Toshinori Kanamori

Interferon potentiates cytotoxic effects of 5-fluorouracil on cell proliferation of established human cell lines originating from neoplastic tissues.

A potentiation of the cytotoxic effects of 5-fluorouracil (5-FU) on human tumor cells by interferon was examined. The human neoplastic cell lines used were HeLa (uterine cervical cancer), MCF-7 (mammary cancer), WI-38 CT-1 (embryonic lung fibroblasts transformed in culture by Co-60 gamma-ray irradiation), KMM-1 (myeloma) and Raji (Burkitts lymphoma). The normal human cell strain used was WI-38 (normal human lung fibroblasts). The cytotoxic effects were determined by colony formation for HeLa, MCF-7, WI-38 CT-1 and WI-38 cells, and by cell growth for KMM-1 and Raji cells. Each cell line was different in sensitivity to interferon or 5-FU. Interferon potentiated synergistically the cytotoxic effects of 5-FU on HeLa, WI-38 CT-1 and KMM-1 cells. In the case of Raji cells, the cytotoxic effects of the combination of interferon and 5-FU were additive. Neither synergistic nor additive lethal effects of the combination of the 2 agents were observed in MCF-7 and WI-38 cells. The present results indicate a possibility that interferon and 5-FU can mutally reduce the amount of the other needed to treat cancer patients.

Molecular design of hybrid tumour necrosis factor alpha with polyethylene glycol increases its anti-tumour potency

This study was conducted to increase the anti-tumour potency and reduce the toxic side-effects of tumour necrosis factor alpha (TNF-alpha). Natural human TNF-alpha was chemically conjugated with monomethoxy polyethylene glycol (PEG) using succinimidyl coupling of lysine amino groups of TNF-alpha. The number-average molecular weight of PEG-modified TNF-alpha (PEG-TNF-alpha) increased with an increase in the reaction time and the initial molar ratio of PEG relative to TNF-alpha. The resulting modified TNF-alpha was separated into fractions of various molecular weights. The specific activity of separated PEG-TNF-alpha s relative to that of native TNF-alpha gradually decreased with an increase in the degree of PEG modification, but the plasma half-life was drastically increased with the increase in molecular weight of modified TNF-alpha. PEG-TNF-alpha s, in which 29% and 56% of lysine residues were coupled to PEG, had anti-tumour activity approximately 4 and 100 times greater than unmodified TNF-alpha in the murine Meth-A fibrosarcoma model. Extensive PEG modification did not increase its in vivo activity. A high dose of unmodified TNF-alpha induced toxic side-effects, but these were not observed with the modified TNF-alpha s. Optimal PEG modification of TNF-alpha markedly increased its bioavailability and may facilitate its potential anti-tumour therapeutic use.

In vitro and in vivo studies on potentiation of cytotoxic effects of anticancer drugs or cobalt 60 gamma ray by interferon on human neoplastic cells

A possibility that interferon may potentiate the cytotoxic effects of anticancer drugs or 60Co gamma ray on human neoplastic cells was studied by in vitro and in vivo experimental procedures. The human neoplastic cells used were HeLa (uterine cervical cancer) and WI‐38 CT‐1 (embryonic lung fibroblasts transformed in culture by 60Co gamma ray) cells. As normal human cells, WI‐38 cells were used. Interferon was a preparation of β‐type produced by human fibroblasts. The cytotoxicity was determined by colony formation for in vitro experiments and by tumor growth for animal experiments. Of 17 anticancer drugs, the cytotoxic effects of six drugs, namely, peplomycin, bleomycin, aclacinomycin, cisplatin, 5‐fluorouracil (5‐FU), and Adriamycin (doxorubicin) were potentiated by concomitant application of interferon. The cytolethal effects of 60Co gamma ray were also enhanced by interferon. The growth of tumor induced by transplantation of HeLa cells into a nude mouse was remarkably reduced by combination therapy of interferon and 5‐FU. The current results indicate a possibility that combined therapy of certain types of anticancer drugs or 60Co gamma ray with interferon may be effective in treatment of cancer patients.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed byN-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

Molecular design of hybrid tumour necrosis factor-alpha. II: The molecular size of polyethylene glycol-modified tumour necrosis factor-alpha affects its anti-tumour potency.

To design hybrid tumour necrosis factor-alpha (TNF-alpha) applicable to systemic anti-tumour therapeutic use, we assessed the relationships among the molecular size of hybrid TNF-alpha, in vitro bioactivity and in vivo anti-tumour potency. Natural human TNF-alpha was covalently modified with polyethylene glycol (PEG) of various number-average molecular weights (Mn = 2000, 5000, 12,000). The in vitro bioactivity of PEG-modified TNF-alpha s decreased with an increase in the degree of PEG modification, irrespective of the molecular weight of PEG. This decrease in the specific bioactivity markedly increased with an increase in the molecular weight of the attached PEG. The in vivo anti-tumour effects of the hybrid TNF-alpha s with a molecular size from 100 to 110 kDa, which had more than 50% of specific bioactivity of native TNF-alpha, were significantly superior to other PEG-TNF-alpha s. These hybrid TNF-alpha s showed over ten times greater anti-tumour effects than native TNF-alpha. Thus, the molecular size, which was determined by the degree of PEG modification and PEG molecular weight, influences the specific activity and anti-tumour effects of hybrid TNF-alpha.

cDNA cloning of a novel trypsin inhibitor with similarity to pathogenesis-related proteins, and its frequent expression in human brain cancer cells

A novel trypsin inhibitor (P25TI) with an apparent molecular size of 25 kDa has previously been purified from the culture medium of human glioblastoma cells. In this study, the cDNA encoding P25TI was isolated by the polymerase chain reaction (PCR) screening system, and its complete amino acid sequence was determined. The cDNA consisted of 1440 nucleotides and encoded a sequence of 258 amino acids. The deduced structure of P25TI seemed to consist of a putative signal peptide sequence (residues 1-25), a propeptide sequence (26-60) and a mature protein (residues 61-258). The P25TI sequence has no homology to other proteinase inhibitors, but has similarity to insect venom allergens, mammalian testis-specific proteins and plant pathogenesis-related proteins. P25TI mRNA was frequently expressed in human neuroblastoma and glioblastoma cell lines. Although Northern blotting analysis failed to detect P25TI mRNA in various human tissues, PCR analysis showed its expression in the brain, placenta and lymphocytes.

In vitro studies on potentiation of cytotoxic effects of anticancer drugs by interferon on a human neoplastic cell line (HeLa)

Experiments were performed to ascertain whether the antitumor effect of various anticancer drugs might be enhanced by interferon, using cultures of HeLa cells originating from a human carcinoma of the uterine cervix. The effects of drugs were assessed by counting cell colonies formed in culture. The drugs studied included 4 metabolic antagonists: cytosine arabinoside (Ara-C), 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP) and methotrexate (MTX), 7 antibiotics: aclacinomycin (ACM), adriamycin (ADM), actinomycin D (ACD), cycloheximide, mitomycin C (MMC), peplomycin (PEP) and puromycin; 2 alkylating agents: nimustine hydrochloride (ACNU) and melphalan, and 3 other drugs, vincristine (VCR), cisplatin (CDDP) and hydroxyurea (HU). Interferon was a preparation of the beta-type produced by human fibroblasts. A specific additive or synergistic potentiation of the cytotoxic effect by concomitant application of interferon was observed with PEP, ACNU, ACM, CDDP, 5-FU and ADM; the drug concentration given a 50% inhibition of cell growth was reduced by one-half or more in cultures with the combination of interferon and these drugs. The treatment of cells with interferon alone caused only 10-30% inhibition of cell proliferation.

Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.

ENHANCEMENT OF THE SENSITIVITY OF A CHEMILUMINESCENT IMMUNOASSAY FOR ESTRADIOL BASED ON HAPTEN HETEROLOGY

OBJECTIVE To develop a highly sensitive chemiluminescent immunoassay (CLIA) that measures the serum estradiol (E2) levels in postmenopausal women. METHODS The previously developed competitive CLIA for E2 consisted of an immobilized antigen and a labeled antibody with an N-functionalized acridinium ester that was modified to enhance its sensitivity. The modifications were changing the immobilized antigen from E2 to its analogues with its expected effect on hapten heterology and selecting optimal incubation conditions. RESULTS The hapten heterology in which estriol was used as the immobilized antigen instead of E2 enhanced the sensitivity of the CLIA about 3-fold. A low incubation temperature and a long incubation time also effectively increased the sensitivity of the CLIA. The combination of these modifications enhanced sensitivity about 10-fold. The proposed CLIA with a 16 pmol/L detection limit, was about 5-fold more sensitive than commercially available immunoassay kits for E2. CONCLUSION The proposed CLIA is sensitive enough to measure serum E2 levels in postmenopausal women.

Competitive chemiluminescent immunoassay for estradiol using an N-functionalized acridinium ester.

We have compared three competitive chemiluminescent immunoassays (CLIA) for estradiol (E2) using an N-functionalized acridinium ester (AE). The assays were a standard competitive assay using immobilized antibody and directly labeled antigen (type A), an immobilized antibody and indirectly labeled antigen (type B), and an immobilized antigen and labeled antibody (type C). In an antibody-immobilized system, the assay using both AE- and E2-labeled thyroglobulin as a tracer (type B) was more sensitive than that using AE directly coupled with E2 (type A). Subsequently, a comparison of the antibody-immobilized system (type B) and an antigen-immobilized system (type C) showed that the latter was slightly more sensitive than the former. The sensitivity of the CLIA (type C) was similar or superior to commercially available CLIA or radioimmunoassays for E2. Thus, the N-functionalized AE proved to be a useful labeling reagent for a competitive CLIA with high sensitivity.