Sadako Yamagata

Gelatinases of metastatic cell lines of murine colonic carcinoma as detected by substrate-gel electrophoresis.

Gelatinases were detected in the conditioned medium of murine colonic carcinoma cells by SDS-polyacrylamide gel electrophoresis using gels copolymerized with gelatin. Several gelatinase activities differing in molecular weight were detected but the major activities migrated with molecular weights of 60,000 and 95,000. The enzymes did not hydrolyze bovine serum albumin or casein, and required calcium for activity. All of the gelatinase activities were inhibited by EDTA, 1,10-phenanthroline and dithiothreitol but not by N-ethylmaleimide and phenylmethylsulfonyl fluoride. The 95,000 dalton gelatinase was separated from the 60,000 dalton gelatinase by affinity chromatography on Ricinus communis agglutinin-agarose, and the former activity was markedly increased in highly metastatic cell lines as compared with its activity in poorly metastatic cell lines.

Gelatinases of murine metastatic tumor cells

The properties of gelatinases secreted in culture medium of murine fibroblasts, macrophages, colonic carcinoma, FBJ virus-induced osteosarcoma, Lewis lung carcinoma and mammary tumor cells were compared. Normal fibroblasts and macrophages secreted gelatinases of 60,000 and 95,000 molecular weights, respectively. Tumor cells secreted both of these gelatinases, although the relative amounts of the 60 kDa and 95 kDa gelatinases differed among the cell lines. The cell lines that had the greatest metastatic potential to the lung secreted the highest amount of 95 kDa gelatinase. The 95 kDa gelatinase produced by tumor cells had properties similar to that of macrophages.

Azido glycoside primer: a versatile building block for the biocombinatorial synthesis of glycosphingolipid analogues

A lactoside primer, 12-azidododecyl beta-lactoside, was synthesized via the Koenigs-Knorr method by glycosylation of 1,12-dodecyldiol with perbenzoylated lactosyl bromide. The presence of the 2-O-acyl substituent in the donor gave the beta-lactoside, and an excess of acceptor ensured monoglycosylation of the diol. Mesylation of the omega-hydroxyl group in the aglycon, followed by displacement of the mesylate with azide and subsequent O-debenzoylation gave the desired omega-azidododecyl beta-lactoside. The azido glycoside primer was examined in mouse B16 melanoma cells for its feasibility as a building block for oligosaccharide biosynthesis. Uptake of the azido glycoside primer by B16 cells resulted in the sialylation of the galactose residue of the primer to give a glycosylated product having the same glycan as in ganglioside GM3. After 24 h incubation of B16 cells with the primers, the amount of sialylated omega-azidododecyl beta-lactoside primer was 75% of the amount of sialylated n-dodecyl beta-lactoside. However, after 48 h incubation, both primers gave equal amounts of the sialylated products. Interestingly, the remaining azido glycoside primer after 48 h incubation was 5.6-fold greater than that of the alkyl primer, indicating degradation of the alkyl primer to a larger extent than the omega-azido glycoside primer. The facile chemical synthesis and the efficient uptake in cells make the azido glycoside primer a versatile building block for the biocombinatorial synthesis of glycolipid oligosaccharides.

Characterization of extracellular matrix-degrading proteinase and its inhibitor in gynecologic cancer tissues with clinically different metastatic form

Background. The authors conducted a comparison study of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase‐1 (TIMP‐1) activities in clinically different metastatic types of ovarian cancer, cervical cancer, and endometrial cancer tissues.

Two-dimensional mapping by the high-performance liquid chromatography of oligosaccharides released from glycosphingolipids by endoglycoceramidase.

A two-dimensional sugar mapping method has been developed by which sensitive, reproducible, and simple analysis can be carried out on the structures and compositions of oligosaccharides released from glycosphingolipids by endoglycoceramidase. The oligosaccharides were labeled quantitatively with an ultraviolet-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-oligosaccharides were separated first on an amide-silica column and then on a C4-silica column by high-performance liquid chromatography. The acidic ABEE-oligosaccharides were eluted as a group at the start of the chromatography while the neutral ABEE-oligosaccharides were separated according to size and structure on an amide-silica column using an eluent without salt. The acidic oligosaccharides were separated according to size and structure when rechromatographed on the same column using an eluent containing KH2PO4. NeuAc-containing ABEE-oligosaccharides were extensively separated from the corresponding NeuGc derivatives. The ABEE-oligosaccharides separated on an amide-silica column were then chromatographed on a column of C4-silica on which lactotriose and neolacto-series oligosaccharides were clearly shown to be separated from the others. On the basis of the retention times of the individual ABEE-oligosaccharides on two separate columns, 9 neutral and 15 acidic oligosaccharides derived from glycosphingolipid standards were two-dimensionally mapped without overlapping. The gangliosides of a human chondrosarcoma tissue and glycosphingolipids of tumor tissue of FBJ virus-transformed murine osteosarcoma cells were analyzed by this method in conjunction with exoglycosidase treatment. At least 11 species of glycosphingolipids were identified in both cases.

Occurrence of an active form of gelatinase in human gastric and colorectal carcinoma tissues

Profiles of gelatinase released during 4 h incubation of explants from human carcinoma tissues (15 gastric and 15 colorectal carcinomas) in serum-free RPMI 1640 medium were compared with those of corresponding normal tissue specimens obtained 2 and 10 cm apart from tumor periphery. All the culture supernatants of both tumor and normal tissues contained gelatinase species of 200, 130, 92 and 72 kDa as detected by zymography with gelatin as a substrate. Besides these gelatinase species, all carcinoma tissue culture supernatants had an additional 66-kDa gelatinase, an active form of the 72-kDa gelatinase. This gelatinase was not detectable in the normal tissues 10 cm apart from the tumor mass, with an exceptional case of gastric carcinoma of Borrmann type 4. These results suggest that the presence of active form of gelatinase might be one of the characteristic properties of these malignant human tumors.

Suppression by ganglioside GD1A of migration capability, adhesion to vitronectin and metastatic potential of highly metastatic FBJ-LL cells.

Ganglioside GD1a, which is highly expressed in poorly metastatic FBJ‐S1 cells, has been shown to inhibit the serum‐induced migration capability of highly metastatic FBJ‐LL cells. In the present study, the capacity of FBJ‐S1 cells to adhere to vitronectin was found to be about half that of FBJ‐LL cells. Pre‐treatment of FBJ‐LL cells with GD1a decreased this capacity by 30% that of the control, whereas GM1‐pre‐treatment caused only a 10% decrease, indicating that GD1a specifically inhibits FBJ‐LL cell adhesion to vitronectin. Since FBJ‐LL cells contain almost no GD1a, transfectants capable of expressing GD1a to varying degrees were produced in this study by transfection of FBJ‐LL cells with GM2/GD2‐synthase cDNA. Decrease in the serum‐induced migration capacity of these transfectants was accompanied by an increment in GD1a expression. Adhesion of the transfectants to vitronectin decreased by 30% as compared with mock‐transfected cells. Within 4 to 5 weeks after GD1a‐expressing transfectant and mock‐transfected cells were transplanted into mice, metastatic nodules were observed in liver, lung, kidney and adrenal glands of mock‐transplanted mice, but not in those with GD1a‐expressing transfectants, indicating that GD1a suppresses the metastasis of FBJ‐osteosarcoma cells, possibly by inhibiting cell migration and cell adhesion. The involvement of the ganglioside in the suppression of metastasis is clearly demonstrated in the present study. Int. J. Cancer 83:685–691, 1999.

Hyaluronidase activity in gynaecological cancer tissues with different metastatic forms

We investigated hyaluronidase activity in gynaecological normal and malignant tissues. Hyaluronidase activity in culture medium of tissue specimens was detected by hyaluronic acid zymography and quantified by densitometry. Hyaluronidase activity was shown as one dominant band (molecular weight 65 kDa) at pH 3.5. Hyaluronidase activity was significantly higher in normal ovary (P < 0.05) and normal endometrium (P< 0.05) than in normal cervix. One dominant 65-kDa hyaluronidase was expressed in 100% (14 out of 14) of ovarian cancer tissues and in 91% (10 out of 11) of endometrial cancer tissues. However, hyaluronidase activity was not observed in cervical cancer tissues. Hyaluronidase activity was significantly higher in ovarian (P < 0.001) and endometrial (P < 0.01) cancer tissues than in cervical cancer tissue and was significantly higher in ovarian cancer tissue than in endometrial cancer tissue (P < 0.05). These facts suggest that the cancer cells make use of the original characteristic of the organ to invade and metastasize. Moreover, these results reflect the difference in metastatic forms and are suggestive of a strong relationship between hyaluronidase activity and invasion and metastasis of ovarian and endometrial cancers compared with cervical cancer.

Different pattern of zymography between human gynecologic normal and malignant tissues

OBJECTIVE Our purpose was to evaluate type IV collagenase in ovarian and endometrial cancer tissues. STUDY DESIGN Tissue specimens were obtained from patients with ovarian and endometrial cancer and uterine myoma. Gelatinase activity was detected by zymography and quantitated by densitometer. RESULTS Four dominant gelatinases were detected in ovarian and endometrial cancer tissues: 200, 130, 92, and 72 kd gelatinase. Other forms observed were 83 kd gelatinase, which is an active form of 92 kd gelatinase, and 66 kd gelatinase, which is an active form of 72 kd gelatinase. Densitometric analysis showed that the 92 kd/72 kd ratio in ovarian cancer tissues was significantly higher than in normal ovarian tissues (p < 0.05) and that the 66 kd/72 kd ratio was higher in ovarian cancer tissues (p = 0.07). Both ratios in endometrial cancer tissues were significantly higher than in normal endometrial tissues (p < 0.05). CONCLUSION Gelatinase activity was remarkably higher in ovarian and endometrial cancer tissues. Especially, 92, 83, and 66 kd gelatinases were clearly detected in cancer tissues, suggesting that these gelatinases were related to the malignant phenotype, because degradation of the components of the basement membrane such as type IV collagen is necessary for cancer cells to metastasize.

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1.