Ken-Ichi Kuroshima

Autocrine transformation by fibroblast growth factor 9 (FGF-9) and its possible participation in human oncogenesis.

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. Cancer 71:442‐450, 1997.

Purification and gene cloning of a chitosanase from Bacillus ehimensis EAG1

Bacillus ehimensis EAG1 (IFO15659) produced and secreted chitosanase in the presence of exogenous chitosan. The chitosanase was purified from the culture filtrate of the bacterium to apparent homogeneity in SDS-polyacrylamide gel electrophoresis. The purified enzyme had a molecular weight of approximately 31,000. A 1.9-kbp DNA fragment containing the chitosanase gene was cloned and the complete nucleotide sequence was determined. The sequence was found to contain a single open reading frame encoding a protein of 302 amino acids. The deduced amino acid sequence showed significant homology with the chitosanase from Bacillus circulans MH-K1.

Stimulation of Thrombopoiesis in Mice by Fibroblast Growth Factor 9

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.

Design and synthesis of a novel series of orally active, selective somatostatin receptor 2 agonists for the treatment of type 2 diabetes

The discovery of a novel series of β-methyltryptophan (β MeTrp) derivatives as selective and orally active non-peptide somatostatin receptor 2 (SSTR2) agonists for the treatment of Type 2 diabetes is described. In our previous research, Compound A, β-MeTrp derivative with highly potent and selective SSTR2 agonistic activity IC50 (SSTR2/SSTR5)=0.3/>100 (nM), was identified asa drug candidate for treatment of Type 2 diabetes which lowers significantly plasma glucose level in Wistar fatty rats in its oral administrations. However, as serious increase in AUC and phospholipidosis (PLsis) were observed in its toxicological studies in rats, follow-up compounds were searched to avoid risk of PLsis with reference to their in vitro PLsis potentials evaluated on the basis of accumulation of phospholipids in HepG2 cells exposed to the compounds. It has been found that introduction of a carbonyl group onto the piperidine and piperazine or aniline moiety of compounds A and B reduced markedly the in vitro PLsis potentials. And further modification of the compounds and their evaluation led to a discovery of compounds 3k with lower in vitro PLsis potentials exhibiting lowering effect of hypoglycemia-induced glucagon secretion in SD rats (ED50=1.1mg/kg) and glucose excursion in meal tolerance test in Wistar fatty diabetic rats (MED=3.0mg/kg) in oral administrations. Compound 3k was selected asa new drug candidate of selective and orally active non-peptide SSTR2 agonists for treatment of Type 2 diabetes with low in vivo PLsis potential.