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Src tyrosine kinase plays key roles in signal transduction following growth factor stimulation and integrin-mediated cell-substrate adhesion. Since src-signal transduction defects are implicated in a multitude of human diseases, we have sought to develop new ways to identify small molecule inhibitors using a yeast-based, activated-src over-expression system. In the present study, we describe the identification of a unique src-signal transduction inhibitor, UCS15A. UCS15A was found to inhibit the src specific tyrosine phosphorylation of numerous proteins in v-src-transformed cells. Two of these phosphoproteins were identified as bona-fide src substrates, cortactin and Sam68. UCS15A differed from conventional src-inhibitors in that it did not inhibit the tyrosine kinase activity of src. In addition, UCS15A appeared to differ from src-destabilizing agents such as herbimycin and radicicol that destabilize src by interfering with Hsp90. Our studies suggest that UCS15A exerted its src-inhibitory effects by a novel mechanism that involved disruption of protein-protein interactions mediated by src. One of the biological consequences of src-inhibition by UCS15A was its ability to inhibit the bone resorption activity of osteoclasts in vitro. These data suggest that UCS15A may inhibit the bone resorption activity of osteoclasts, not by inhibiting src tyrosine kinase activity, but by disrupting the interaction of proteins associated with src, thereby modulating downstream events in the src signal transduction pathway.

Alpha-fetoprotein (AFP) producing gastric cancer (AFP-GC) is very malignant and highly metastatic compared with common gastric cancer. However, the causal relationship between AFP production and the high malignancy of AFP-GC is unclear. We investigated AFP gene regulation in AFP-GC by an active transcription factor, HNF1 (hepatocyte nuclear factor 1) and a repressive transcription factor, ATBF1 (AT motif binding factor 1). RNase protection assays revealed that the production of AFP in gastric cancer cells did not directly associate with HNF1 expression. An inverse relation between the expressions of ATBF1 and AFP was clearly observed in gastric cancer cells. CAT assays showed the direct inhibition of AFP gene expression by ATBF1. Methylation analysis of the AFP promoter region in gastric cancer cells suggested that methylation itself could not explain the silencing of the AFP gene. Immunohistochemistry of resected clinical samples revealed that AFP producing cells lacked ATBF1 immunoreactivity. Our data suggests that the absence of ATBF1 is responsible for AFP gene expression in gastric cancer, and the absence of ATBF1 is a distinct characteristic of AFP-GC and might be important for its highly malignant nature.

Suicide gene therapy using ganciclovir (GCV) with transfection of the herpes thymidine kinase (HSVtk) gene has been studied for cancer therapy. The present study demonstrates an efficient method of suicide gene therapy for multiple hepatic tumors, involving repetitive transfection of the HSVtk gene driven by the alpha-fetoprotein (AFP) promoter using hemagglutinating virus of Japan (HVJ)-liposomes. AFP-producing cells (HUH7) and AFP-nonproducing cells (LS180) were injected subcutaneously (s.c.) to establish tumors in nude mice. Two plasmid constructs, bacterial LacZ gene driven by the AFP promoter (AFPLacZ), and HSVtk gene driven by the AFP promoter (AFPTK1) were encapsulated into the HVJ-liposome and used. When AFPLacZ was injected into the s.c. tumors, expression of LacZ gene was confined to HUH7 tumors. Repeated transfection of AFPTK1 followed by GCV treatment markedly suppressed growth of HUH7 tumors, and apoptosis of HUH7 cells was recognized in the tumor. Next, HUH7 cells were injected into the portal vein in severe combined immunodeficiency mice to establish a hepatic tumor model. After inoculation with the tumor, HVJ-liposomes containing the AFPTK1 plasmid vector were injected into the portal vein via the splenic hilum, followed by GCV treatment. This gene therapy significantly inhibited the growth of tumors in the liver and markedly improved survival. Three injections of the AFPTK1 plasmid vector completely inhibited tumor growth. This procedure seems to have great potential for the treatment of multiple hepatic tumors.

We have previously reported that a retrovirus vector (LNAF0.3TK) carrying a herpes simplex virus thymidine kinase gene regulated only by the 0.3-kb human alpha-fetoprotein (AFP) promoter provides ganciclovir (GCV)-mediated cytotoxicity in high AFP-producing human hepatoma cells but not in low AFP-producing cells. In the present study, a retrovirus vector (LNAF0.3(E+)TK), in which herpes simplex virus thymidine kinase gene expression is under the control of a human AFP enhancer directly linked to its promoter, was constructed and compared with LNAF0.3(E+)TK. In the intermediate and low AFP-producing human hepatoma cells PLC/PRF/5 and huH1/cl.2, respectively, as well as in the high AFP-producing human hepatoma cells (HepG2), LNAF0.3(E+)TK sensitized these cells to GCV in vitro but did not affect cell growth in nonhepatoma cells (HeLa). In an animal model using athymic mice harboring PLC/PRF/5 cells, GCV treatment resulted in more pronounced growth inhibition in the LNAF0.3(E+)TK virus-infected cells than in the LNAF0.3(E+)TK virus-infected cells. These results indicate that the human AFP enhancer that is directly linked to its promoter involves selective and enhanced tumoricidal activity in gene therapy for hepatocellular carcinoma.

In the course of screening for inhibitors of the lymphocyte kinase, Lck (p56lck), aiming at novel immunosuppressants, we isolated a novel alkaloid, lymphostin (LK6-A), from the culture broth of Streptomyces sp. KY11783. Lymphostin was produced in a fermentation medium supplemented with a highly porous polymer resin, which prevented the degradation of this compound in the culture broth. Lymphostin inhibited the kinase activity of Lck with an IC50 value of 0.05 microM, and exhibited potent inhibitory activity against the mixed lymphocyte reaction (MLR) with an IC50 value of 0.009 microM.

The mode of action of gilvocarcins was studied. Gilvocarcins V, M and A possessed antibacterial activities decreasing in that order. Gilvocarcin V inhibited DNA synthesis in Bacillus subtilis through strong interaction with DNA and resulting cleavage. Gilvocarcin M showed interaction with DNA and a small change in DNA mobility upon electrophoresis in agarose gel, while gilvocarcin A showed no interaction with DNA, thus reflecting their relative biological activities.

Novel antitumor antibiotics, tetrocarcin complex composed of three components (A, B and C) were isolated from the culture broth of Micromonospora chalcea KY11091 through various procedures. Tetrocarcins showed marked activity against experimental tumors such as mouse sarcoma 180 and mouse leukemia P388. Multiple injections showed better therapeutic effect against tumors. Mode of action of tetrocarcin A against Bacillus subtilis was found to be through the inhibition of RNA synthesis.

A novel antitumor antibiotic complex has been obtained from the culture broth of Actinomycete strain DO-11 (KY11091) isolated from a soil sample collected in Sendai, Miyagi, Japan. On the taxonomic studies the producing organism is described as Micromonospora chalcea KY11091. For the production of the antibiotics, soluble starch served as a good carbon source and yeast extract was the best nitrogen source tested. The antibiotic complex designated as tetrocarcins is active against Gram-positive bacteria, but is not active against Gram-negative bacteria. Tetrocarcin A showed bacteriocidal activity against Bacillus subtilis.

New components of tetrocarcins (E1, E2, F and F-1) were found in the culture broth of Micromonospora chalcea KY 11091 that was known to produce tetrocarcins A, B and C. Tetrocarcin F-1 consisted of tetronolide and nitro sugar (tetronitrose). Tetrocarcins E1 and E2 consisted of F-1 and deoxy sugar (L-digitoxose). Tetrocarcin F consisted of F-1 and two deoxy sugars (their structures were not yet determined). They all showed antibacterial activities against Gram-positive bacteria and the specific activity decreased with decrease in the numbers of deoxy sugars attached to the aglycone.

A novel complex of calphostin (UCN-1028), which specifically inhibits protein kinase C (PKC) has been isolated from the culture broth of a fungi Cladosporium cladosporioides. Purification of individual components was carried out by silica gel, non-porus resin Diaion HP-20SS and Sephadex LH-20 chromatography, leading to isolation of five closely related components. A, B, C, D and I. Calphostins showed cytotoxic activities against various tumor cells, and these cytotoxicities were proportional to their inhibitory activities against PKC.

In the course of continued screening program for new selective inhibitors of protein kinase C (PKC), fermentation broths from over 5,000 soil isolates were screened for their inhibitory activity of PKC. HPLC analysis of active cultures revealed that five different strains (N-71, N-115, N-126, N-128 and N-139) of Streptomyces isolated from various local soil samples were found to produce staurosporine and related compounds. Of these strains, N-126, a high producing strain, was found to produce new selective inhibitors of PKC, UCN-01 and its stereoisomer, UCN-02. The pH control of fermentation resulted in an increase of the production of UCN-01 and UCN-02.

A new inhibitor of protein kinase C (PKC), UCN-01, was isolated from the culture broth of Streptomyces sp. N-126. We have found that this strain also produces UCN-02 which is a stereoisomer of UCN-01. The inhibitors have the molecular formula C28H26N4O4 and have an indolo[2,3-alpha]carbazole chromophore. Their structures have been elucidated by mass and NMR spectra. UCN-01 has been shown to inhibit PKC and protein kinase A (PKA) with IC50 values of 0.0041 and 0.042 microM, respectively, and UCN-02 has been shown to inhibit PKC and PKA with IC50 values of 0.062 and 0.25 microM, respectively. UCN-01 and UCN-02 also showed the cytotoxic effect on the growth of HeLa S3 cells.

Tetrocarcin A, isolated from a Micromonospora culture showed activity against experimental i.p. inoculated tumors such as Ehrlich carcinoma, MH134 hepatoma, B16 melanoma. But it was not active against solid tumors such as sarcoma 180 and Ehrlich carcinoma. It was marginally active against the growth of solid Lewis lung carcinoma without prolonging the life span of the tumor-bearing mice. It was active against P388 leukemia (i.v.-i.v. system). It did not show myelosuppression and nephrotoxicity in mice. DNA and protein synthesis of P388 cells in culture were more significantly suppressed than RNA synthesis by tetrocarcin A.