Myoken Y, Myoken Y, Okamoto T, Kan M, McKeehan W L, Sato J D, Takada K
Department of Oral and Maxillofacial Surgery 1, Hiroshima University School of Dentistry, Japan.
Int J Cancer. 1996 Mar 1;65(5):650-7. doi: 10.1002/(SICI)1097-0215(19960301)65:5<650::AID-IJC15>3.0.CO;2-B.
Fibroblast growth factor-1 (FGF-1) and FGF-2 are heparin-binding polype ptides which express potent mitogenic properties in neoplastic cells. In the present study, we have examined the contribution of endogenous FGF-1 and FGF-2 to the autocrine growth of HSY human salivary-gland adenocarcinoma cells in vitro. Using specific monoclonal antibodies against FGF-1 and FGF-2, immunohistochemical analysis of HSY cells revealed strong expression of both FGF-1 and FGF-2 in the cytoplasm and nucleus. Consistent with these data, 2 molecular mass species of FGF-1 (16 and 18 kDa) and 3 FGF-2 (18, 24 and 27 kDa) were identified in HSY cells by Western-blot analysis. Scatchard analysis of FGF binding sites on HSY cells indicated the presence of 23,000 [125I]FGF-1 binding sites/cells with a dissociation constant (KD) of 178 pM and 13,000 [125I]FGF-2 binding sites/cell with a KD of 102 pM. In addition, HSY cells were shown to express the mRNA for FGF receptor-1 (FGFR-1) by reverse transcription-polymerase chain reaction (RT-PCR), confirming the existence of high-affinity FGF binding sites. The influence of endogenous FGF-1 and FGF-2 on HSY cell growth was evaluated by suppressing the expression and activity of FGF by using anti-sense oligonucleotides and neutralizing antibodies. The addition of 50 micron FGF-1-specific anti-sense oligonucleotides to HSY cells resulted in a 61% inhibition of cell growth, while 50 microM FGF-2-specific anti-sense oligonucleotides resulted in a 76% inhibition. These effects were dose-dependent and specific, since sense oligonucleotides were ineffective in inhibiting HSY cell growth at the same concentration. Furthermore, HSY cell growth was suppressed in the presence of anti-FGF-1 or anti-FGF-2 neutralizing antibody, resulting in a 58% inhibition at 8 micromilligrams/ml. Our observations suggest that FGF-1 and FGF-2 may act as autocrine regulators by interacting with FGF receptors on HSY cells.
成纤维细胞生长因子-1（FGF-1）和FGF-2是肝素结合多肽，在肿瘤细胞中表现出强大的促有丝分裂特性。在本研究中，我们检测了内源性FGF-1和FGF-2对体外HSY人涎腺腺癌细胞自分泌生长的作用。使用针对FGF-1和FGF-2的特异性单克隆抗体，对HSY细胞进行免疫组织化学分析，结果显示FGF-1和FGF-2在细胞质和细胞核中均有强表达。与这些数据一致，通过蛋白质印迹分析在HSY细胞中鉴定出2种分子量的FGF-1（16 kDa和18 kDa）和3种FGF-2（18 kDa、24 kDa和27 kDa）。对HSY细胞上FGF结合位点的Scatchard分析表明，存在23,000个/细胞的[125I]FGF-1结合位点，解离常数（KD）为178 pM，以及13,000个/细胞的[125I]FGF-2结合位点，KD为102 pM。此外，通过逆转录-聚合酶链反应（RT-PCR）显示HSY细胞表达FGF受体-1（FGFR-1）的mRNA，证实存在高亲和力FGF结合位点。通过使用反义寡核苷酸和中和抗体抑制FGF的表达和活性，评估内源性FGF-1和FGF-2对HSY细胞生长的影响。向HSY细胞中添加50 μM FGF-1特异性反义寡核苷酸导致细胞生长抑制61%，而50 μM FGF-2特异性反义寡核苷酸导致细胞生长抑制76%。这些作用具有剂量依赖性和特异性，因为正义寡核苷酸在相同浓度下对抑制HSY细胞生长无效。此外，在存在抗FGF-1或抗FGF-2中和抗体的情况下，HSY细胞生长受到抑制，在8 μg/ml时抑制率为58%。我们的观察结果表明，FGF-1和FGF-2可能通过与HSY细胞上的FGF受体相互作用而作为自分泌调节因子。
