Yoshihiro Mogi

Regulation of asialoglycoprotein receptor synthesis by inflammation-related cytokines in HepG2 cells

The asialoglycoprotein receptor (AGPR) is responsible for the catabolism of acute phase proteins. The effects of inflammation-related cytokines on the expression of AGPR were investigated in HepG2 cells derived from a human hepatoblastoma cell line. Binding studies, using a [125I]-labeled asialo-orosomucoid ligand, revealed that AGPR numbers on cell surfaces were increased by interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). In cells treated with IL-1, IL-6, or TNF, immunohistochemical staining with an anti-AGPR monoclonal antibody demonstrated augmented expression. Pulse labeling analysis, using [35S]-labeled methionine, showed newly synthesized AGPR in both the precursor and the mature forms. When IL-1, IL-6, and TNF were added to the culture medium, total synthesis of AGPR (sum of the mature and precursor forms) was increased. In addition, the relative proportion of the synthesized precursor form of AGPR was higher in cytokine-treated than in untreated cells, suggesting that these cytokines augment the synthesis of AGPR, particularly in the stage prior to glycosylation.

Kinetics of Internalization and Cytotoxicity of Transferrin‐Neocarzinostatin Conjugate in Human Leukemia Cell Line, K562

ABSTRACT Human serum transferrin was conjugated with an anticancer‐active polypeptide, neocarzinostatin, by using N‐succinimidy1‐3‐(2‐pyridyldithio)propionate. The conjugate consisted of 1.8 mol of neocarzinostatin per 1 mol of transferrin on average and retained cytotoxic activity against human tumor cells. This conjugate was capable of binding to the transferrin receptor of human myelogenous leukemia K562 cells and was internalized by endocytosis. The LD50 values of the conjugate and neocarzinostatin alone in the presence of excess native bovine transferrin were 0.20 μ/ml and 1.80 μ/ml, respectively, suggesting that the effect of the conjugate was greater than that of neocarzinostatin alone. A pulse‐chase experiment using 125I‐labeled conjugate revealed that 25% of the internalized conjugate was degraded in lysosomes and the rest was recycled back to the cell surface without degradation. About 75% of this conjugate recycled back to the cell surface in 18.3 min (3.4 min for receptor binding and 14.9 min for recycling to the cell surface through the acidosomes), while the rest was delivered from the cell surface to the lysosome in 19.6 min. This phenomenon was confirmed by chasing the radioactivity in subcellular fractions separated by Percoll density gradient centrifugation. Therefore, it was concluded that this conjugate is internalized specifically by transferrin receptors and is at least partly transferred to and accumulated in lysosomal compartments, resulting in the inhibition of cellular DNA synthesis.

Platelet aggregation induced by adenosine diphosphate released from cloned murine fibrosarcoma cells is positively correlated with the experimental metastatic potential of the cells.

We established five clones (ML‐01, ML‐02, MH‐01, MH‐02, MH‐03) from murine 3‐methylcholan‐threne‐induced fibrosarcoma A (Meth A), and investigated their experimental metastatic potentials in relation to their platelet‐aggregating activities. A clone with a high metastatic potential (MH‐02) showed a characteristic biphasic pattern of platelet aggregation, of which the first peak was not present in the aggregation patterns of the clone with low metastatic potential (ML‐01). The first peak was eliminated by treatment of the cells with apyrase, indicating that adenosine diphosphate (ADD was the causative substance of this particular peak. The metastatic potential of clones correlated well with the ADP concentration of the culture media. These results suggest that the increased ADP production and consequential enhancement of platelet‐aggregating activity are closely related to the increment of pulmonary metastatic potential of MH‐02 clone.

The near-infrared electronic endoscope for diagnosis of esophageal varices

We developed an electronic endoscope sensitive to light delivered by an infrared laser, which makes it possible to visualize submucosal vessels of the gastrointestinal tract. The system consists of a modified electronic endoscope and an image processing unit. A high output laser diode was chosen as the source of the infrared ray with a wavelength of 815 nm, an output of 200 milliwatts, and a band width of 30 nm. Using this instrument, esophageal varices were classified into three categories: venous dilation, dark spot, and diffuse dark area. The venous dilation and dark spot appearances correlated with early esophageal varices, which were rarely seen with visible light. On the other hand, the diffuse dark area appearance was considered to be a high risk sign of future bleeding. This system is useful not only for the early diagnosis of esophageal varices, but also for the localization of optimal puncture sites and the evaluation of the effect of sclerotherapy.

Retroperitoneal extrarenal angiomyolipoma with early gastric carcinoma

Abstract: We present a very rare case of a retroperitoneal extrarenal angiomyolipoma accompanied by early gastric cancer. A 41-year-old Japanese man, who had undergone surgery for a type IIc early gastric cancer 2 years earlier, was admitted to hospital presenting with back pain and abdominal fullness. Computed tomographic scanning and magnetic resonance imaging of the abdomen disclosed a massive fatty tumor extending from the hepatic hilus to the retroperitoneum. A large retroperitoneal tumor mass with no sign of involvement in the kidney was totally resected by radical surgery. Histologically, the tumor was classified as an angiomyolipoma.

Close Correlation between the Dephosphorylation of p53 and Growth Suppression by Transforming Growth Factor-β1 in Nasopharyngeal Carcinoma Cells Transduced with Adenovirus Early Region Genes

The mechanism of growth inhibition by transforming growth factor (TGF)‐β1 was investigated. We examined the growth inhibitory effects of TGF‐β1 on human nasopharyngeal carcinoma (KB) cells which constitutively expressed p53. TGF‐β1 suppressed the DNA synthesis of KB cells in a dose‐dependent manner. It had minimal effect on adenovirus‐2‐transduced KB cells expressing either adenovirus early region 1B (E1B) or 1A (E1A) product, which respectively binds to p53 or Rb product and inhibits its function, and no growth inhibition at all was observed with KB cells expressing both E1B and E1A products. Dephosphorylation of the p53 was promoted by TGF‐β1 stimulation in KB cells, but not in E1B‐producing KB cells, which sequestrate the function of p53. The growth inhibition of KB cells by TGF‐β1 was significantly reduced by treatment with okadaic acid. These results suggest that p53 transduces the antiproliferative signal of TGF‐β1 possibly through its dephosphorylation.

Suppressive effect of ethanol on the expression of hepatic asialoglycoprotein receptors augmented by interleukin-1β,interleukin-6, and tumor necrosis factor-α

Abstract: Blood levels of inflammatory-related cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, are elevated in patients with alcoholic liver diseases. We investigated the effects of these cytokines and ethanol on the expression of hepatic asialoglycoprotein receptors (AGPRs) in a human hepatoblastoma cell line, HepG2. An [125I]-asialo-orosomucoid binding assay showed significant increases in surface AGPR numbers in HepG2 cells by treatment with IL-1β, IL-6, and TNF-α, to levels which were approximately 130% of the values in untreated control cells. However, the enhanced AGPR numbers induced by treatment with these cytokines were markedly suppressed, to 70%–80% of the number in the untreated cells, by treatment with ethanol. Immunological detection of AGPR with a specific antibody demonstrated that the modulation of surface AGPR numbers was correlated with the cellular expression levels of AGPR. These results suggest that, although IL-1β, IL-6, and TNF-α stimulate the synthesis of hepatic AGPR, ethanol suppresses the expression of AGPR augmented by these cytokines. This leads to an increase in serum asialo-orosomucoid levels caused by the disordered catabolism mediated by AGPR in patients with alcoholic liver disease.

Ethanol inhibits asialoglycoprotein receptor synthesis but augments its mRNA expression in a human hepatoma cell line

The effect of ethanol on the expression of asialoglycoprotein receptor protein and its mRNA was studied in a human hepatoma cell line, HepG2. The number of asialoglycoprotein receptors on the cell surface was decreased to 60% of the control level, without a loss in affinity, by incubating the cells with 100 mM ethanol. The decrease in cell surface asialoglycoprotein receptors was paralleled by a decrease in total receptor numbers, including intracellular and surface receptors. The internalization of asialoglycoprotein was also diminished, to 44% of that in control cells. The decreases in cell surface receptors and total receptor numbers were partially restored by 2 mM 4-methylpyrazole, suggesting that ethanol metabolites participated in the diminution of asialoglycoprotein receptor expression. However, the steady-state expression of asialoglycoprotein receptor mRNA was increased in ethanol-treated cells and further augmented by a longer ethanol exposure. These paradoxical results, i.e., the decrease of asialoglycoprotein receptor protein and the increase of its mRNA expression, suggest that the reduction in the asialoglycoprotein receptor protein is a post-transcriptional event and that a possible feedback regulatory mechanism may control asialoglycoprotein receptor gene transcription and/or impair the degradation of its mRNA.

Augmented Expression of a Type IV Collagen-binding Protein in a Highly Metastatic Murine Fibrosarcoma Clone

The adhesive properties of highly and weakly metastatic murine sarcoma (Meth A) clones were investigated. A highly metastatic clone, MH‐02, preferentially adhered to type IV collagen‐coated plastic dishes and to bovine pulmonary arterial endothelial cell‐coated plastic dishes as compared to a weakly metastatic clone, ML‐01. Pretreatment of MH‐02 and ML‐01 cells with antisera against MH‐02 cells resulted in almost equivalent adhesiveness to type IV collagen. Preincubation of 125I‐radiolabeled tumor cells with the antisera against MH‐02 significantly reduced the arrest of MH‐02 cells in the lung, but ML‐01 cells were not affected. The number of pulmonary metastatic nodules of MH‐02 cells was reduced to the same level as that of ML‐01 cells by preincubation of the tumor cells with the antisera in an experimental metastasis experiment. These results indicated that the high metastatic ability of MH‐02 can be attributed to its preferential adhesiveness to type IV collagen. The type IV collagen‐binding proteins of MH‐02 and ML‐01 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and autoradiography. Among several proteins which bound to type IV collagen, expression of a protein with a molecular weight of 29 kD was significantly greater in MH‐02 than in ML‐01. These results suggest that the greater adhesion of highly metastatic MH‐02 cells to type IV collagen is due to enhanced expression of the type IV collagen‐binding 29 kD protein.