The localization of fibroblast growth factor-1 (FGF-1) and FGF-2 in human oral squamous cell carcinoma (SCC) was examined by immunohistochemical techniques using anti-FGF-1 and anti-FGF-2 monoclonal antibodies. Immunofluorescence staining of two oral SCC cell lines revealed that growing cancer cells were intensely positive for both FGF-1 and FGF-2, but confluent cells showed a faint immunostaining. In addition, two molecular mass species of FGF-1 (16 and 18 kDa) and one of FGF-2 (18 kDa) were identified by Western blot in cell extracts derived from growing SCC cells, but not from confluent SCC cells. The growing cell extracts significantly stimulated the proliferation of human umbilical vein endothelial cells. Immunoperoxidase staining of 13 oral SCC cases showed that both well-differentiated and poorly-differentiated cancer cells were positive for FGF-1 and FGF-2 with high frequency and intensity as compared to normal oral epithelium. These results indicate that SCC cells express high levels of endogenous FGF-1 and FGF-2, and suggest that these growth factors may contribute to cancer cell growth.

We have developed a serum-free medium for the growth and differentiation of periodontal ligament-derived cells (PLC). In addition, the expression of both fibroblast growth factor (FGF) and FGF receptor (FGFR) in the PLC was investigated by immunohistochemical examination, heparin affinity chromatography (HAC), and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Optimal growth of the cells was achieved in Iscove's modified Dulbecco's medium supplemented with insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, sodium selenite, and oleic acid in type-I collagen-coated dishes. Both FGF-1 and FGF-2 stimulated cell growth and inhibited differentiation as measured by inhibition of alkaline phosphatase activity of the cells. An immunohistochemical analysis of FGF-1 and FGF-2 revealed that immunoreactive FGF-1 and FGF-2 were detected predominantly in the cytoplasm of growing cells. In addition, perinuclear FGF-1 staining and nuclear FGF-2 staining were observed in the same growing cells. In contrast, a faint diffuse staining of FGF-1 and FGF-2 was detected in cytoplasm of the confluent differentiated cells. The 2.15 M NaCl eluate from HAC of the cell extracts exhibited growth-promoting activities for the PLC, and it also stimulated the growth of human umbilical vein-derived endothelial cells and inhibited binding of [125I]-FGF to its receptors, indicating the cells produced FGFs or FGF-like growth factors. RT-PCR analysis revealed that the cells expressed FGFR-1 mRNA but not mRNAs for FGFR-2, FGFR-3 and FGFR-4 mRNA. These results suggest that the FGF-FGFR-1 system plays an important role in the growth and differentiation of periodontal ligament-derived cells.

A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd = 360 pM, 28,000 sites/cell). The results of cross-linking with [125I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 microM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 micrograms/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.

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