The Histone Deacetylase Inhibitor Trichostatin A Sensitizes Human Renal Carcinoma Cells to TRAIL-Induced Apoptosis through Down-Regulation of c-FLIP(L)

Histone acetylation plays a critical role in the regulation of transcription by altering the structure of chromatin, and it may influence the resistance of some tumor cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) by regulating the gene expression of components of the TRAIL signaling pathway. In this study, we investigated the effects and molecular mechanisms of trichostatin A (TSA), a histone deacetylase inhibitor, in sensitizing TRAIL-induced apoptosis in Caki human renal carcinoma cells. Our results indicate that nontoxic concentrations of TSA substantially enhance TRAIL-induced apoptosis compared with treatment with either agent alone. Cotreatment with TSA and TRAIL effectively induced cleavage of Bid and loss of mitochondrial membrane potential (MMP), which was associated with the activation of caspases (-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase (PARP), contributing toward the sensitization to TRAIL. Combined treatment with TSA and TRAIL significantly reduced the levels of the cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (c-FLIP), whereas those of death receptor (DR) 4, DR5, and FADD remained unchanged. The synergistic effect of TAS and TRAIL was perfectly attenuated in c-FLIP(L)-overexpressing Caki cells. Taken together, the present study demonstrates that down-regulation of c-FLIP contributes to TSA-facilitated TRAIL-induced apoptosis, amplifying the death receptor, as well as mitochondria-mediated apoptotic signaling pathways.

beta-lapachone-Induced Apoptosis of Human Gastric Carcinoma AGS Cells Is Caspase-Dependent and Regulated by the PI3K/Akt Pathway

beta-lapachone is a naturally occurring quinone that selectively induces apoptotic cell death in a variety of human cancer cells in vitro and in vivo; however, its mechanism of action needs to be further elaborated. In this study, we investigated the effects of beta-lapachone on the induction of apoptosis in human gastric carcinoma AGS cells. beta-lapachone significantly inhibited cellular proliferation, and some typical apoptotic characteristics such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells were observed in beta-lapachone-treated AGS cells. Treatment with beta-lapachone caused mitochondrial transmembrane potential dissipation, stimulated the mitochondria-mediated intrinsic apoptotic pathway, as indicated by caspase-9 activation, cytochrome c release, Bcl-2 downregulation and Bax upregulation, as well as death receptor-mediated extrinsic apoptotic pathway, as indicated by activation of caspase-8 and truncation of Bid. This process was accompanied by activation of caspase-3 and concomitant with cleavage of poly(ADP-ribose) polymerase. The general caspase inhibitor, z-VAD-fmk, significantly abolished beta-lapachone-induced cell death and inhibited growth. Further analysis demonstrated that the induction of apoptosis by beta-lapachone was accompanied by inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K inhibitor LY29004 significantly increased beta-lapachone-induced apoptosis and growth inhibition. Taken together, these findings indicate that the apoptotic activity of beta-lapachone is probably regulated by a caspase-dependent cascade through activation of both intrinsic and extrinsic signaling pathways, and that inhibition of the PI3K/Akt signaling may contribute to beta-lapachone-mediated AGS cell growth inhibition and apoptosis induction.

Re-engineering the mitochondrial genomes in mammalian cells / 대한해부학회지

Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.

Modulation of Apoptosis and Cell Cycle by Synthetic Bile Acid Derivatives / 대한해부학회지

This paper outlines the current understanding of cell cycle modulation and induction of apoptosis in cancer cells by natural and synthetic bile acid. Bile acid homeostasis is tightly regulated in health, and the cellular and tissue concentrations of bile are restricted. However, when pathophysiological processes impair biliary secretion, hepatocytes are exposed to an elevated concentration of bile acids, which triggers cell death. In this context, we have synthesized several new bile acid derivatives. These synthetic bile acids modulate the cell cycle and induce apoptosis in several human cancer cells similar to the effects of natural bile acids. In human breast and prostate cancer cells with different tumor suppressor p53 status, synthetic bile acid induced growth inhibition and apoptosis, and these changes were associated with upregulation of Bax and p21WAF1/CIP1 through a p53-independent pathway. In Jurkat human T cell leukemia cells, the synthetic bile acids induced apoptosis through caspase activation. The synthetic bile acids induced apoptosis in a JNK-dependent manner in SiHa human cervical cancer cells through the induction of Bax and activation of caspases in PC3 prostate cancer cells and induction of G1 phase arrest of the cell cycle in HT29 colon cancer cells. The synthetic bile acids also induced apoptosis in four human glioblastoma multiform cell lines (e.g., U-118MG, U-87MG, T98G, and U-373MG) and one human TE671 medulloblastoma cell line. A chenodeoxycholic acid derivative, called HS-1200, significantly decreased the growth of TE671 medulloblastoma tumor size and increased lifespan in nonobese diabetic and severe combined immunodeficient (NOD/SCID) mice. These findings suggest that these new synthetic bile acids, which are novel apoptosis mediators, might be applicable to the treatment of various human cancer cells.

The expression of TRAIL and its receptors in human osteoarthritic cartilages / 대한내과학회지

BACKGROUND/AIMS: The apoptosis of chondrocytes is assumed to be involved in the pathogenesis of osteoarthritis (OA), and the TNF related apoptosis inducing ligand (TRAIL) is thought to have a pivotal role in the apoptosis of chondrocytes. We investigated the expression of TRAIL and its receptors in human osteoarthritic cartilages. METHODS: Human OA cartilage tissues were obtained from the medial side of the cartilage in the knee joints of 25 patients who underwent total knee replacement surgery, and the normal human cartilages of the knee joint were obtained at autopsy from seven young adults who had no history of joint diseases. The expressions of TRAIL and the death receptor were analyzed by immunohistochemistry or immunofluorscent staining. The concentration of TRAIL in the synovial fluid was measured by enzyme linked immunosorbent assay. RESULTS: TRAIL and its receptors were expressed in the OA cartilage, but not in the normal cartilage. TUNEL staining and immunohistochemistry for TRAIL on the serial sections showed that most TRAIL positive cells were TUNEL positive. The OA joint fluid contained concentrations of TRAIL that were readily detectable (80 and 120 microgram/ppm in the synovial fluid of each, respectively). However, the synovial fluid of the knee joint obtained at autopsy from the seven young adults contained low concentrations of detectable TRAIL (0~2 microgram/ppm). CONCLUSIONS: These results support the notion that TRAIL and its receptors are involved in the pathogenesis of human OA. A better understanding of TRAIL induced apoptosis in chondrocytes might lead to the development of a new therapeutic strategy for OA.

The Increase in Hepatic Uncoupling by Fenofibrate Contributes to a Decrease in Adipose Tissue in Obese Rats

Fenofibrate is a drug that has been suggested to inhibit weight gain by increasing the catabolism of fatty acid in the hepatic mitochondria. We hypothesized that fenofibrate induces an increase in energy expenditure in the hepatic mitochondria, which results in the reduction of adipose tissue. In this study we measured hepatic uncoupling protein (UCP)-2, -3, core temperatures and abdominal fat composition with MRI in Otsuka Long-Evans Tokushima Fatty rats. The fenofibrate group (n=7) was fed fenofibrate (320 mg/kg) mixed chow. The control group (n=7) was fed chow only. The body weight (531.6+/-7.6 g) of the fenofibrate group was significantly lower than that (744.3+/-14.9 g) of the control group (p<0.005). The areas of visceral and subcutaneous fat in the fenofibrate group (11.0+/-0.9 cm2, 4.2+/-0.3 cm2) were significantly less than those in the control group (21.0+/-0.7 cm2, 7.4+/-0.4 cm2) (p=0.046, respectively). The esophageal and rectal temperatures of the fenofibrate group (37.7+/-0.1 degrees C, 33.1+/-0.2 degrees C) were significantly higher than those of the control group (37.3+/-0.1 degrees C, 32.2+/-0.1 degrees C) (p=0.025, p=0.005). There was de novo expression of UCP-3 in the liver of the fenofibrate group. These data suggest that increased energy dissipation, via hepatic UCP-3 by fenofibrate, contribute to decreased weight gain in obese rats.

Prevention of IL-3 Deprivation Induced Apoptosis in Mast Cells by LPS / 대한해부학회지

Mast cells play a protective role in host defense against bacteria, and are found in high number at the host-environment interface. Stimulation of mast cells by bacterial cell wall components is essential for their protective effects against entero-bacterial infection. Mast cells have an extraordinarily long longevity in peripheral tissues compared to other types of blood cells. We undertook this study to reveal the mechanism underlying the prevention on IL-3 deprivation-induced apoptosis in mast cells by LPS. Bone marrow derived mast cells (BMMCs) were obtained from femurs of BALB/c mice and cultured under IL-3 stimulation for 4-6 weeks. At the same point when IL-3 was depleted, BMMCs were treated with LPS. To examine the effect of LPS, DNA electrophoresis, Western blotting, immunoflourescense staining, confocal microscopy, RT-PCR and immunoprecipitation were conducted. IL-3 deprivation reduced cellular viability and induced nuclear condensation and translocation of apoptosisassociated factors. However, LPS treatment prevented these IL-3 deprivation-induced apoptotic events. IL-3 deprivation downregulated representative antiapoptotic factors such as XIAP, 14-3-3, Bcl-2 and Bcl-xL. Expression level of these factors was maintained in BMMCs treated by LPS, which was similar to the level in the control cells. The Bim to Bcl-2 interaction was increased in IL-3 depleted cells, which was prevented by LPS treatment. It was also demonstrated that LPS induced the new expression of a cytokine-induced antiapoptotic factor anamorsin gene in BMMCs under IL-3 deprived condition. Taken together, LPS prevents IL-3 deprivation induced apoptosis in BMMCs via several antiapoptotic factors. This preventive mechanism of LPS on BMMCs apoptosis may contribute the long longevity of mast cells in the peripheral tissue.

Comparison of Na+/I- Symporter Expression Rate in Malignant and Benign Thyroid Diseases: Immunohistochemical Study / 핵의학분자영상

PURPOSE: Previous studies have not showed consistent results for the level of expression of sodium/iodide symporter (NIS) in thyroid diseases, especially malignant tumor. We undertook this study to evaluate the distribution of NIS expression in malignant thyroid diseases and compare with that in benign thyroid disease. MATERIALS AND METHODS: Total patients were 119 cases (Men 15, 48+/-13 yrs). Total number of samples were 205 pieces. In malignant thyroid disease, there were 153 samples: 90 in papillary carcinoma, 4 in follicular carcinoma, 2 in medullary carcinoma and 57 in metastatic lymph node. In benign thyroid disease, there were 52 samples: 36 in goiter/cyst, 11 in thyroiditis and 5 in follicular adenoma. Using immunohistochemical methods, we probed 205 samples with monoclonal anti-NIS Ab. Grading of staining was scored as 0 (negative or absent), 1 (weakly positive), 2 (moderately positive) or 3 (strongly positive). Expression rate (ER) of NIS positivity in individual disease entity was expressed as percentage of total number divided by number in 2 plus 3 grade. RESULTS: ERs of malignant thyroid diseases were 63% in papillary carcinoma, 81% in metastatic lymph node, 71% in follicular carcinoma and 100% in medullary carcinoma. ERs of benign thyroid disease were 53% in goiter/cyst, 64% in thyroiditis and 40% in follicular adenoma. ER of malignant thyroid diseases was higher than benign thyroid diseases (71% vs 54%). Grading of NIS expression in papillary carcinoma or goiter/cyst was heterogeneously distributed in considerable cases. Normal tissue also showed heterogeneous distribution of NIS expression, which was not correlated with that of primary lesion. CONCLUSION: In papillary thyroid carcinoma, distribution of NIS expression was heterogeneous and increased, and not different compared with that of benign thyroid disease.

zVAD-fmk, unlike BocD-fmk, does not inhibit caspase-6 acting on 14-3-3/Bad pathway in apoptosis of p815 mastocytoma cells

In a preliminary study, we found that benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD- fmk), unlike Boc-aspartyl(OMe)-fluoromethylketone (BocD-fmk), at usual dosage could not prevent genistein-induced apoptosis of p815 mastocytoma cells. This study was undertaken to reveal the mechanism underlying the incapability of zVAD-fmk in preventing this type of apoptosis. We observed that 14-3-3 protein level was reduced in genistein-treated cells and that BocD-fmk but not zVAD-fmk prevented the reduction of 14-3-3 protein level and the release of Bad from 14-3-3. We also demonstrated that truncated Bad to Bcl-xL interaction in genistein- treated cells was prevented by BocD-fmk but not by zVAD-fmk treatment. Our data indicate that BocD- fmk, compared to zVAD-fmk, has a certain preference for inhibiting 14-3-3/Bad signalling pathway. We also elucidated that this differential efficacy of BocD-fmk and zVAD-fmk resulted from the different effect in inhibiting caspase-6 and that co-treatment of zVAD-fmk and caspase-6 specific inhibitor substantially prevented genistein-induced apoptosis. Our data shows that caspase-6 plays a role on Bad/14-3-3 pathway in genistein-induced apoptosis of p815 cells, and that the usual dose of zVAD-fmk, in contrast to BocD-fmk, did not prevent caspase-6 acting on 14-3-3/Bad-mediated event.

Ad-TRAIL Induces Apoptosis in Chondrocytes in Vitro and Neutralizing Antibody to TRAIL Prevents The Induction of Apoptosis in Vitro and in Vivo / 대한해부학회지

We undertook this study to investigate whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)implicates in the pathogenesis of osteoarthritis (OA). Expression of TRAIL, and death and decoy receptors was examined on the primary cultured chondrocytes and the cartilage obtained from experimentally-induced OA rat model by immunohistochemistry. In vitro and in vivo blocking experiments with anti-TRAIL neutralizing antibody were conducted, and the prevention of cell death was determined by TUNEL assay. Ad-TRAIL infection induced expression of TRAIL and increased expression of death receptor DR4, and decreased expression of DR5 and DcR1 in primary cultured rat chondrocytes. Ad-TRAIL, at 10 and 100 MOI doses, decreased viability of chondrocytes on 4 days after infection. The cartilage obtained from chemically induced OA rat model showed the increased expression of TRAIL and DR4, and the decreased expression of DR5 and DcR1. Whereas surgically induced OA cartilage in vivo showed the increased expression of TRAIL, and DR4 &5 and the decreased expression of DcR1, compared to the control cartilage. In vitro blocking experiment prevented partially Ad-TRAIL induced death of chondrocytes. Furthermore, in vivo blocking experiment also partially prevented apoptosis in two in vivo OA models. In conclusion, TRAIL-induced chondrocyte apoptosis may play a role in pathogenesis of osteoarthritis.

Antitumor Activity of TRAIL Recombinant Adenovirus in Human Malignant Glioma Cells

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.

Characteristics of Mitochondrial Events in Synthetic Bile Acids-induced Apoptosis of Human Osteosarcoma Cells / 체질인류학회지

ABSTRACT: Apoptosis of osteosarcoma cells induced by bile duct derivates, HS-1200 was investigated with relation to mitochodria. HS-1200 induced cytochrome c and Smac/DIABLO release from mitochondria which are major factors related to apoptosis. In these apoptosis processes, release of cytochrome c was not blocked by caspase inhibitor, but release of Smac/DIABLO was blocked. BKA, a kind of PTP (permeablity transition pore) inhibitor, did not block both of them. Interestingly, the alteration of MMP was not observed by means of using JC-1 dye. Although MitoTracker, DiOC-6 and Rhodamine123 were used to confirm previous results, the decrease of MMP was not observed. In order to investigate whether this phenomenon is apoptosis-specific or cell-specific process, genistein was added to cells which usually decreased MMP. After adding genistein, MMP was not decreased, suggesting this phenomenon is cell-specific process. Conclusionally, HS-1200 induced apoptosis of osteosarcoma cells via mitochondria, cytochrome c and Smac/DIABLO were released from mitochondria without decrease of MMP. The release of Smac/DIABLO was dependent of caspase.

Synthetic Bile Acid Derivative HS-1200-induced Apoptosis of Human Osteosarcoma Cells / 대한해부학회지

Bile acids and synthetic its derivatives induced apoptosis in various kinds of cancer cells and had anticancer effects. However, it wasn`t discovered those materials have apoptosis induced effects on osteosarcoma cells. The present study was done to examine the synthetic bile acid derivatives induced apoptosis on osteosarcoma cells and such these apoptosis events. The synthetic bile acid derivatives, chenodeoxycholic acid (CDCA) induced the cell death on human osteosarcoma (HOS) cells contrary to ursodeoxycholic acid (UDCA). HS-1200, a synthetic derivative of CDCAs, was chosen to experiment apoptosis events in HOS cells. HOS cells treated with HS-1200 showed nucleus condensation, cytochrom c release, Bax/Bcl-xL alteration, activation of caspase-3 and caspase-activated deoxyribonuclease (CAD), and degradation of poly (ADP-ribose) polymerase (PARP). Though this study needs more investigations, these in vitro data suggest that treatment of the synthetic bile acid derivatives can give medical therapy on HOS cells.

A Novel Chenodeoxycholic Derivative HS-1200 Induces Apoptosis in Human HT-29 Colon Cancer Cells / 대한방사선종양학회지

PURPOSE: To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). MATERIALS AND METHODS: Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a trypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. RESULTS: The dose required for the half maximal inhibition (IC50) of the HT-29 cell growth was 100~150 micro M of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with 100 micro M of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential (deltaxm) was also prominently decreased in the HS-1200 treated cells. CONCLUSION: These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

Mast Cell Apoptosis Induced by Cyclosporin A / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Mast cell is a key cell in the pathogenesis of allergic rhinitis. It is expected that apoptosis of mast cell is an important step that can lead to treatment of allergic rhinitis. Meanwhile, the cyclosporin A (CsA) is immunosuppresant agent to inhibit the action of various immune cells. The purpose of this study is to identify whether CsA directly induces apoptosis of mast cells in vitro. MATERIALS AND METHOD: After the culture of p815 cells, mouse mastocytoma cells, the cells were treated with 1 micrometer, 2 micrometer, 5 micrometer, and 10 micrometer CsA, and then LD50 of p815 cells were calculated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. For identification of apoptosis of p815 cells, electrophoresis and flow cytometry after CsA treatment were done and morphological changes in light microscope was observed. We also quantified apoptotic cells by TUNEL assay and Hoechst stain. RESULTS: The LD50 of p815 cells is 9.87 micrometer after CsA treatment during 24 hours, 6.11 micrometer during 48 hours and 6.21 micrometer during 72 hours. With the higher concentration of CsA treatment, the greater effect on apoptosis of p815 cells was revealed. We observed laddering pattern for DNA fragmentation in electrophoresis. Nuclear fragmentation and chromatin condensation of p815 cells was observed under the light microscope. Results of flow cytometry were similar to the MTT assay results. Quantification of apoptotic p185 cells by TUNEL assay and Hoechst stain were calculated. CONCLUSION: Apoptosis of mast cells can be induced by CsA treatment in vitro.

The Effect of ECMs on MHC Class II Molecule Expression in Retinal Pigment Epithelium

Expression of MHC class II molecule by retinal pigment epithelial cells and the interaction of the cell with extracellular matrix molecules involve in the pathogenesis of proliferative vitreoretinopathy and chorioretinitis. In this study interferon-gammainduced the expression of MHC class II molecules on RPEJ cells. Extracellular matrix molecules increased the interferon-gamma-i n d u c e d expression of MHC class II molecules. TGF-beta2 inhibited the interferon-gammainduced expression of MHC class II molecules. However, there was no significant effect on such inhibitory function according to the types of extracellular matrix molecules. Blocking the autocrine effect of TGF-beta2 by the specific antisense oligonucleotides decreased its inhibitory function. PLC-gamma1-specific antisense oligonucleotide inhibited the effect of TGF-beta2, which suggests that PLC-gamma1 involves in the signal transduction of TGF-beta2 on the expression of MHC class II molecules. In conclusion, the present study provides further understandings to the previous knowledge of pathogenesis of immunologic retinal disorders.

The Role of CD44 in the Adhesion of Human Osteosarcoma to Hyaluronic Acid / 체질인류학회지

Osteoblast expresses a sequence of extracellular matrix during differentiation, suggest that multiple adhesion mechanisms regulate osteoblast differentiation and bone development. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) which mainly distribute in cartilage and extracellular matrix (ECM). HA has very high molecular weights and their structures occupy large solvent domains which give solution of high viscosity. During early development and before tissue differentiation, HA can constitute the major structural macromolecule in the ECM, where it can promote both cell proliferation and migration. CD44 is a cell surface receptor for HA. The polymorphic family of integral membrane glycoproteins CD44 is found on a wide variety of cells. CD44's function in the cell membrane is transmembrane signalling between extracellular matrix and acin filament. In present study, HOS was used as a model to examine whether HOS adhere to HA, CD44 and GAGs on cell surface participate in the adhesion to HA and there is difference in CD44 expression at that time. HOS adhered to HA in the manner of time -dependent. After incubation for 180 minutes, about 90% of cells adhered to HA. When HOS was pretreated with anti -CD44 antibody, hyaluronidase and genistein, the adhesion rate was significantly decreased. CD44 was more expressed in HOS plated on HA -coated wells than BSA -coated wells. Taken together, HOS has a adhesive affinity to HA. CD44, GAGs on cell surface and tyrosine kinases play an important role in the adhesion of HOS to HA.

Immunohistochemical Characterization on the Effect of Immunomodulating Factor (IMF) from Actinobacillus actinomycetemcomitans on Dendritic Cells, Lymphocytes and Macrophages in the Mouse Spleen / 대한해부학회지

This study was performed to investigate the effects of Immunomodulating factor (IMF), derived from Actinobacillus actinomycetemcomitans, on various immune cells in the mouse spleen. A single dose of IMF (10 microgram/kg) was administer-ed to BALB/c mice by intraperitoneal injection. After the mice were sacrificed in groups of five at 6 h and 24 h, the spleens were removed. The immunocytochemical characterization of the immune cells was carried out using the various monoclonal antibodies in cryostat-cut sections. We demonstrated in this study a strong stimulating effect of IMF on dendritic cells and B lymphocytes in the mouse spleen after IMF administration. The MOMA-1(+) immunoreactivity on the marginal metallophilic macrophages in the splenic marginal zone disappeared 6 h and reappeared 24 h after IMF treatment. However, various subpopulations of T lymphocytes, CD3(+), CD4(+), CD8(+), TCRalpha, beta(+) and Vbeta8(+) T cells in the mouse spleen did not show any significant change in their distributional pattern after IMF treatment. Dendritic cells were found to be increased in number in the periarterial lymphatitc sheath, and B lymphocytes were also increased in number in the lymphoid follicles of the spleen after IMF injection. In conclusion, IMF exhibited a potent stimulative effect on dendritic cells and B lymphocytes in vivo.

Immunohistochemical Study of Immune Cells, with a Special Emphasis on Macrophage Subpopulations in the Rat Spleen after Cyclophosphamide Treatment / 대한해부학회지

This study was undertaken to investigate the in vivo effects of cyclophosphamide (CY) on subpopulations of macrophages and other types of immune cells including dendritic cells (DCs) as well as on ICAM-1 expression in the spleen of rats. After a single dose of CY (150 mg/kg) was administered to Sprague-Dawley rats by intraperitoneal injection, the rats were sacrificed at 1, 3, 7, 14 and 28 days. The immunocytochemical characterization of the tissues were carried out using the monoclonal antibodies W3/25, OX8, HIS24, 8A2, OX6, OX62, ED1, ED2, ED3, and TLD-4C9 for analysis of macrophage subpopulations, DC(s), CD4(+) and CD8(+) T cells, B cells and ICAM-1 expression in cryostat-cut sections. CY exhibited a profound immunosuppressive effect on CD4(+) and CD8(+) T cells as well as B cells as was expected. However, it was found that CY induced an increase in number of certain subpopulations of macrophages, including ED1(+), ED2(+) and ED3(+) macrophages. Contrarily, CY elicited a decrease in number of DCs. CY induced a conspi-cuous upregulation of ICAM-1 on certain populations of leukocytes. This increased expression of ICAM-1 after CY treatment appears to be related with the recruitment of certain populations of leukocytes. Most of these features began to appear from the first day and reached the maximun on the third and especially, the seventh day, but two weeks after CY administration, these phenomena declined. In conclusion, the present study provided a new insight into the differential effects of CY on various populations and subpopulations of immune cells in the rat spleen.

Change of Surface Carbohyd rate during Trans differentiation of Retinal Pigment Epithelial Cell

This study was conducted to study the changes of cell surface carbohydrates during transdifferentiation of retinal pigment epithelial(RPE)cells. RPE cells were cultured from adult pig eyes. Surface carbohydrates of RPE cells from 1st, 3rd, 5th, 7th and 9th passages were assayed by lectin histochemistry and enzyme immunoassay. Changes in binding affinities to the lectins employed were demonsrated during trasdifferentiation of RPE cell. Whereas binding affinities of ConA, ECL, PNA, WGA, and UEA-I decreased graudally as the number of culture passage increased, binding properties to LCA, STL and DBA fluctuated depending on the number of passages. The results demonstrate changes of surface carbohydrates of RPE cell during trasdifferentiation. We suggest that changes of surface carbohydrates of RPE cell during trasdifferentiation may be close relations with the functional changes during transdifferentiation.