Ley glycolipid-recognizing monoclonal antibody inhibits procoagulant activity and metastasis of human adenocarcinoma.

Tumor procoagulant is associated with cancer at advanced stages of malignancy such as infiltration and metastasis. In the present study, we investigated the role of Ley glycolipid in the mechanism of cancer metastasis. Ley glycolipid acts as an important cofactor in the expression of the blood-coagulating activity of cancer cell-derived coagulating activity 1 (CCA-1), which is one of the known tumor procoagulants. Monoclonal antibody (MoAb) FS01, which serves as the Ley-recognizing epitope, inhibits the procoagulant activity of CCA-1 was found to dose-dependently inhibit the procoagulant activity of normal plasma induced by the human lung adenocarcinoma cell line, HAL8, which shows a high level of Ley expression. It did not, however, inhibit the procoagulant activity of the human colon cancer cell line, RPMI4788, which does not express Ley. Administration of FS01 MoAb inhibited lung metastasis of HAL8 cells, but not that of RPMI4788. The absence of antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity of FS01 MoAb against the HAL8 cell line suggests that the inhibition of HAL8 metastasis by FS01 MoAb derives from the inhibition of blood-coagulating activity of the latter. These findings indicate that Ley glycolipid plays an important role in the mechanism of cancer metastasis via the procoagulant activity of CCA-1.

Apoptosis is promoted by the dsRNA-activated factor (DRAF1) during viral infection independent of the action of interferon or p53.

An apoptotic cellular defense mechanism is triggered in response to viral dsRNA generated during the course of infection by many DNA and RNA viruses. We demonstrate that apoptosis induced by dsRNA or a paramyxovirus is independent of the action of interferon as it can proceed in a variety of cell lines and primary cells deficient in an interferon response. Initiation of apoptosis appears to be triggered by activation of a cellular transcription factor, the dsRNA-activated factor (DRAF1). DRAF1 is composed of interferon regulatory factor 3 (IRF-3) and the transcriptional coactivators CREB binding protein (CBP) or p300. We find that activation of IRF-3 in the absence of viral infection stimulates apoptosis. In addition, a negative interfering mutant blocks both target gene induction and apoptosis, demonstrating a requirement for gene expression by IRF-3/DRAF1 to promote apoptosis. IRF-3/DRAF1 target gene expression is also induced in response to a distinct apoptotic stimulus, the DNA damaging agent etoposide. The activity of the p53 tumor suppressor does not appear to be required for IRF-3/DRAF1-mediated apoptosis.

Deficient Th1-type immune responses via impaired CD28 signaling in ultraviolet B-induced systemic immunosuppression and the restorative effect of IL-12.

A single large dose (15 kJ/m(2)) of UVB-irradiation induces systemic immunosuppression and tolerance. We previously reported that IL-12 promotes the accessory cell function of epidermal Langerhans cells. In this study we have examined whether IL-12-pretreated antigen-presenting cells (APC) could restore the diminished T-cell response in mice irradiated with a single large dose of UVB. Spleen cells from UVB-irradiated mice showed reduced IFN-gamma production in a hapten-specific response but the function of APC in non-exposed skin of UVB-irradiated mice was not impaired. The pretreatment of APC with IL-12 did not restore the impaired IFN-gamma production by T cells from UVB-irradiated mice. Neither IL-10 nor TGF-beta was found to be involved in the suppression. Instead, we observed that anti-CD3 mAb-induced IFN-gamma production by T cells from UVB-irradiated mice was not augmented in the presence of anti-CD28 mAb, whereas IL-4 production was enhanced by the addition of anti-CD28 mAb. Furthermore, the reduced IFN-gamma production by T cells from UVB-irradiated mice in response to antigen plus APC could be restored by adding IL-12 to the culture. Our results thus indicate that UVB-induced systemic immunosuppression involves impaired Th1-type responses of T lymphocytes through CD28 stimulation, and that IL-12 compensates for the impaired CD28 costimulatory signaling in T cells resulting in the restoration of Th1-type responses.

Processing glucosidase inhibition by 1-azafagomine.

Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.

High expression of glycosphingolipids involved in procoagulant activity of cancer cells.

To investigate the involvement of tumor-associated antigens such as sLe(a) and sLe(x), as well as Le(y) glycosphingolipid in the procoagulant activity of cancer cells, we examined the relationship between the inhibitory effect of anti-sLe(a) or anti-sLe(x) antibodies on the procoagulant activity of cancer cells, and cell surface expression of sLe(a) and sLe(x) antigens. We observed that the procoagulant activity of cancer cells which highly expressed sLe(a) or sLe(x) antigens (expression rate >70%) was significantly inhibited by the relevant antibodies. These data suggested that not only Le(y) but also sLe(a) and sLe(x) antigens play an important role in coagulation induced by various cancer cells.

IL-12 promotes the accessory cell function of epidermal Langerhans cells.

The objective of this study was to investigate the effects of cytokines regulating cutaneous immune responses, on the accessory cell function of epidermal cells (EC). EC were treated with various cytokines, and the accessory cell function of the cytokine-pretreated EC was examined by the allogeneic mixed epidermal cell-lymphocyte reaction (MECLR). Among the cytokines examined, IFN-gamma- and IL-12-pretreated EC augmented IFN-gamma production in the MECLR, while none of the other cytokines was effective. However, the cytokine-pretreated EC did neither affect T cell proliferation nor IL-4 production in the MECLR. Next we attempted to analyze the mechanisms by which IL-12-pretreated EC increase IFN-gamma production in the MECLR. Endogenous IFN-gamma produced during the IL-12 pretreatment of EC was found to play only a minor role in modulating the function of EC. The expression of MHC class II, CD80 and CD86 on EC was not affected by IL-12. On the other hand, soluble mediators that induce IFN-gamma production during the MECLR containing IL-12-pretreated EC were identified as endogenously produced IL-12 (the major mediator) and IL-18 (the minor mediator). Furthermore, the results of depletion experiments indicate that IL-12 promotes the accessory cell function of Langerhans cells to responder T cells in inducing IFN-gamma production in the MECLR.

Correlation of prognosis of breast cancer patients and expression of Ley which acts as a cofactor of tumor procoagulant.

Association between Ley expression and prognosis of breast cancer was investigated using monoclonal antibody (MoAb) FS01, which recognizes Ley as an epitope, inhibits the procoagulant activity of cancer cell-derived coagulating activity 1 (CCA-1). Expression intensity and procoagulant activity of CCA-1, tissue factor and HLA-DR on breast cancer cell lines were also examined. Immunohistochemical staining of Ley was performed on primary lesions of 223 breast cancer patients who received absolute curative operation. Flow cytometric analysis and clot timer was used to detect expression and activity of each procoagulant on cancer cell lines. The Ley expression was 73.5%, and no significant relation was observed between clinicopathological factors and intensity of Ley expression. The group showing strong Ley positivity had a significantly poorer prognosis than the Ley-negative group in 5-year disease-free survival (p=0.019). Multivariate analysis using the Cox's proportional hazards' regression model showed that Ley expression is an independent prognostic factor (p=0.018), following tumor size and lymph node metastasis. Ley expression on cancer cell surface is higher than tissue factor and HLA-DR. FS01 and anti-tissue factor MoAb inhibited the coagulating activity of tissue factor-expressing lines, but no cells were inhibited by staphylococcal enterotoxin A, which is known to inhibit the coagulating activity of HLA-DR. CCA-1 and tissue factor plays a important role in the blood coagulating activity of breast cancer cell lines. Breast cancer patients are thought to have a poor prognosis because Ley expression on the surface of the cancer cell induces blood coagulation via CCA-1.

Generation of a monoclonal antibody that inhibits the procoagulant activity of various cancer cell lines.

BACKGROUND: Tumor procoagulant is one of the factors responsible for disseminated intravascular coagulation and metastasis. The authors found procoagulant activity in LK52 human squamous cell carcinoma cells, which they designated cancer cell-derived blood coagulating activity 1 (CCA-1). A monoclonal antibody (MoAb) was generated to characterize this CCA-1 procoagulant activity. To date, antibodies that show an inhibitory effect on procoagulant activity as well as high reactivity in cancer cells are well known for their tissue factor specificity. METHODS: Characterization of the procoagulant activity of CCA-1 was performed and an anti-CCA-1 MoAb, FS01, was generated. CCA-1 expression on the cancer cell surface was examined by flow cytometry. Procoagulant activity of various cancer cell lines and the inhibitory effect of the FS01 MoAb on this procoagulant activity was monitored by a clot timer. RESULTS: The enzymologic character differed from that of cancer procoagulant (CP). The FS01 MoAb inhibited the procoagulant activity of CCA-1, but did not inhibit that of tissue factor. A positive correlation was observed between the expression intensity of CCA-1 and the inhibitory effect of the FS01 MoAb on the procoagulant activity of cancer cell lines. Expression of CCA-1 was observed more frequently than that of tissue factor in human cancer cell lines. CONCLUSIONS: The FS01 MoAb generated in the current study is a new antibody that reacts with various cancer cell lines, but not with normal cells. FS01 inhibits cancer cell-derived procoagulant activity and does not react with tissue factor and CP. CCA-1, which is recognized by the FS01 MoAb, appears to play a major role in cancer cell-derived procoagulant activity.

Reduction of glycogen in eggs of the silkworm, Bombyx mori, by use of a trehalase inhibitor, trehazolin, and diapause induction in glycogen-reduced eggs.

A new trehalase inhibitor, trehazolin, caused a potent inhibition of ovary trehalase in the silkworm, Bombyx mori. A single injection of trehazolin into pupae (40&mgr;g/animal) did not interfere with the accumulation of proteins and lipids, but markedly reduced glycogen content in eggs accompanied by a remarkable increase in hemolymph trehalose levels. The most potent effect of trehazolin was expressed in eggs that developed at the mid-stage of pupal-adult development. In these eggs glycogen content was reduced to a trace level, less than 3% of that of the control. The reduced glycogen content was almost restored to the control level by injection of glucose but not by trehalose. Trehazolin treatment influenced oviposition and larval hatching, whereas embryogenesis went on normally in glycogen-reduced eggs. Injection of synthetic diapause hormone into non-diapause type hosts induced an incidence of 45% diapause in the eggs and increased their glycogen content. Surprisingly, injection of trehazolin never affected diapause induction by the hormone, despite considerably reduced glycogen content in these eggs. Thus, our findings provide a new method for production of eggs containing various amounts of glycogen, and a novel system for analyzing diapause-associated metabolism besides the well-known glycogen-sorbitol metabolism.

Le(y) glycolipid acts as a co-factor for tumor procoagulant activity.

We have generated a monoclonal antibody (MAb), FS01, which inhibits the procoagulant activity (CCA-1) produced by a human squamous cell carcinoma cell line, LK52. Expression of the antigen recognized by FS01 MAb in various cancer cell lines correlated well with the procoagulant activities of the expressing cell lines. Our objective was to characterize the molecule reacting with FS01 MAb and to analyze its involvement in the CCA-1 procoagulant activity. The molecule was identified as a glycolipid and found to be involved in the procoagulant activity because both procoagulant activity and reactivity to FS01 MAb were lost after endoglycoceramidase treatment of CCA-1. Furthermore, FS01 MAb recognized the Lewis Y (Le[y]) antigen. To confirm the involvement of a glycolipid incorporating the Le(y) antigen in the procoagulant activity, we attempted to purify CCA-1 from LK52 culture supernatant. In one of the purification steps, a fraction containing low procoagulant activity (CCA-1p) separated from the Le(y)-positive fraction (CCA-1c). Although CCA-1c alone did not show procoagulant activity, the procoagulant activity of CCA-1p was augmented by CCA-1c and this augmentation was inhibited by FS01 MAb. Furthermore, CCA-1c enhanced the procoagulant activity of 33 cell lines tested as well as CCA-1p. In addition, purified Le(y) glycolipid from canine intestine augmented the procoagulant activity of CCA-1p, and this augmentation also could be inhibited by FS01 MAb. We conclude that Le(y) glycolipid is a co-factor for the procoagulant activity derived from cancer cells.

Trehazolin, a slow, tight-binding inhibitor of silkworm trehalase.

Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-alpha-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 microM. Other glycosidases tested were not inhibited by 500 microM trehazolin. The inhibition of trehalase by trehazolin was competitive with respect to trehalose. A notable feature of the inhibition was a slow progression of the association and dissociation of the enzyme-inhibitor complex. Preincubation of the enzyme and the inhibitor at 37 degrees C potentiated the inhibition by 10-times in a time-dependent manner up to 6 h. Dialysis of the inactivated enzyme recovered the enzymatic activity very slowly, and the rate constant for the dissociation at 37 degrees C was (7.3).10(-2) h-1. Trehalamine, a deglucosylated form of trehazolin, inhibited both silkworm trehalase and exo-alpha-glucosidases only weakly. The inhibition of trehalase by trehalamine was reversible. Rat isomaltase inhibition by trehazolin and sucrase inhibition by trehalamine were also reversible. Taken together, trehazolin is a specific slow, tight-binding inhibitor of trehalase, and the glucose moiety of the inhibitor is essential to the tight binding.

Establishment of new interferon-gamma-resistant mutant cells with dominant phenotypes.

We established interferon-gamma-resistant (IGR) cells from a human colon adenocarcinoma cell line, LoVo. Their resistance was extremely high, and the ED50 values of IFN-gamma were > 10(5) IU/ml. Interestingly, although IGR-5 cells were still sensitive to the antiproliferative effect of IFN-alpha, the cells lost responsiveness to the antiviral effects of both IFN-alpha and gamma. Another clone, IGR-53, was unresponsive to both the antiproliferative and antiviral effects of either IFN-alpha or gamma. Furthermore, the IFN-gamma-resistant phenotypes of IGR cells were apparently dominant to the parental LoVo cells based on complementation tests. Although IGR-53 cells lack IFN-gamma receptors, IGR-5 cells seemed to have functional IFN-gamma receptors and processing mechanisms of IFN-gamma bound to the receptors. Northern analysis showed that IGR-5 cells responded to IFN-gamma and alpha, but the enhancement of IRF-1 expression by IFN-gamma was markedly suppressed.

Analyses of mixed melanogenesis in tyrosinase cDNA-transfected human amelanotic melanoma cells.

In the pigment cell the synthesis of tyrosinase and the formation of premelanosomes are independent, yet coordinated, processes. However, the interrelationship between the two processes has not been elucidated previously. In this study, an expression plasmid for human tyrosinase cDNA was constructed and transfected into a human amelanotic melanoma cell line, G-361. Stable transfected cells (G-CMHT-3) were obtained with high tyrosinase activity and distinct melanization occurred. As for the type of melanin, both pheo- and eumelanin contents increased in G-CMHT-3 cells. Interestingly, catalase activity as one of the other melanogenic enzymes was decreased in G-CMHT-3 cells. The decrease of catalase activity was considered to play a role in melanin-polymer formation, resulting in the increase of both pheo- and eumelanin contents. Under electron microscopic observation, dopa-oxidase-positive Golgi-associated endoplasmic reticulum of lysosome, coated vesicles, and premelanosomes were observed in pigmented G-CMHT-3 cells, and the expressed tyrosinase was considered to be well translocated to these organelles. In addition, the number of premelanosomes (stages I-III) as well as melanosomes (stage IV) increased in G-CMHT-3 cells compared to those in G-361 cells. It is also noted that G-CMHT-3 cells showed more normal phenotype premelanosomes with occasional transitional premelanosomes exhibiting partial melanin polymer formation within their concentric whorl-like internal membranes. Furthermore, the number of eumelanosomes in G-CMHT-3 cells was much larger than that in G-361 cells. These results suggest that the tyrosinase introduced by its cDNA transfection induced active and structurally non-aberrant premelanosome formation resulting in the upregulated pheo- and eumelanogenesis.

Isolation of trehalamine, the aglycon of trehazolin, from microbial broths and characterization of trehazolin related compounds.

Trehalamine, (3aR,4R,5S,6S,6aS)-2-amino-4-(hydroxymethyl)-3a,5,6,6a- tetrahydro-4H-cyclo-pent[d]oxazole-4,5,6-triol (1) and D-glucose were obtained by acid hydrolysis of trehazolin (3), a trehalase inhibitor produced by actinomycetes. More vigorous hydrolytic treatment of trehazolin afforded an aminocyclitol, (1R,2S,3R,4S,5R)-5-amino-1- (hydroxymethyl)cyclopentane-1,2,3,4-tetraol (2). Trehalamine, the aglycon of trehazolin, was also found in the culture broths of two trehazolin producing strains, Micromonospora sp. SANK 62390 and Amycolatopsis sp. SANK 60791. These trehazolin related compounds trehalamine (1) and 2 were poor inhibitors of trehalase (1; IC50 1.8 x 10(-4) M, 2; > 5.0 x 10(-4) M). On the other hand they inhibited more potently rat intestinal sucrase (1; IC50 6.8 x 10(-5) M) and sweet almond beta-glucosidase (2; IC50 5.6 x 10(-6) M) than trehazolin.

Tyrosinase gene transcription and its control by melanogenic inhibitors.

The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1(178) (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1(178) cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1(178) cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1(178) cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression.

Synerazol, a new antifungal antibiotic.

Synerazol, a new antifungal antibiotic, was isolated from cultured broth of Aspergillus fumigatus SANK 10588. The structure was determined based on NMR and mass spectral evidences. Synerazol was found to be a related substance to pseurotin A. Synerazol was active against Candida albicans and other fungi, and showed marked synergistic activity with azole-type antifungal agents.

Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini.

The existence of a mycoplasmal arginine deiminase which catalyzes the conversion of L-arginine to L-citrulline has been postulated. Here we show the partial amino acid sequence of arginine deiminase of Mycoplasma arginini and the complete nucleotide sequence of the arginine deiminase gene of M. arginini. The open reading frame deduced from this sequence consists of 1,230 bp encoding 410 amino acids. The mature form of this enzyme contains 409 amino acids after the deletion of the first methionine. In this open reading frame, TGA nonsense codons are used as tryptophan codons; this usage was verified by determination of the amino acid sequence. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 46,372. Recently, the nucleotide sequence of the arginine deiminase gene of M. arginini was reported by Kondo et al. (K. Kondo, H. Sone, H. Yoshida, T. Toida, K. Kanatani, Y.-M. Hong N. Nishino, and J. Tanaka, Mol. Gen. Genet. 221:81-86, 1990). However, their sequence differed from ours in several places and especially at the C terminus.

Monoclonal antibodies as probes for monitoring the denaturation process of bovine beta-lactoglobulin.

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.