Transfection of human fibroblast growth factor 9 (FGF-9) cDNA into mouse BALB/c 3T3 clone A31 cells led to morphological transformation of the cells and foci formation 4 weeks later. Isolated transformants had a higher saturation density than parental A31 cells, could grow in soft agar, and secreted FGF-9 into the culture supernatant. The introduction of FGF-9 N33 cDNA, which encodes a truncated protein that has 33 N-terminal amino acids deleted and has the same mitogenic potency as FGF-9, failed to lead to foci formation. Although FGF-9 is a secretory protein, it does not have a typical secretory signal sequence, and the secreted protein retains the full sequence coded in the cDNA except for the initiating methionine. The produced FGF-9 N33 was not secreted and remained within the cell. It is possible that FGF-9 has an uncleavable signal sequence within the first 33 N-terminal amino acids. All of the phenotypes acquired by transformation could be arrested by treatment with a neutralizing anti-human FGF-9 monoclonal antibody (MAb) 150-59. Additionally, transformants formed tumors in nude mice. Injection of MAb 150-59 suppressed tumor formation in nude mice and caused existing tumors to regress. Our results suggest that the cellular transformation mediated by FGF-9 is produced by autocrine stimulation. We have detected FGF-9 production in the human tumor cell lines glioma NMC-G1, from which FGF-9 was originally purified, and stomach carcinoma AZ-521. The growth of NMC-G1 was not affected by MAb 150-59, but that of AZ-521 was arrested by MAb 150-59 in the presence of heparin. Moreover, the growth of the AZ-521 cell tumor in nude mice could be partially arrested by antibody treatment. The possibility of a participation of FGF-9 in the formation of human tumors is suggested. Int. J.

Transfection of human fibroblast growth factor 9 (FGF-9) cDNA into mouse BALB/c 3T3 clone A31 cells led to morphological transformation of the cells and foci formation 4 weeks later. Isolated transformants had a higher saturation density than parental A31 cells, could grow in soft agar, and secreted FGF-9 into the culture supernatant. The introduction of FGF-9 N33 cDNA, which encodes a truncated protein that has 33 N-terminal amino acids deleted and has the same mitogenic potency as FGF-9, failed to lead to foci formation. Although FGF-9 is a secretory protein, it does not have a typical secretory signal sequence, and the secreted protein retains the full sequence coded in the cDNA except for the initiating methionine. The produced FGF-9 N33 was not secreted and remained within the cell. It is possible that FGF-9 has an uncleavable signal sequence within the first 33 N-terminal amino acids. All of the phenotypes acquired by transformation could be arrested by treatment with a neutralizing anti-human FGF-9 monoclonal antibody (MAb) 150-59. Additionally, transformants formed tumors in nude mice. Injection of MAb 150-59 suppressed tumor formation in nude mice and caused existing tumors to regress. Our results suggest that the cellular transformation mediated by FGF-9 is produced by autocrine stimulation. We have detected FGF-9 production in the human tumor cell lines glioma NMC-G1, from which FGF-9 was originally purified, and stomach carcinoma AZ-521. The growth of NMC-G1 was not affected by MAb 150-59, but that of AZ-521 was arrested by MAb 150-59 in the presence of heparin. Moreover, the growth of the AZ-521 cell tumor in nude mice could be partially arrested by antibody treatment. The possibility of a participation of FGF-9 in the formation of human tumors is suggested. Int. J.

Fibroblast growth factor 9 (FGF-9), a novel member of the FGF family, was found to have thrombopoietic activity in vitro and in vivo. In an in vitro megakaryocyte colony-stimulating factor assay, anti-mouse interleukin-6 (IL-6) monoclonal antibody neutralized FGF-9 activity. This suggests that the activity may be exerted via IL-6 induction. BALB/c mice that received subcutaneous FGF-9 injections of 4 to 100 micrograms/day for 2 weeks showed a dose-dependent transient increase in peripheral platelet counts 10 to 12 days after the first treatment. Histologic studies showed a marked increase in megakaryocytes in bone marrow and extramedullary hematopoiesis in the spleen and the liver. Examination of changes in the DNA content of bone marrow megakaryocytes revealed that the ploidy distribution underwent a marked shift 3 days after FGF-9 injection, with a large increase in the 2N megakaryocyte population. The major modal ploidy shifted from the normal 16N to 2N. The number of megakaryocyte progenitor cells in FGF-9-treated mice increased up to 1.5-fold in the bone marrow and 10-fold in the spleen on day 6. These results indicate that FGF-9 acts on the in vivo proliferation of megakaryocytes.

Hybridomas secreting monoclonal antibodies (MAbs) against human fibroblast growth factor 9 (FGF-9) were established using recombinant human (rh) FGF-9 N33 as the immunogen. Among these, MAb 150-59 demonstrated a potent neutralizing activity against FGF-9. It arrested the FGF-9-induced growth of BALB/c 3T3 A31 cells at an equimolar dose of the factor. It also abrogated the in vivo thrombopoietic activity of FGF-9. Mitogenic activity of several other FGF family members such as FGF-1, FGF-2, and FGF-4 was not neutralized by this MAb. A sensitive sandwich enzyme immunoassay for FGF-9 was developed employing MAbs 150-59 and 13-3. The detection limit of this system was 3 pg/well. In this assay system, FGF-1 and FGF-2 were not cross-reactive up to 1 microgram/well. Using this system, the distribution of FGF-9 in rat organs was examined. FGF-9 could be detected only in the extract of rat cerebellum. Also, we detected a high amount of FGF-9 in the culture supernatant of certain cell lines originated from human tumor. These findings suggest that this enzyme immunoassay system may be used to clarify biological meaning of FGF-9.