Effect of trapidil on effector functions of monocytes related to atherosclerotic plaque.

The infiltration and activation of inflammatory cells play an important role in the formation and stability of coronary atherosclerotic plaque in patients with acute coronary syndrome. In this study, we evaluated the effect of trapidil, an anti-platelet agent, on atheroma-related functions of human T cells and monocytes. Trapidil and anti-CD154 (CD40 ligand) antibody inhibited the increase of procoagulant activity in the mixed lymphocyte reaction; trapidil also suppressed the induction of tissue factor, monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 in the mixed lymphocyte reaction. Trapidil did not alter CD154 expression on isolated T cells, but it diminished CD40 expression on isolated monocytes and human monocytic leukemia THP-1 cells stimulated with interferon-gamma. Moreover, trapidil reduced MCP-1 production of isolated monocytes and THP-1 cells stimulated with interferon-gamma plus CD154-transfected cells. This effect was not seen with other tested anti-platelet agents and coronary vasodilators. In conclusion, trapidil directly acts on monocytes/macrophages to lower their susceptibility to CD154 on T cells.

Protection of mice from LPS-induced shock by CD14 antisense oligonucleotide.

CD14 is a pattern recognition receptor on myeloid cells and plays a pivotal role in an innate immune system that is responsible for Gram-negative and Gram-positive bacteria infection. Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, can induce production of a large quantity of proinflammatory cytokines into the circulation mediated by CD14-mediated macrophages and monocytes. These cytokines eventually cause septic shock. Several in vitro and in vivo studies have shown that suppression of a CD14 function by a CD14 antibody led to an inhibition of the production of proinflammatory cytokines such as TNF-alpha, IL-1 beta, and IL-8. In the present study, we found that CD14 antisense oligonucleotide (ODN) can prevent lethal LPS shock in D-galactosamine-sensitized mice. This ODN inhibited CD14 expression in a mouse macrophage cell line, RAW264.7, and suppressed production of TNF-alpha in LPS-stimulated RAW264.7 cells. Furthermore, we designed a consensus antisense ODN that could hybridize human and mouse CD14 RNA, and we evaluated its efficacy. The consensus antisense ODN rescued mice primed with Mycobacterium bovis bacillus Calmette-Guerin (BCG) from the LPS-induced lethal shock. In this model, the CD14 antisense ODN down-regulated LPS-elicited CD14 expression in the liver, resulting in a decrease in LPS-induced TNF-alpha production. These findings suggest that the CD14 antisense ODN is distributed in the liver and efficiently suppresses LPS-induced TNF-alpha production by reducing CD14 expression on Kupffer cells. This CD14 antisense ODN may be useful for the development of a therapeutic agent against sepsis and septic shock.

Establishment of a human fibroblast cell line producing tumor necrosis factor alpha (KMST-6/TNF) and growth inhibitory effects of its conditioned medium on malignant cells in culture.

To develop a new gene therapy model for cancer, a clonal cell line (KMST-6/TNF) which produces human tumor necrosis factor alpha (hTNF-alpha) has been developed by introducing hTNF-alpha cDNA into a human immortal fibroblast cell line (KMST-6). The conditioned medium (CM) of KMST-6/TNF cells inhibited the growth of various malignant human cell lines, but not that of normal human fibroblasts. Although the growth inhibitory effects of KMST-6/TNF CM were neutralized to a considerable degree by anti-TNF-alpha antibody, its inhibitory effects were more marked than the purified human natural TNF-alpha itself in the same units, suggesting that KMST-6/TNF CM contains some growth inhibitory substances other than TNF-alpha. However, interferons alpha, beta, and gamma were undetectable in the KMST-6/TNF CM.

Cyclin E overexpression responsible for growth of human hepatic tumors with p21WAF1/CIP1/SDI1.

We examined a relationship between p21WAF1/CIP1/SDI1 and cell-cycle-related proteins in 12 human liver tumor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2, -5, -6, -7; Hep3B; HepG2) out of eight cell lines having p21WAF1/CIP1/SDI1 protein overexpressed cyclin E protein, although one of them (JHH-5) overexpressed a reduced size of cyclin E. The rest (HuH-6) of the 8 cell lines with p21WAF1/CIP1/SDI1 showed a decreased expression of cyclin E. Four cell lines (JHH-4; HLE; HuH-7; PLC/PRF/5) deficient of p21WAF1/CIP1/SDI1 protein did not overexpress cyclin E protein. As to expression of the other cell-cycle-related proteins, cyclin A, cyclin D1, CDK2 or CDK4, no significant difference was detected among the 12 cell lines. These findings indicate that the human liver tumor cell lines which have the p21WAF1/CIP1/SDI1-inducible barriers of the cell cycle progression can go through the G1/S checkpoint by overexpressing cyclin E.

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.

Human fibroblasts (KMST-6/RAS line) transformed with 60Co gamma-rays and c-Ha-ras oncogene constitutively produce a large amount of human granulocyte-colony stimulating factor (G-CSF).

Human fibroblasts (KMST-6/RAS) transformed with 60Co gamma-rays and the Ha-ras oncogene formed tumors in nude mice. These mice showed splenomegaly and an increase in granulocytes in the peripheral blood. There was a direct correlation between tumor size and spleen size. Histologically, prominent proliferation of granulocytes was observed in the enlarged spleen. These findings indicated that KMST-6/RAS cells might have been producing granulocyte colony-stimulating factor (G-CSF) in the nude mice. In fact, in vitro studies demonstrated that the cells produced G-CSF in the culture medium and that production of G-CSF was greater during the logarithmic growth than during the stationary phase. Nearly equal amounts of G-CSF were produced by cells grown in serum-free or 10% serum-supplemented medium. Neither expression of the ras oncogene nor the tumorigenicity of cells correlated with the production of G-CSF. G-CSF production in KMST-6/RAS cells was significantly stimulated by butyrate, but not by dexamethasone or 5-azacytidine.

An immunoenzymometric assay for a CA125-like antigen, CA602.

An immunoenzymometric assay (IEMA) for a new CA125-like antigen, CA602, was developed. Five monoclonal antibodies raised against a human ovarian carcinoma cell line could detect their respective antigens in the sera of ovarian carcinoma patients. The antigen levels detected in serum by the various antibodies correlated significantly to each other, and to CA125 levels. The results of epitope analyses and combined IEMAs suggested that the epitopes recognized by these antibodies are not same, but exist on the same antigen which bears the CA125 epitope. A sensitive IEMA was developed with 602-1 and 602-6 antibodies which showed high reactivity to the CA125-like antigen. The antigen defined by these two antibodies was designated as CA602, and serum CA602 levels correlated well with CA125 levels. The CA602 antigen is a CA125-like antigen. Furthermore, the serum CA602 levels did not correlate to CA54/61 levels. The combined assays of CA602 and CA54/61 may increase the detection of ovarian carcinoma.

A new CA125-like antigen (CA602) recognized by two monoclonal antibodies against a newly established ovarian clear cell carcinoma cell line (RMG-II).

A cell line designated RMG-II was established from the ascites of a patient with ovarian clear cell carcinoma. The chromosomal analysis revealed aneuploidy with a hypertetraploid modal number and 8 marker chromosomes. Radioimmunoassay and immunocytochemical staining showed that RMG-II cells produced some tumor markers such as CA125 and TPA. Two monoclonal antibodies, designated MA602-1 and MA602-6, were generated by immunization of mice with an extract prepared from the culture supernatant of RMG-II cells. The epitopes recognized by these two monoclonal antibodies were proved to differ from the CA125 epitope, but to exist on the molecule bearing CA125. We developed a double-determinant sandwich enzyme immunoassay using these two monoclonal antibodies, and the antigen defined by this assay was termed CA602. CA602 was frequently found in the sera of ovarian cancer patients; the positive rates were 92%, 38%, 60%, and 80% for serous, mucinous, clear cell, and endometrioid ovarian carcinomas, respectively, when the cut-off value was set at 60 U/ml (= mean + 3SD of healthy females). CA602 levels in serum were also high in endometriosis patients and in early pregnancy, as is the case for CA125, and the correlation coefficient between CA602 and CA125 was high (r = 0.88). Our preliminary evidence suggests that this CA602 assay system has higher sensitivity than the CA125 one.

Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase.

1. Polyamine oxidase was purified from the soluble fraction of porcine liver by more than 70,000-fold to electrophoretic homogeneity using N8-acetylspermidine-Sepharose 4B affinity chromatography. 2. The molecular weight and isoelectric point of this enzyme were 62,000 and pH 4.5, respectively. 3. Optimal pH for the catalytic activity was close to 10.0. 4. The enzyme activity was enhanced by 5 mM dithiothreitol or 5 mM benzaldehyde. 5. Preferential substrates for this cytoplasmic PAO were N1-acetylspermine, N1-acetylspermidine and spermine. 6. Spermidine was not virtually the substrate for this enzyme. 7. The present results suggested the physiological roles of cytoplasmic PAO, being coupled with the reaction of spermidine/spermine N1-acetyltransferase, in recycling the cellular polyamines to putrescine.

Induction of spermidine/spermine N1-acetyltransferase in needle-punctured rat lens as a model of traumatic cataract.

Isolated rat lens was punctured with a needle at a single point in the equatorial region and was incubated at 37 degrees C. Spermidine/spermine N1-acetyltransferase activity was increased about 5-fold at 8 h after the puncture. Concomitantly, putrescine content in the lens increased markedly at 8-16 h after the puncture, while spermidine levels were slightly depressed. Pretreatment of the lens with actinomycin D or cycloheximide blocked the increases of spermidine/spermine N1-acetyltransferase activity and putrescine content. Ornithine decarboxylase, on the other hand, was not induced to a detectable degree by this stimulus and 5 mM difluoromethylornithine could not block the increase of putrescine content. Polyamine oxidase showed a relatively constant activity that was sufficient for the metabolism of newly formed N1-acetylspermidine. These results suggested that, in the punctured lens, the polyamine levels were regulated predominantly by the activity of spermidine/spermine N1-acetyltransferase, but not by the induction of ornithine decarboxylase.