expression analysis during Dasoku formation, it was concluded that FGF10 seems to be an endogenous initiation factor of limb bud outgrowth and to act as an inducer of Fgf8 expression in the prospective apical ectoderm. One of receptors for FGF10 is likely to be FGFR2IIIb, which is expressed in the epithelial cells of the limb bud. Xu et al. (1998) have mutated FGFR2 by deleting the entire immunoglobin-like domain III of the receptor. Fgfr2DIgIII/ DIgIII mouse embryos do not form limb buds. From this genetic result and others, they concluded that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction. Recently, mouse embryos homozygous for null mutation in Fgf10 was obtained by KatoÕs group of Tokyo University. Their preliminary results indicated that the mutant mice have no limb and no lung (personal communication). These facts support an idea that FGF10 is an endogenous factor inducing outgrowth of the limb bud.

A model for roles of FGFs in initiation of limb bud formation and determination of limb identity is proposed. So far, genes which may be involved in determination of limb identity are Hox-9 genes, Ptx1, Tbx4 and Tbx5. Hoxd-9 may regulate expression of Tbx5, while Hoxb-9, Hoxc-9 and Hoxd-9 may regulate expression of Ptx1 and Tbx4, judged from their expression patterns. Since members of the FGF family can change expression of Hox-9 genes in Dasoku formation, FGF may also be involved in regulation of Hox-9 expression in normal limbs, although it would appear that FGF does not determine limb identity by itself. Since initial endogenous FGF is most likely to be FGF10, FGF10 is a candidate for a factor regulating Hox-9 expression in the LPM. However, even if it is the case, for the main targets of FGF10 appear to be epithelial cells, effects of FGF10 on the mesenchymal cells might be indirect. Thus, there is a possibility that other unidentified FGF which regulates directly expression of Hox-9. Subsequently, Tbx5 and Tbx4 activate their own sets of target genes to establish individual phenotypes with each limb identity. In determination of bone patterning, some factors may regulate Hox-9-Hox-13 codes for establishing cartilage pattern formation. For determination of epithelial phenotypes, mesenchymal cells expressing Tbx5/4 may produce some secreting factors such as BMP and FGF which act on epithelial cells. The consequent epithelial-mesenchymal interaction would result in epithelial pattern formation. The presence of a signaling cascade is speculated, in which Tbx5 and Tbx4 activate the FGF10-FGF8 positive feedback loop to initiate the outgrowth of both limbs.

Regulation of FGF Activity by Heparin or Heparan Sulfates and Role of FGF System in Cancer Progression

Fibroblast growth factors (FGFs) constitute a family of structurally related but genetically distinct polypeptides that share strong affinity for heparin or heparan sulfates (HS), act as intrinsic regulators of cell to cell communication within all tissues including the developing embryo, have impact on practically all known cellular functions and therefore are important in associated tissue pathologies. Among them, FGF-1 (acidic FGF) and FGF-2 (basic FGF) are widely distributed in most adult tissues and show strong angiogenic activity in vivo. Both FGF-1 and FGF-2 stimulate the proliferation of stromal cells such as fibroblasts, vascular endothelial and smooth muscle cells in vitro. In contrast, the expression of FGF-7(keratinocyte growth factor, KGF) is limited to the stromal compartment of parenchymal tissues containing a well-defined stroma and epithelium. Of all the FGF ligands, FGF-7 exhibits the strictest range of specificity for receptor isotype. It acts specifically on epithelial cells which express a specific splice variant of receptor in epithelium makes FGF-7 a directionally-specific paracrine factor underlying communication of epithelial cells with their stroma.

FGF action is mediated through binding and activation of receptors with tyrosine kinase activity. FGF receptors (FGFR) consist of four different gene products that have two or three immunoglobulin (Ig)-like loop structures in the extracellular domain and a tyrosine kinase in the intracellular domain. Regulated combinatorial alternate splicing resulting in coding sequence for cassettes of receptor subdomains generates a large degree of functional diversity of the monomeric product from one FGFR gene. The FGFR type 1 gene in general, and its splice variant FGFR1IIIc in particular, appears to be the member of the four gene family whose expression is limited to stromal cells. The IIIb splice variant of FGFR2, which arises by mutually exclusive alternate splicing of exon cassettes coding for the second half of the third Ig loop of the receptor ectodomain, appears limited to epithelial-like cells.

In addition to ligand and thyrosine kinase, our results along with others show that heparan sulfate proteoglycans (HSPG) on the cell surface or within the extracellular matrix play an integral role in the FGF signal transduction complex. In addition to their role as a reservoir or sequestering agent for FGF ligands, HS chains interact with the ectodomain of FGFR through a specific amino acid sequence in Ig loop II which requires divalent cations and potentially anchors the ectodomain in a ligand-dependent conformation for dimerization and activation of receptor kinase. Through the combined requirement for a single bivalent HS chain that will react with both an FGF ligand and FGFR ectodomain plus the length sufficient to the two together, HS chains may exhibit an element of ligand- and receptor-related specificity. In fibroblasts and endothelial cells, exogenous heparin potentiates and, in some cases, is absolutely required for the activity of FGF-1, but not FGF-2. This and our results suggest that endothelial cells express an HSPG which is specific for FGF-2 and FGFR1. In contrast, nonmalignant immortalized and tumor-derived epithelial cells appear to express HS which support the interaction of both FGF-1 and FGF-2 under the same conditions. These results raise the possibility that specific HS chains of specific classes of HSPG may complement different combinations of FGF ligands and FGFR ectodomains in assembly and activation of the FGF signal transduction complex.

Transplantable rat prostate tumors exhibit characteristics of human tumors as they progress from the slow-growing, androgen-responsive, well-differentiated state to the malignant, androgen-independent, undifferentiated state. Dunning tumors exhibit well-differentiated stromal and epithelial compartments. Cloned epithelial cell lines (DT-E) derived from the R3327PAP tumors express exclusively FGFR2IIIb and respond to stromal-derived FGF-7 (or FGF-1). The derived stromal cells (DT-S) express FGFR2IIIc and respond to FGF-2 (and FGF-1). Cells derived from malignant R3327AT3 tumors which arise from tumors similar to the R3327PAP tumor after castration of male hosts and prolonged passage in castrated hosts or females or tumors that eventually emerge from the DT-E cells in absence of stroma kill hosts after several weeks, are completely insensitive to androgen, are undifferentiated and express FGFR2IIIc and FGFR1IIIc. The AT3 cells are completely insensitive to FGF-7 and stromal cells. Increasing evidence suggests that the control of epithelial cell functions by androgen is partitioned between the stroma and epithelium. Androgenic regulation of the communication between stroma and epithelium underlies the indirect control of epithelial cell functions (growth and differentiation ) by androgen. The partitioning of the expression of FGF-7 in stromal cells and its receptor FGFR2IIIb on epithelial cells appears to be a directionally-specific paracrine communication system in which signal arises in stroma and reception lies in the epithelium. In prostate and other steroid-responsive tissues, the FGF-7 signal is under control of androgen and, although not proven to date, it is predicted that the reception end in epithelium may be regulated by androgen. Subversion of the system from multiple directions may underlie the diverse properties of prostate tumors as they progress eventually to a state of independence on androgen and stroma, to a state of growth autonomy and to the undifferentiated state. Androgen-independence may occur by loss of dependence of expression of FGF-7 on androgen without loss of stromal independence and differentiation. Exon switching from FGFR2IIIb to FGFR2IIIc and activation of FGFR1 or loss of the FGFR2 gene entirely which has been observed to occur during progression of model prostate tumors potentially confers both androgen-independence and lack of differentiation concurrent with independence on stroma. Activation of abnormally-expressed FGF ligands (e.g. FGF-2, FGF-3, FGF-5) may confer growth autonomy by the autocrine loop formed between the tumor-associated ligands and the FGFR2IIIc and FGFR1IIIc isoforms. Indeed, introduction of FGFR1 by tranfection in nonmalignant DT-E cells accelerates their progression in vivo and introduction of FGFR2 in malignant AT3 cells inhibits their growth in vitro and in vivo. Most recently, increasing evidence suggests that FGF action occurs through much more tightly regulated steps than simply binding of ligands to the ectodomain of tyrosine kinase receptors. HS chains attached to the cores of HSPG appear to be bivalent and potentially specific integral components of the FGFR signal transduction complex. The HSPG component plays a critical role in both restriction and regulated activation of the oligomeric FGFR signal transduction complex. In this integral role, HSPG are expected to play an equally important role to ligand and receptor kinase isotype in normal prostate tissues and prostate tumors.

Signal Transduction and Transcriptional Regulation of Endothelial Cells during Angiogenesis

Angiogenesis is a process by which new blood vessels are formed from pre-existing ones. Angiogenesis is thought to be regulated by the balance between angiogenic factors and angiogenesis inhibitors. A number of factors have been identified as angiogenic factors . Among them. fibroblast growth factor (FGF) family proteins including acidic FGF and basic FGF have potent angiogenic activities. When vascular endothelial cells are stimulated with these factors, angiogenesis takes place.

Angiogenesis is a complex phenomenon which includes at least four distinct properties of ECs; degradation of vascular basement membrane and interstitial matrices by proteases, migration, proliferation, and tube formation. Among them, the coordinate induction of proteases and cell migration is a principal feature of ECs in the initial step of angiogenesis. Plasminogen activator (PA) and matrix metalloproteinases (MMPs) are proteases which are involved in the degradation of extracellular matrices. ECs express urokinase-type PA (u-PA), tissue-type PA (t-PA), MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin l), MMP-9 (gelatinase B), and MTl (membrane type I) -MMP. Cell migration requires actin-regulated cell motility as well as cell adhesion to extracellular matrix. Integrins, a and b heterodimeric adhesion molecules, play a pivotal role in cell adhesion to extracellular matrix. ECs express various integrins. Among them, integrin avb3 plays an important role in angiogenesis.

Recent studies in our laboratory focused on the signal transduction and transcriptional regulation of ECs during the course of angiogenesis. When ECs were stimulated with basic FGF, MAP kinase homologs; ERKl/2 and p38 MAP kinase were activated. The specific inhibition of either ERK1/2 or p38 MAP kinase could inhibit angiogenesis in vitro. It appeared that the activation of ERKl/2 was involved in cell proliferation as well as induction of transcription factor ETS-1, whereas the activation of p38 MAP kinase was involved in actin reorganization, which was responsible for cell motility. To clarify the role of ETS-1 in ECs, we established both high and low ETS-1 expressing EC lines, and compared angiogenic properties of these cell lines with a parental murine EC line. The growth rate was almost identical with each cell lines. The gene expressions of matrix metalloproteinases (MMP-1, MMP-3. and MMP-9) as well as gelatinolytic activity of MMP-9 were significantly increased in high ETS-1 expressing cells. Moreover, low ETS-1 expressing cells could not spread on vitronectin substratum and the phosphorylation of focal adhesion kinase was markedly impaired because of the reduced expression of integrin b3. As a result, the invasiveness was enhanced in high ETS-1 expressing cells and reduced in low ETS-1 expressing cells compared with parental cells, and high ETS-1 expressing cells made more tube-like structures in type I collagen gel. These results indicate that ETS-1 regulates angiogenesis by inducing MMP-1, MMP-3, MMP-9, and integrin b3 in ECs.

Role of Basic Fibroblast Growth Factor (bFGF) in Tumor Angiogenesis

Angiogenesis, the development of new blood vessels, is essential for rapid growth and metastasis of solid tumors. Tumor angiogenesis is regulated by the angiogenic stimulators produced by tumor cells. The first molecule identified as a purified angiogenic factor was basic fibroblast growth factor (bFGF or FGF-2) which is the prototype member of a family of FGF. bFGF is almost ubiquitously distributed in tumor cells derived from solid tumors (In Vitro Cell. Dev. Biol., 28A:419-428,1992). This was followed by the identification of various angiogenic factors. Among these factors, recent attention has focused on vascular endothelial growth factor (VEGF). Strong evidences in support of in vivo role of these angiogenic factors have been provided by the observations that the neutralizing antibodies against bFGF and VEGF suppress tumor growth and angiogenesis produced by transplantation of a variety of tumor cell lines into athymic mice. However, the inhibitory effects were variable and relative contribution of these growth factors in tumor angiogenesis is unclear. In the present study, the role of bFGF in tumor growth and angiogenesis is demonstrated using a neutralizing monoclonal antibody against bFGF that we have generated (Proc. Natl. Acad. Sci. USA, 86:9911-9915, 1989).

Properties of Neutralizing Monoclonal Antibody against bFGF


We produced a neutralizing monoclonal antibody against bFGF (bFM-1) which is highly specific for bFGF from bovine, human, rabbit and murine sources and also for high molecular weight form of human bFGF. bFM-1 did not cross-react with inactivated bFGF, suggesting that the antibody recognizes the conformation of the bFGF molecule necessary for its biological activity. The antibody inhibited the growth of bovine capillary endothelial (BCE) cells in culture not only in the presence but also in the absence of exogenous bFGF, indicating that it blocks the biological activity of bFGF and also the autocrine action of bFGF which is produced and secreted by BCE cells. When BCE cells were incubated in culture medium supplemented with bFM-1, viability as measured by plating efficiency decreased, indicating that endogenous bFGF also acts as a survival factor for the cultured endothelial cells. It has been also shown that bFM-1 had neutralizing activity in some in vivo experiments. For example, the proliferation of peritubular endothelial cells during compensatory renal growth after unilateral nephrectomy in mice was inhibited by i.v. injection of bFM-1 (Am. J. Physiol., 265:F712-F716, 1993). During the repair process of the defects of articular cartilage in rabbit, the local administration of bFM-1 inhibited the endochondral repair response (Develop. Growth Differ., 39:143-156, 1997). Therefore, bFM-1 should modulate in vivo tumor angiogenesis and growth that are regulated by bFGF due to its growth inhibitory and/or anti-survival effect(s) on the endothelial cells.

Antitumor Effect of bFM-1

bFM-1 inhibited solid tumor growth in athymic mice to a variable extent possibly depending on the expression levels of bFGF and VEGF. Among various human tumor cell lines, growth of solid tumor developed by s.c. injection of a colon cancer cell line (RPMI4788) into the backs of athymic mice was almost completely inhibited by the i.p. administration of bFM-1 twice a week. Enzyme-linked immunosorbent assay (ELISA) studies were performed on the plasma and various tissue extracts of the treated mice to examine the circulating antibody level and its distribution in the organs. High level of the immunoreactive bFM-1 in the circulation and the tissues including solid tumors was achieved by the present administration schedule. On the other hand, growth of xenografts derived from human epidermoid carcinoma cell line (A431) and human cervical carcinoma cell line (HeLa) was partially inhibited by the treatment with the antibody. These three cell lines all expressed bFGF not only in cultures but also in xenografts. Growth of mouse myeloma cell (P3U1) xenograft, which expressed bFGF to a lesser extent, was not inhibited by the treatment with the antibody. In vitro growth of these cell lines was not inhibited by the antibody, suggesting that the in vivo growth inhibition is due to anti-angiogenic effect of the antibody. RPMI4788 cells and A431 cells also expressed VEGF and growth of these xenografts was inhibited by the neutralizing monoclonal antibody against VEGF. Both antibodies had synergistic inhibitory effects on xenograft growth, suggesting that bFGF and VEGF act in a concerted manner for angiogenesis in the solid tumors developed from these cell lines producing both growth factors.

Effect of bFM-1 on Angiogenesis in Matrigel

In order to elucidate further the function of bFGF in tumor angiogenesis, Martigel (mouse basement membrane extract) containing bFGF or tumor cells with or without bFM-1 was injected s.c. into near abdominal midline of athymic mouse. The gels were removed after 7 days and the hemoglobin concentration was spectrophotometrically measured to quantitate the angiogenesis. Hemoglobin content increased in Matrigel supplemented with RPMI4788 cells or A431 cells, paralleling the increase in vessels observed histochemically. The angiogenesis induced by these cells was suppressed by the addition of bFM-1. When bFGF was retained in Matrigel, the angiogenesis in the gel also increased. The angiogenesis induced by bFGF was inhibited not only by bFM-1 but also by the neutralizing monoclonal antibody against VEGF, indicating that the angiogenic activity of bFGF is at least in part mediated by VEGF.

Conclusion

In solid tumors that express bFGF, the growth factor probably has key role in the tumor angiogenesis depending on an expression balance between bFGF and VEGF. It is highly probable that the angiogenic activity of bFGF is mediated by VEGF in some cases.

Functional dichotomy and regulation of the human FGF-binding protein HBp17

HBp17 is a heparin-binding protein, which was initially purified from culture medium conditioned by A431 epidermoid carcinoma cells. This protein interacts with and modulates the activities of at least three members of the fibroblast growth factor family. When HBp17 cDNA was transfected into a non-tumorigenic A431 variant cell line that expresses FGF-2 (bFGF) but not HBp17, the transfectants formed squamous cell carcinomas in nude mice. The tumorigenic potential of the transfectants correlated with the level of HBpl7 expression while FGF-2 1evels remained relatively constant. We used representational display analysis to identify additional changes in gene expression that occurred as the A431 variant cell line became tumorigenic. Northern hybridization was performed to detect HBp17 mRNA in normal and tumor-derived cells. The results showed that HBp17 mRNA transcription was restricted to normal and malignant squamous epithelial cells. These results suggested that HBp17 has a physiological role in normal stratified squamous epithelial tissue, and it is also involved in the development of squamous cell carcinomas. We believe that the normal function of HBp17 as an FGF-binding protein that promotes cell growth and differentiation is subverted as squamous epithelial cells undergo malignant transformation and acquire the ability to produce FGFs de novo. In this abnormal physiological environment HBp17 promotes the paracrine and autocrine activities of the tumor-derived FGFs.

An understanding of HBp17 gene structure and the mechanisms that regulate HBp17 expression will help to further clarify HBp17 function in normal epithelial tissue and its role during the development of squamous cell carcinomas. To this end we cloned the human HBp17 gene from a WI-38 genomic library using human HBp17 cDNA as a probe. A comparison of the DNA sequence of a relevant 5 kb genomic fragment with the known full-length cDNA sequence indicated that HBp17 was encoded by a single exon following introns of 214 bp and 1.7 kb. Thus, the HBp17 gene consists of three exons, two of which are not translated, and two introns. Functional analysis of the HBp17 promoter was performed by transfection of A431, HeLa-S3 and HepG2 cells with promoter-1uciferase gene constructs containing 725 bp, 646 bp, 520 bp, 306 bp or 206 bp of 5'-flanking DNA. Two regions with promoter activity were identified: the region upstream of the first exon had weak activity; while strong promoter activity resided between the two introns. The strong promoter activity was localized to a 126 bp DNA fragment that was sufficient to initiate HBp17 gene transcription in A431 cells. The 126 bp fragment contained a TATA box approximately 30 bp upstream of a transcription start site and several response elements for known transcription factors. Electrophoretic mobility shift assays with A431 nuclear extracts in conjunction with promoter mutation analyses demonstrated that HBp17 gene expression was modulated by both positive and negative regulators of transcription.

This work was supported by grant 3276R from the Council for Tobacco Research-U.S.A., Inc. and grant CN-101 from the American Cancer Society.