Yasuhisa Naito

Expression of high-mobility group-1 mRNA in human gastrointestinal adenocarcinoma and corresponding non-cancerous mucosa

An 1194‐nucleotide complementary DNA clone, FMI, encoding a human high‐mobility group‐I protein (HMG‐I) was isolated from a well‐differentiated human gastric‐carcinoma cell line complementary DNA library by a differential screening method. FMI is similar to the published human HMG‐I in mature protein, with only 3 different codons at positions 11, 149, and 190. We analyzed 33 gastric and colorectal adenocarcinomas for expression of the FMI gene. Northern‐blot analysis revealed that all of the cancers expressed FMI at a higher level than in corresponding non‐cancerous mucosa, with 2 transcripts of approximately 1.4 and 2.4 kilobases. The FMI expression level in the non‐cancerous tissues increased with the depth of accompanying cancer invasion. Only 18.2% of well‐differentiated cancers showed a higher expression level in corresponding non‐cancerous tissues, whereas the expression in corresponding non‐cancerous tissues was significantly higher in moderately (60%) and poorly differentiated (83.3%) cancers. In situ hybridization demonstrated the location of FMI mRNA in well‐ and poorly differentiated gastric‐cancer cells as well as in non‐cancerous tissue adjacent to poorly differentiated gastric cancer, but no hybridization was detected in normal epithelial cells adjacent to well‐differentiated gastric cancer. These findings may provide new information on HMG‐I mRNA expression in human gastrointestinal cancer and suggest a correlation between FMI mRNA expression to the differentiation and the stage of human gastrointestinal adenocarcinomas. Int. J. Cancer 74:1–6.

Antitumor Effect of a Neutralizing Antibody to Vascular Endothelial Growth Factor on Liver Metastasis of Endocrine Neoplasm

Distant metastasis of gastrointestinal endocrine neoplasm is resistant to currently available treatments. Because hematogenic metastasis is dominant, anti‐angiogenic drugs are expected to be a novel therapy for this neoplasm. In the present study, the therapeutic effect of vascular endothelial growth factor neutralizing antibody (VEGFAb) on liver metastasis of an endocrine neoplasm was investigated experimentally. Cecal transplantation into nude mice of small pieces of EN‐1, a xenotransplanted human intestinal endocrine neoplasm, resulted in liver metastasis. A treated group (n=19) received 100 μg/mouse of VEGFAb intraperitoneally on alternate days from day 10 after tumor transplantation, and the control group (n=19) received saline. Five of the 19 control mice died of tumor progression, of which 2 could not be evaluated. The cecal tumor weighed 6316±2333 mg (n=17) in the control group and 1209±837 mg (n=19) in the treated group (P<0.01) 6 weeks after transplantation. Liver metastasis developed in 16 of 17 control mice and in 2 of 19 treated mice (P<0.01). The VEGF level of the whole cecal tumor in the control group was significantly higher than that in the treated group (305.1±174.1 vs. 54.7±41.2 mg; P<0.001). VEGFAb did not cause any body weight loss (28.52±1.63 in the control vs. 28.44±1.71 g in the treated group). These results indicate that VEGFAb may be a novel therapeutic agent for endocrine neoplasm with distant metastasis.

Cell culture of small round cell tumor originating in the thoracopulmonary region. Evidence for derivation from a primitive pluripotent cell

The authors describe a 14‐year‐old girl with small round cell tumor originating in the chest wall analyzed by the extensive studies including light and electron microscopic examination, histochemical study, immunochemical study, cytogenetics, and gene analysis. A cell line producing carcinoembryonic antigen (CEA) and neuron‐specific enolase (NSE) has been established from pleural effusion of the pulmonary metastatic tumor. Cytogenetic analysis disclosed a reciprocal translocation (11;22)(q24;q12). Additionally, immunocytochemical studies demonstrated that CEA, NSE, vimentin, cytokeratin, and epithelial membrane antigens are positive, but desmin and S‐100 protein are negative. Although neurofilament was negative in the pulmonary metastatic tumor cells, it became positive in cell line in vitro. These results suggest that this tumor may be derived from the primitive and pluripotential cells, differentiating into mesenchymal, epithelial, and neural features in variable proportions.

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

SummaryTo elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture.Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers.For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested.These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.

Effects of pyrroloquinoline quinone (PQQ) and PQQ-oxazole on DNA synthesis of cultured human fibroblasts

The effects of pyrroloquinoline quinone (PQQ) and PQQ-oxazole (PQQ-glycine adduct) on DNA synthesis were examined using cultured human fibroblasts. Confluent fibroblasts were cultured in serum-free Dulbeccos modified Eagles media, and various concentrations of PQQ and PQQ-oxazole were added to the media. After incubation for 24 h, [3H]thymidine was added to the media as an indicator for DNA synthesis of the cells. The thymidine incorporation into the cells was significantly enhanced even in the presence of very low concentrations of PQQ (0.003-0.03 microM); it remained significantly enhanced, up to 30 microM PQQ. However, the incorporation remarkably decreased at 750-1500 microM of PQQ. In contrast to the results for PQQ, DNA synthesis was not stimulated by addition of 0.003-3 microM PQQ-oxazole, but it was slightly enhanced at concentrations 15-750 microM. In morphological examination of the cultured human fibroblasts, cell density was increased by addition of 0.003-30 microM PQQ when compared with that of the control, supporting the above biochemical data. However, there were no distinct differences in morphological effect between PQQ and PQQ-oxazole.

Interaction of EphB2-tyrosine kinase receptor and its ligand conveys dorsalization signal in Xenopus laevis development.

The Eph class is the largest family of receptor tyrosine kinases and has been shown to play various roles in neural development including axon pathfinding and neural crest migration. EphB2 associates with transmembrane ligands Ephrin-B1 and -B2, which leads to tyrosine phosphorylation of both the ligands and receptor and is presumed to regulate cell-to-cell interactions by bidirectional signaling. We have investigated the biological effects of the EphB2-induced signal in the early stage of Xenopus laevis development. Xenopus EphB2 transcripts were detected maternally and were expressed at equal levels between the ventral and dorsal halves of the gastrulae, with expression increasing after the late gastrula stage. EphB2 mRNA expression in dorsal marginal zone explants from gastrulae increases during later development while that in ventral explants does not. We show here that microinjection of RNA encoding EphB2 into a ventral blastomere of embryos induced a partial secondary dorsal axis which consisted of neural tissues, notochord and somites. Analysis with molecular markers verified that the microinjected EphB2 dorsalized the mesoderm of ventral marginal zone explants. These dorsalizing effects of EphB2 in both the whole embryo and ventral explants were inhibited by the coinjection of RNA encoding the soluble form of Ephrin-B1. Furthermore, co-injection of EphB2 and Ephrin-B1 RNAs synergistically enhanced the dorsalization effect. These data show that the interaction between EphB2 and its ligands including Ephrin-B1 causes signaling events which lead to dorsal development, and strongly suggests the existence of proteins which mediate the dorsalization induced by EphB2 in early stage embryos of Xenopus laevis.

l-158,809 and (d-Ala7)-angiotensin I/II (1–7) decrease PAI-1 release from human umbilical vein endothelial cells

The endothelium is a major source of plasminogen activator inhibitor-1 (PAI-1), which plays a critical role in the regulation of fibrinolysis. There are many reports on the increase in the expression of PAI-1 by angiotensin II (Ang II). In the present study, we investigated the effects of angiotensin-related substances on the release of PAI-1 from human umbilical vein endothelial cells (HUVECs). Ang II increased PAI-1 and tissue plasminogen activator (t-PA) release, while its metabolite angiotensin-(1-7) (Ang-(1-7)) amino acid fragment decreased them. Angiotensin Type 1 (AT1) receptor antagonist, L-158,809 (L-1), and Ang-(1-7) receptor antagonist, (D-Ala(7))-angiotensin I/II (1-7) (D-Ala), decreased PAI-1 and t-PA release; angiotensin Type 2 (AT2) antagonist, PD123,319 (PD), however, did not have any effects on the release of PAI-1 and t-PA. The addition of the equal concentration or 10-times-higher concentration of L-1 to Ang II did not change PAI-1 release compared to that by Ang II. Although Ang-(1-7) and L-1 decreased PAI-1 release, there were no additional effects on the decrease of the amounts of PAI-1 by the mixture of Ang-(1-7) and the equal concentration or 10-times-higher concentration of L-1 compared to those by Ang-(1-7). The equal concentration of D-Ala to Ang II did not change the amounts of PAI-1, but the addition of the 10-times-higher concentration of D-Ala to Ang II resulted in significant decrease of the amounts of PAI-1 compared to those by Ang II. The addition of equal concentration or 10-times-higher concentration of D-Ala to Ang-(1-7) showed the significant decrease of the amounts of PAI-1 compared to those by Ang-(1-7). In conclusion, L-158,809 and (D-Ala(7))-angiotensin I/III (1-7) may be used as profibrinolytic drugs.

Morphological analysis of renal cell culture models of calcium phosphate stone formation

Cell culture models of calcium phosphate renal stone formation were established using the MDCK cell line. Renal microliths were detected within pseudocysts in three-dimensional soft agar cultures, and were also observed in the basal region of cells lining the cell sheet, and immediately beneath domes or blisters in monolayers and collagen gel cultures. Light and scanning electron microscopy indicated that these microliths had a similar lamellated and spherical appearance to those in humans. These microliths were first detected microscopically after 21 days of culture, and were found to be composed of calcium phosphate by X-ray and microinfrared spectroscopic analyses. These culture models may provide a powerful new tool to study the pathogenesis of renal stone diseases and/or calcium phosphate stone formation in humans and animals.

Microlith Formation In Vitro by Madin Darby Canine Kidney (MDCK) Cells

Background: The mechanism of renal stone genesis as well as the location of stone crystal formation in the kidney remains unclear. Possible sites of stone generation are either in the tubular lumen or tubular cell.

Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat

Specific expression of the structure-specific recognition protein (SSRP) gene was investigated in rat fetal, adult, and tumor tissues using a 2.0-kb partial sequence of rat SSRP cDNA isolated from a cDNA library of rat renal cell carcinoma. The results revealed that it was rather specifically expressed in rat fetal kidney and renal cell carcinoma induced by Fenitrilotriacetate, but not in adult kidney, when various organs were tested by Northern blot analysis. In situ hybridization further demonstrated that it was located in the neoplastic cells of renal cell carcinoma and in the epithelial cells of fetal kidney but undetectable in any cells of normal adult kidney. These observations seem to imply the involvement of SSRP gene, which is believed to recognize structural alterations of DNA, in kidney development and carcinogenesis of certain types of kidney cancer.