Pharmacokinetic study of darbepoetin alfa: absorption, distribution, and excretion after a single intravenous and subcutaneous administration to rats.

KRN321 is a hyperglycosylated analogue of recombinant human erythropoietin (rHuEPO, epoetin alfa), and its absorption, distribution, and excretion have been studied after a single intravenous and subcutaneous administration of 125I-KRN321 at a dose of 0.5 microg kg-1 to male rats. The half-lives of immunoreactive radioactivity in the terminal phase after intravenous and subcutaneous administration were 14.05 and 14.36 h, respectively, and the bioavailability rate after subcutaneous administration was 47%. The total radioactivity in tissues was lower than that in the serum in all tissues excluding the thyroid gland and skin at the injection site (subcutaneous administration). The maximum concentrations were observed in the bone marrow or skin at the injection site followed by the thyroid gland, kidneys, adrenal glands, spleen, lungs, stomach and bladder. The radioactivity found in trichloroacetic acid-precipitated fractions suggested that a high-molecular weight compound, unchanged or mixed with endogenous protein, distributed to the tissues after administration. The whole-body autoradiographic findings in both groups were in agreement with the tissue distribution mentioned above. The blood cell uptake of KRN321 was low for both groups. The excretion ratios of radioactivity into urine and faeces up to 168 h were 71.4 and 14.1% after the intravenous administration and 74.9 and 12.0% after the subcutaneous administration. There was no difference in the excretion profile of radioactivity between the two groups.

Blue light inhibits melanin synthesis in B16 melanoma 4A5 cells and skin pigmentation induced by ultraviolet B in guinea-pigs.

BACKGROUND: Little has been known about the effects of visible light in mammalian cells. We recently found that blue light not only suppressed the growth of B16 melanoma cells in a time-dependent manner but also inhibited metastasis of the B16 melanoma cells to the lung. These findings suggest that exposure to blue light modifies the functions of B16 melanoma cells. The present study investigated the effects of blue light on B16 melanoma 4A5 cells and Weiser-Maple guinea-pigs to confirm the biological effect of blue light on melanin formation. METHODS: The effect of red, green, and blue light on melanin synthesis in B16 melanoma 4A5 cells was measured. The back skin of brown Weiser-Maple guinea-pigs was exposed to ultraviolet B (UVB; 588 mJ/cm(2) (0.7 mW/cm(2)x 14 min) three times a week for 2 weeks to induce melanin deposition. Thirty minutes after each UVB exposure, blue light was applied for 30 min. Pigmentation of the exposed areas of skin was checked once a week, and photographs of the skin were taken by digital camera. Observation was continued for 18 days after the final UVB exposure. RESULTS: Melanin synthesis in B16 melanoma 4A5 cells was selectively suppressed by blue light, but blue light did not induce decolorization of previously produced melanin. In the back skin of brown guinea-pigs, the brightness of the sites exposed to UVB began to decrease on the fifth day of the experiment, decreasing further from the 12th day to the 18th day after UVB exposure. The brightness of the sites exposed to UVB and blue light decreased in a manner similar during the UVB exposure, but remained relatively unchanged from the 12th day to the 30th day. CONCLUSIONS: These results suggest that blue light suppresses melanin formation following repeated UVB exposure. Further investigation with various light such as blue light may lead to a new approach to the care of ultraviolet-affected skin such as hyperpigmentation.

Paired tumor marker of soluble E-selectin and its ligand sialyl Lewis A in colorectal cancer.

BACKGROUND: Better diagnosis of metastatic disease has been pursued by oncologists: however, many of the tumor markers have been still controversial. Our purpose was to estimate the usefulness of soluble E-selectin and its ligand sialyl Lewis A for more accurate diagnosis as a combined tumor marker for metastases in colorectal cancer. METHODS: E-selectin and sialyl Lewis A, collected from preoperative blood, were measured of its levels in 54 patients with colorectal cancer classified according to Dukes' stage. E-selectin was assayed by enzyme-linked immunosorbent assay, whereas sialyl Lewis A was quantified by enzyme immunoassay using immunoclone kit. RESULTS: The elevation in the level of E-selectin was significantly higher in Dukes' D group than that of healthy volunteers (P < 0.001, Fisher's procedure of least significance test), Dukes' A (P = 0.01), B (P = 0.025) and C (P < 0.01). Significantly higher level of sialyl Lewis A was shown in the group of metastases than that of non-metastases (P < 0.0068. Student's t-test). Paired elevation of E-selectin and sialyl Lewis A was significantly higher in the hematogenous metastases than non-metastases (P < 0.001, Fisher exact test). CONCLUSIONS: These results suggest that E-selectin could play some role in the progress of hematogenous metastases. The elevation of E-selectin alone or both E-selectin and sialyl Lewis A may be one of the useful indexes for more precise diagnosis of hematogenous metastases of human colorectal cancer.

P- and E-selectins recognize sialyl 6-sulfo Lewis X, the recently identified L-selectin ligand.

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.

Expression of sialyl 6-sulfo Lewis X is inversely correlated with conventional sialyl Lewis X expression in human colorectal cancer.

Sialyl 6-sulfo Lewis X determinant has been described recently as a major ligand for L-selectin on high endothelial venules of human peripheral lymph nodes. From our investigation of its distribution in human colorectal cancer tissues and cultured colon cancer cells, the sialyl 6-sulfo Lewis X determinant was preferentially expressed in the nonmalignant colonic epithelia rather than cancer cells (P < 0.001; n = 23). This was in contrast to the distribution of conventional sialyl Lewis X, which was preferentially expressed in cancer tissues rather than nonmalignant epithelia (P = 0.007; n = 23), indicating that 6-sulfation predominantly occurs in nonmalignant tissues and is suppressed upon malignant transformation. In confirmation of this, a nonsialylated determinant 6-sulfo Lewis X was also found to be preferentially localized in the nonmalignant epithelia. Significant expression of sialyl 6-sulfo Lewis X was observed in only 2 lines, whereas 8 were positive for conventional sialyl Lewis X, among 13 cultured colon cancer cell lines. Transfection of cells with fucosyltransferase (Fuc-T) VI induced expression of sialyl 6-sulfo Lewis X, whereas transfection of Fuc-T III did not, suggesting that the determinant was synthesized mainly by Fuc-T VI in colonic epithelia. Members of the sialic acid cyclase pathway, the de-N-acetyl sialyl 6-sulfo Lewis X and cyclic sialyl 6-sulfo Lewis X determinants, were also preferentially expressed in the nonmalignant epithelia rather than colonic cancer cells (P < 0.001; n = 23). Stimulation of the sialyl 6-sulfo Lewis X-positive colon cancer cell line with a calcium ionophore ionomycin markedly reduced sialyl 6-sulfo Lewis X and induced cyclic sialyl 6-sulfo Lewis X expression. These results suggested that the metabolic conversion of sialyl 6-sulfo Lewis X into cyclic sialyl 6-sulfo Lewis X by a calcium-dependent enzyme, sialic acid cyclase, as we hypothesized for human leukocytes previously (C. Mitsuoka et al., Proc. Natl. Acad. Sci. USA, 96: 1597-1602, 1999), also occurs in nonmalignant colonic epithelia.

Spatially and temporally regulated expression of N-acetylglucosamine-6-O-sulfotransferase during mouse embryogenesis.

GlcNAc-6-O-sulfotransferase is involved in formation of 6-sulfo-N -acetyllactosamine-containing structures such as 6-sulfo sialyl Lewis x. We investigated the mode of expression of GlcNAc-6-O-sulfotransferase during postimplantation embryogenesis in the mouse by in situ hybridization. Sulfotransferase mRNA was not detected on embryonic day (E) 6.5, while on E7.5 it was detected in the mesoderm, ectoderm, and ectoplacental cone. On E10.5, the sulfotransferase signals were mainly observed in the nervous tissue. On E12.5 and 13.5, various tissues in the process of differentiation expressed this mRNA. Several epithelial and mesenchymal tissues undergoing epithelial-mesenchymal interactions strongly expressed the mRNA. For example, in the developing tooth strong sulfotransferase mRNA expression was found only in the condensing mesenchyme on E13.5. On E13.5 and 15.5, the sites showing intense expression of the sulfotransferase again became restricted. In the brain, sulfotransferase mRNA was frequently found as discrete signals in narrow regions. These results suggest that 6-sulfo-N-acetyllactosamine structures have important roles in development. On E13.5 and 15.5, G152 (6-sulfo sialyl Lewis x antigen) was expressed in the neocortex, and AG223 (6-sulfo Lewis x antigen) in the thalamus and neocortex where the sulfotransferase signal was detected. However, in other organs, expression of these antigens did not correlate with the sulfotransferase mRNA, implicating complex nature of regulation of expression of the fucosyl 6-sulfo antigens.

Reconstitution of functional L-selectin ligands on a cultured human endothelial cell line by cotransfection of alpha1-->3 fucosyltransferase VII and newly cloned GlcNAcbeta:6-sulfotransferase cDNA.

Recently, we proposed sialyl 6-sulfo Lewis X as a major carbohydrate-capping group of the L-selectin ligands on high endothelial venules in human lymph nodes. In this study we succeeded in reconstituting functional L-selectin ligands on a cultured human endothelial cell line, ECV304, by transfecting the alpha1-->3fucosyltranseferase VII (Fuc-T VII) and newly cloned GlcNAcbeta:6-sulfotransferase (6-Sul-T) cDNAs. The ECV304 cells transfected with Fuc-T VII cDNA expressed conventional sialyl Lewis X detected with specific antibodies including 2H5, whereas the cells transfected with 6-Sul-T cDNA expressed sialyl 6-sulfo lactosamine as well as MECA-79-defined carbohydrate determinants, but these singly transfected cells failed to express sialyl 6-sulfo Lewis X, as detected with the antisialyl 6-sulfo Lewis X mAb G152. Sialyl 6-sulfo Lewis X appeared only on the cells that were cotransfected with both 6-Sul-T and Fuc-T VII cDNAs. Significant adhesion of L-selectin-expressing cells was seen only to the doubly transfected ECV304 cells and was inhibited by G152. No adhesion was observed to the cells transfected either with 6-Sul-T or with Fuc-T VII cDNA alone. The mRNAs of the two enzymes were expressed or were inducible upon interleukin 1 stimulation in human endothelial cells. These results indicate that a set of carbohydrate determinants synthesized by the concerted action of the two enzymes, as typically represented by the sialyl 6-sulfo Lewis X-capping group, serves as an essential component of the ligand for L-selectin and that the reagents 2H5 and MECA-79, utilized in earlier studies to detect L-selectin ligand on high endothelial venules, recognize two different aspects of the same set of synthetic products.

Regulation of selectin binding activity by cyclization of sialic acid moiety of carbohydrate ligands on human leukocytes.

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form "cyclic sialic acid." The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral-alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-gammadelta T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines and O-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called "sialic acid cyclase," and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.

Human N-acetylglucosamine-6-O-sulfotransferase involved in the biosynthesis of 6-sulfo sialyl Lewis X: molecular cloning, chromosomal mapping, and expression in various organs and tumor cells.

N-Acetylglucosamine-6-O-sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of a non-reducing N-acetylglucosamine (GlcNAc) residue. We have cloned human GlcNAc-6-O-sulfotransferase cDNA, based on the sequence homology to cloned cDNA of mouse GlcNAc-6-O-sulfotransferase. The predicted protein sequence of the human enzyme was highly homologous to that of the mouse enzyme; in the 363 amino acid stretch of the catalytic region, the two proteins were nearly identical except for conservative changes in 3 amino acid residues. The expressed enzyme transferred sulfate to GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc. Co-transfection of the enzyme cDNA and fucosyltransferase VII cDNA into COS-7 cells resulted in cell surface expression of 6-sulfo sialyl Lewis X. Fluorescence in situ hybridization analysis revealed that the GlcNAc-6-O-sulfotransferase gene is located on human chromosome 7q31. mRNA of the human enzyme was strongly expressed in the bone marrow, peripheral blood leukocytes, spleen, brain, spinal cord, ovary, and placenta, and moderate levels of expression were observed in many organs including lymph nodes and thymus. In situ hybridization with the mouse system showed that the transcript was localized in specific regions of the brain, i.e. pyramidal cells in the CA3 subregion of the hippocampus, cerebellar nucleus and Purkinje cells. Among human tumor cells, strong expression of the mRNA was found in MOLT-4 and Jarkat lymphoblastic leukemia cells, Raji lymphoma cells, K-562 chronic myelogeneous leukemia cells, U251 glioma cells, and G361 melanoma cells. Carbohydrate structures synthesized by the sulfotransferase may be involved in various aspects of the differentiation and behavior of blood cells, their progenitor cells, and neurons in the central nervous system.

Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase.

We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.

Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.

We investigated the molecular species of sulfated sialyl Lewis X determinants, the putative L-selectin ligand, expressed on high endothelial venules (HEV) in human lymph nodes. Comparison of the reactivity pattern of HEV with the reactivity of the pure 6-sulfo, 6'-sulfo, or 6,6'-bissulfo sialyl Lewis X determinant with hitherto known anti-sialyl Lewis X antibodies strongly suggested 6-sulfo sialyl Lewis X to be the best candidate for the major sulfated sialyl Lewis X determinant on HEV, followed by 6,6'-bissulfo sialyl Lewis X, whereas 6'-sulfo sialyl Lewis X was unlikely. We newly generated monoclonal antibodies (mAbs) G152 and G72 directed against 6-sulfo sialyl Lewis X, which intensely labeled HEV in immunohistochemical examination and inhibited binding of recombinant L-selectin-IgG to HEV, suggesting that the determinant serves as a ligand for L-selectin. To test the concomitant expression of 6, 6'-bissulfo sialyl Lewis X, specific mAbs (G2706, G27011, G27037, and G27039) were generated, but all antibodies failed to react to HEV. Next, we established mAbs (AG97 and AG273) directed against 6-sulfo Lewis X, the asialo form of 6-sulfo sialyl Lewis X. The antibodies were not reactive to untreated HEV, but strongly reacted to sialidase-treated HEV. This indicated the predominance of the sialylated form of 6-sulfo sialyl Lewis X and minimal expression of its asialo form, corroborating that it was synthesized by fucosyltransferase VII, the isoenzyme that preferentially produces the sialylated form of the determinant.

Specific detection of sialyl Lewis X determinant carried on the mucin GlcNAcbeta1-->6GalNAcalpha core structure as a tumor-associated antigen.

Sialyl Lewis X serves as a ligand for selectins and is proposed to be implicated in hematogenous metastasis of cancers. When a cultured human breast cancer cell line, MCF-7, which does not express sialyl Lewis X, was transfected with human fucosyltransferase VI cDNA, a strong expression of sialyl Lewis X was induced on transfectant cells. The transfectant cells were found to be also reactive to the antibody NCC-ST-439, which was initially raised against human gastric cancer cells and later was shown to recognize a tumor-associated carbohydrate antigen in breast, gastric, and colon cancers. This suggested that the antigen recognized by NCC-ST-439 is closely related to sialyl Lewis X. Subsequent studies indicated that NCC-ST-439 specifically reacts to NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6GalNAcalpha1 -->R, the sialyl Lewis X on the mucin GlcNAcbeta1-->6 GalNAcalpha structure. The antibody was not reactive to the conventional sialyl Lewis X determinants on straight and/or branched polylactosamine structures including NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->3Galbeta1-->4 Glcbeta1-->R and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6Galbeta1-->4 Glcbeta1-->R. This was in clear contrast to most of the known anti-sialyl Lewis X antibodies, which do not discriminate internal structures carrying the sialyl Lewis X determinant. On the other hand, the newly generated monoclonal antibody GSC154-27 had a specificity completely the reverse of the specificity of NCC-ST-439 in that it was strongly reactive to the conventional sialyl Lewis X determinants in straight and branched polylactosamine structures, while far less reactive to the sialyl Lewis X determinant on the mucin GlcNAcbeta1-->6GalNAcalpha core structure. A set of these two antibodies would be useful in discriminating the molecular species of sialyl Lewis X expressed by malignant cells and in studying their functional significance.

Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases.

The adhesion of circulating cancer cells to the vascular endothelium is an important at step in the hematogenous metastasis of cancer. E-selectin expressed on endothelial cells and carbohydrate ligands expressed on cancer cells mediate this adhesion. We investigated the clinical significance of such cell adhesion molecules in breast cancer. The cytosol concentration of sialyl Lewis(x) was found more elevated in cancerous tissue than that in adjacent non-cancerous tissue. In the serum, sialyl Lewis(x) and soluble E-selectin were seen elevated in patients with advanced and recurrent breast cancer, especially in those with distant metastases. From the above, we have concluded that sialyl Lewis(x) and soluble E-selectin could be used as tumor markers with a close relationship to the metastasis of breast cancer.

Increased concentration of soluble E-selectin in the sera of breast cancer patients.

E-selectin which is expressed on endothelial cells plays an important role in the adhesion of cancer cells to the vascular endothelium, being the ligand for a carbohydrate antigen expressed on cancer cells. In this study, the clinical usefulness of this protein was examined. E-selectin was expressed on the endothelial cells of the small vessels adjacent to the cancer nests in 63 of the 104 (60.6%) primary tumors of breast cancer. The expression of E-selectin in locations adjacent to the cancer nests was more pronounced than that in distant ones. The mean value of serum soluble E-selectin (ng/ml) was 38.3 in benign breast disease, 47.8 in those with no evidence of recurrence, 49.4 in stage I/II primary breast cancer, 75.8 in stage III/IV primary breast cancer, and 93.7 in recurrent breast cancer. The mean value of serum soluble E-selectin was 106.2 ng/ml in patients with distant metastases, and 50.4 ng/ml in those with no evidence of distant metastases. Thus, the concentration of soluble E-selectin was significantly elevated in the sera of patients with distant metastases. These findings suggested that cancer cells induced the expression of E-selectin on endothelial cells and, that serum soluble E-selectin is useful as a tumor marker having a close relationship to metastasis in breast cancer.

[Effects of carcinostatic agents in the hematogenous metastasis of cancer].

The experimental studies in vitro and in vivo were performed to investigate the effects of carcinostatic agents in the adhesion of cancer cells to endothelial cells. Certain carcinostatic agents induce the expression of E-selectin on endothelial cells and enhance the expression of carbohydrate ligands on cancer cells. Consequently, increased adhesion of cancer cells to endothelial cells was observed by the treatment of carcinostatic agents. In the trans-splenic hepatic metastasis model using nude mice, the augmentation of cancer metastasis was observed by the treatment of carcinostatic agents. From the above, we concluded that carcinostatic agents may increase the metastatic ability of cancer cells in patients with cancer.

Sulfated sialyl Lewis X, the putative L-selectin ligand, detected on endothelial cells of high endothelial venules by a distinct set of anti-sialyl Lewis X antibodies.

Endothelial cells of high endothelial venules (HEV) in human peripheral lymph nodes expressed a distinct type of sialyl Lewis X antigen, which was detected preferentially with a set of anti-sialyl Lewis X antibodies, 2F3, 2H5 and HECA-452 in immunohistochemistry, while another set of anti-sialyl Lewis X antibodies, FH-6 and CSLEX-1, failed to detect it. The adhesion of cells expressing L-selectin to HEV was inhibited by members of the former set of antibodies in Stamper-Woodruff assays performed on frozen sections of human peripheral lymph nodes. Transfection of a cultured endothelial cell line with a human alpha1-->3 fucosyltransferase, Fuc-T VII, resulted in the expression of a distinct type of sialyl Lewis X antigen having the reactivity similar to that of HEV; i.e., the antigen appearing on the transfectant clone was detectable only with the set of 2F3, 2H5 and HECA-452, but not with the set of FH-6 and CSLEX-1. Treatment of transfectant cells with sodium chlorate, a metabolic inhibitor of sulfation, resulted in reactivity to the members of the latter set of antibodies, suggesting that sulfation of sialyl Lewis X moiety was the cause of the discrepancy in the reactivity of the anti-sialyl Lewis X antibodies. When tested against various authentic sulfated sialyl Lewis X determinants, 6-sulfo sialyl Lewis X and 6,6'-bis-sulfo sialyl Lewis X were found to be reactive to the antibodies, 2F3, 2H5 and HECA-452, but not with antibodies FH-6 and CSLEX-1, suggesting that the distinct type of sialyl Lewis X determinant on the HEV endothelial cells and Fuc-T VII-transfected endothelial cell clone are mainly 6-sulfo and/or 6,6'-bis-sulfo sialyl Lewis X determinants.

Biological and clinical significance of hepatocyte growth factor in breast cancer.

Hepatocyte growth factor (HGF) is a mesenchyme- or stroma-derived multipotent factor that regulates the growth, motility, and morphogenesis of various types of cells, including cancer cells. We investigated the effect of HGF on human breast cancer cells, and measured the concentration of HGF in the sera of breast cancer patients. When BT-20 cells were stimulated with HGF, the transmigration of cancer cells was markedly accelerated. In a checkboard assay, pronounced chemotaxic locomotion of BT-20 cells is expressed, corresponding to HGF concentrations. HGF treatment of BT-20 cells resulted in enhanced expression of alpha 2, alpha 3 and beta 1 integrin subunits, and augmented the binding activity to immobilized collagen. The c-met protein was expressed on the cancer cells in 48 of the 97 (49.5%) breast cancer primary tumors. In the serum, the advanced and recurrent cancer group showed a high level of this protein in comparison with the other patient groups. The mean value of serum HGF was 0.65 ng/ml in patients with distant metastases and 0.27 ng/ml in those with no such evidence. Thus, the HGF concentration becomes significantly elevated in the sera of patients with distant metastases. These findings suggest that HGF is involved in invasion and metastasis of breast cancer, and that serum HGF is useful as a tumor marker with a close correlation to the metastatic state of breast cancer.

Increased concentration of hepatocyte growth factor in the sera of patients with gastrointestinal cancer.

We measured the concentration of hepatocyte growth factor (HGF) in the sera of 138 patients with gastrointestinal cancer. The mean value and positive rate for this protein (assuming 0.4 ng/ml as the cut-off value) were 0.29 ng/ml (11.4%) in healthy donors (n=44), 0.90 ng/ml(70.8%) in esophageal cancer (n=24), 0.39 ng/ml (33.3%) in gastric cancer (n=39), and 0.40 ng/ml (42.7%) in colorectal cancer (n=75). Thus, the mean values of the esophageal and colorectal cancer group were significantly higher than that of healthy donors. There was a tendency for elevated mean value of serum HGF in the sera of patients with distant metastases, compared to those with no such evidence, i.e., 1.36:0.77 ng/ml in esophageal cancer, 0.69:0.30 ng/ml in gastric cancer, and 0.66:0.37 ng/ml in colorectal cancer. This difference was statistically significant in gastric and colorectal cancer. These findings suggest that serum HGF is useful as a tumor marker with a close correlation to the metastatic state of gastrointestinal cancer.