Do Young Yoon

Enhanced IL-18 expression in common skin tumors

Interleukin-18 (IL-18) has been found to have multiple effects upon various cells involved in inflammatory response. Recently we reported that B16 murine melanoma cells are able to produce IL-18, which is involved in the regulation of intracellular reactive oxygen intermediates (ROI) and Fas-ligand expression, indicating that IL-18 plays key role in the tumor activity of melanoma. In this study, we investigated the pattern of IL-18 expression in the human system. IL-18 production was tested by enzyme linked immunosorbent assay (ELISA) assay in various tumor cell lines, including Raji (Burkitts lymphoma), IM-9 (B lymphoblast), Jurkat (acute T cell leukemia), SK-MES-1 (squamous cell carcinoma (SCC) cell line), SK-MEL-2, G-361, DM-4, and DX-3 (melanoma cell lines). ELISA tests showed that IL-18 was highly expressed in malignant skin tumors such as SK-MES-1, SK-MEL-2, G-361, DM-4, and DX-3 cell lines, thus suggesting that IL-18 production may be associated with the malignancy of skin tumors. Here, we report that enhanced IL-18 expression is positively correlated with malignant skin tumors such as SCC and melanoma, suggesting the importance role of IL-18 in malignancy of skin tumors. Taken together, expression of IL-18 by tumor cells in human skin tissue may provide an important clue to understand the pathogenesis of malignant skin tumors.

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.

Tetramethoxy hydroxyflavone p7F downregulates inflammatory mediators via the inhibition of nuclear factor κB

Abstract: Artemisia has been traditionally used in Korean herbal medicine to clear damp heat and to treat uteritis and jaundice. Flavonoids isolated from Artemisia are also known to possess anti‐inflammatory activities. In this study, 5,6,3′,5′‐tetramethoxy 7,4′‐hydroxyflavone (p7F) was isolated from Artemisia absinthium. We examined in vitro and in vivo regulatory functions of p7F on the production of nitric oxide (NO), prostaglandin E2 (PGE2), and tumor necrosis factor‐α (TNF‐α) as well as the expression of inducible NO synthase (iNOS), cyclooxygenase‐2 (COX‐2), and collagen‐induced arthritis. p7F inhibited the expression or production of proinflammatory mediators such as COX‐2/PGE2 and iNOS/NO in lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells. p7F also suppressed the serum level of TNF‐α in mice treated with collagen and inhibited nuclear factor‐κB (NF‐κB) activation as well as NF‐κB promoter activity in RAW 264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in hydrogen peroxide‐stimulated RAW 264.7 cells. p7F has antioxidant activity and inhibits NF‐κB activation. Taken together, these results suggest that p7F can be clinically applied to the treatment of inflammatory diseases.

Ryanodine receptor-mediated interference of neuronal cell differentiation by presenilin 2 mutation.

Neuronal cell differentiation alterations induced by mutant presenilin 2 (PS2) were investigated in transgenic mice expressing wild‐type or mutant‐type PS2. Progressive increases in differentiation and marker protein expression were found in neuronal cells expressing wild‐type PS2, whereas these processes were much perturbed in mutant‐type PS2 with elevated ryanodine‐receptor (RyR) expression and intracellular calcium levels. Moreover, dantrolene, a blocker of RyR reduced the PS2 mutation‐induced interference of cell differentiation and calcium release, but caffeine, an activator of RyR, exacerbated PS2 mutation‐induced interference with cell differentiation. Our results indicate that mutant PS2 inhibits normal neuronal cell differentiation and that RyR‐mediated calcium overrelease may be a significant factor.

Stimulation of Cell Growth by Erythropoietin in RAW264.7 Cells: Association with AP-1 Activation

Erythropoietin (EPO), a hematopoietic factor, is required for normal erythrocyte developments, but it has been demonstrated to have many other functions, and its receptor is localized in other tissues. In the present study, we investigated whether EPO can promote other cell proliferation and possible molecular mechanisms. EPO restored the inhibition of the RAW264.7 and PC12 cell growth by fetal bovine serum (FBS) withdrawal in a dose dependent manner but not that of other cell types tested. The restoring effect of EPO was completed when the RAW264.7 cells were cultured in the medium containing as low as 3% of FBS, and 10 U/mL EPO could replace FBS. The restoring effect of EPO in the RAW264.7 cells was associated with the increased of c-Fos and c-Jun expression as well as AP-1 activation. These data demonstrate that EPO can stimulate RAW264.7 cell as well as PC12 cell growth even when the cells were cultured without FBS or in the presence of small amounts of FBS in the medium, and this stimulating effect is associated with the activation of AP-1 transcription factor.

Mutant presenilin 2 increases acetylcholinesterase activity in neuronal cells

A presenilin 2 mutation is believed to be involved in the development of Alzheimers disease. In addition, transgenic mice with a presenilin 2 mutation have been reported to have learning and memory impairments. In this study, exposing PC12 cells expressing mutant presenilin 2 to 50 μM Aß25–35, 30 mMl-glutamate and 50 μM H2O2 caused a significant increase in acetylcholine esterase activity. Anin vivo study revealed high levels of this enzyme activity in the mutant presenilin 2 transgenic brains compared with the wild type presenilin 2 transgenic and non-transgenic samples. These results suggest that a mutant presenilin 2-induced neurodegeneration in Alzheimers disease might be involved in the increase in acetylcholinesterase activity. These findings might help in the development of an appropriate therapeutic intervention targeting mutant presenilin 2-induced Alzheimers disease.

Immunosuppression of xenograft rejection in porcine kidney PK15 cells by porcine IL-18.

Xenotransplantation, the transplantation of cells, tissues or organs between individuals of different species, would resolve the current shortage of organs, but rejection remains the major hurdle to successful xenotransplantation. In the present study, we analyzed mixed lymphocyte reactions (MLRs) and used 51Cr release assays in order to identify the proliferation and expansion of mouse CD8+ cytotoxic T lymphocyte cells against PK15, PK15/pIL-18 or PK15/mIL-18 cells. In addition, we identified T cell populations in mouse splenocytes and lymph node cells using two-color flow cytometry. It was found that the CD8+T cells of xenograft recipients proliferated extensively and that the survival rates of populations of PK15/mIL-18 or PK15/pIL-18 cells were higher than untransfected controls. Moreover, CD3+T cells were increased in mice injected with PK15 cells or PK15/pIL-18 cells but PK15/pIL-18 cell numbers were lower in lymph nodes than untransfected controls. CD8+T cells numbers were reduced in the lymph nodes of PK15/pIL-18 injected mice. These results suggest that porcine IL-18 regulates anti-pig cellular rejection in C57BL/6 mice.

Immunofluorescent quantification of tyrosine phosphorylated proteins by flow cytometric analysis

To measure the quantity of tyrosine phosphorylated proteins in cells, we have used a flow cytometry technique with fluorescein isothiocyanate-labeled anti-phosphotyrosine antibody (FITC-αPY mAb). The analysis was applied to the phosphotyrosine titration and showed an optimum amount of FITC-αPY mAb (30μg/1×10cells). The staining specificity of our assay was tested by the addition of exogenous competitors, showing a specific inhibition by phosphotyrosine but not by phosphoserine, phosphothreonine, or tyrosine. The assay was also able to elucidate the inhibitory effect on tyrosine phosphorylation of genistein, a protein tyrosine kinase inhibitor. These results imply that immunofluorescent quantification assay using a flow cytometer could be a useful technique to determine the intracellular level of tyrosine phosphorylated protein.