Expression of TIMP1, TIMP2 Genes by Ionizing Radiation / 대한방사선종양학회지

PURPOSE: Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. MATERIALS AND METHODS: Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity (D0), radiation cytotoxicity and metastatic potential were measured by clonogenic assay, MTT assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promotor region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was performed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promotor. RESULTS: D0 of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. MTT assay confirmed cell viability, over 94% at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. CONCLUSIONS: We observed the difference of expresson and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.

Enhanced Effect of Iodide - Uptake in Thyroid Carcinoma Cells by Infecting Adenovirus - Human Sodium Iodide Symporter (Ad - hNIS) / 대한내분비학회지

BACKGROUND: The sodium-iodide-symporter (NIS) is a plasma membrane glycoprotein with 13 putative transmembrane domains, which is responsible for concentrating iodide into the thyroid by an active transport and provides the mechanism for radioactive-iodine (RAI) therapy for thyroid cancer. However, undifferentiated thyroid cancers and about 2050% of differentiated thyroid cancers do not take up the RAI at therapeutic dose. The NIS has been cloned from rat and human (hNIS) and characterized recently. In an attempt to develop a new therapeutic strategy using hNIS gene for improving the efficacy of RAI therapy in thyroid cancers, we have constructed a recombinant adenovirus encoding the hNIS (Ad-hNIS) and tested its function by an iodide uptake by infecting human thyroid cancer cells. METHODS: RT-PCR was performed to measure an intrinsic hNIS expression in thyroid cancer cell lines, such as NPA, FRO and ARO. To generate the hNIS adenovirus, hNIS cDNA was isolated and ligated into Swa I site of cosmid shuttle vector (pAxCAwt). We have produced recombinant adenovirus by co-transfecting the cosmid with DNA-TPC to 293 cell line. Adenovirus that express (beta-Galactosidase (LacZ) was also prepared by the similar strategy. Adenovirus infection efficiency was measured in three thyroid cancer cell lines. Finally, 24 hours after infection of ad-hNIS into the cells, I125-uptake was measured. RESULTS: Endogenous hNIS expression was detected only in FRO cells but not in NPA, ARO and Hela cells by RT-PCR. X-Gal staining after infection of Ad-LacZ to thyroid cancer cell (NPA, ARO, FRO) showed that an infection rate in ARO cells was 98.5+0.5%, 97.0+0.2% in FRO cells and 75.5+5.0% in NPA cells. We selected ARO cells for the infection of Ad-hNIS due to the highest infection efficiency and the absence of endogenous hNIS expression. When ARO cells were infected with the ad-hNIS, I125 uptake was increased 504+6.4%. CONCLUSION: Overexpression of hNIS gene in thyroid cancer cells elicited over 5 fold increase in I-uptake, suggesting that the Ad-hNIS infection to the thyroid cancer cells may improve the efficiency of radioactive iodine therapy.

Inhibition of Interleukin-1 Signal by Annexin-1 in Phorbol Myristate Acetate Stimulated Lymphocytes / 대한면역학회지

Annexin-1 (ANX1) is a 37 kDa protein that is induced and secreted by glucocorticosteroid hormone. The secreted ANX1 has been believed to exert its function by binding to its putative rnembrane receptor. In this report we demonstrate that ANXl receptor (ANX1R) signal blocks the interleukin-1B (IL-1B) receptor signal pathway in human peripheral blood mononuclear cells (PBMCs). When PBMCs were treated with both IL-1B (100 ng/ml) and PMA (10 ng/ml) in the absence or presence of dexamethasone for 5 days, dexamethasone (100 nM) suppressed lymphocyte proliferation to 24% of the control. However addition of anti-ANX1 polyclonal antibody of 1:200 and 1:1,000 dilution to this system induced recovery of proliferation to 80% and 40%, respectively, when compared to the control. In the mixed lymphocyte reaction, dexamethasone suppressed lymphocyte proliferation to 9% of that of control when stimulated with IL-1B (100 ng/ml) and phorbol myristate acetate (10 ng/ml). Addition of anti-ANX1 polyclonal antibody (1:1,000) to this system also recovered the proliferation to 20% of that of the control system. In the ANX1 receptor induction experiment using flow cytometry, ANX1 receptor expression on lymphocytes, CD4+ T cells, CD8+ T cells and monocytes increased depending on the externally added IL-1B ranging from 10 to 1,000 ng/ml. From these results, it is evident that dexamethasone induces ANX1 secretion into the culture medium and anti-ANX1 polyclonal antibody abolishes the effects of dexamethasone. Furthermore these results imply that extracellular ANX1 exerts its effects by binding to the receptor on the cell membrane and the activated signal(s) of ANX1R block IL-1B receptor signal in the lymphocytes.

Effect of Protein Kinase C Inhibitor (PKCI) on Radiation Sensitivity and c-fos Transcription Activity / 대한방사선종양학회지

PURPOSE: The human genetic disorder ataxia-telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. The recent identification of the gene mutated in AT, ATM, and the demonstration that it encodes a homologous domain of phosphatidylinositol 3-kinase (PI3-K), the catalytic subunit of an enzyme involved in transmitting signals from the cell surface to the nucleus, provide support for a role of this gene in signal transduction. Although ionizing radiation was known to induce c-fos transcription, nothing is known about how ATM or PKCI mediated signal transduction pathway modulates the c-fos gene transcription and gene expression. Here we have studied the effect of PKCI on radiation sensitivity and c-fos transcription in normal and AT cells. MATERIALS AND METHODS: Normal (LM217) and AT (AT5BIVA) cells were transfected with PKCI expression plasmid and the overexpression and integration of PKCI was evaluated by northern blotting and polymerase chain reaction, respectively. 5 Gy of radiation was exposed to LM and AT cells transfected with PKCI expression plasmid and cells were harvested 48 hours after radiation and investigated apoptosis with TUNEL method. The c-fos transcription activity was studied by performing CAT assay of reporter gene after transfection of c-fos CAT plasmid into AT and LM cells. RESULTS: Our results demonstrate for the first time a role of PKCI on the radiation sensitivity and c-fos expression in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells but reduced apoptosis in AT cells. The basal c-fos transcription activity is 70 times lower in AT cells than that in LM cells. The c-fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells CONCLUSION: Overexpression of PKCI increased radiation sensitivity and repressed c-fos transcription in LM cells but not in AT cells. The results may be a reason of increased radiation sensitivity of AT cells. PKCI may be involved in an ionizing radiation induced signal transduction pathway responsible for radiation sensitivity and c-fos transcription. The data also provided evidence for novel transcriptional difference between LM and AT cells.

EFFECTS OF EXTRACTS OF DRYNARIAE RHIZOMA ON THE CHARACTERISTICS OF RAT CALARIA AND BONE MARROW CELLS / 대한치주과학회지

This study was performed to evaluate the effects of extracts of Drynariae Rhizoma on the characteristics of rat calvaria cells(RCV) and bone marrow cells(RBM) which have the important role on the bone formation in vitro. Drynariae Rhizoma has been known as the useful herbal medicament for treatment of the wound healing including regeneration of bone fracture, and also has been used to treat the periodontal lesions, tooth mobility, gingival bleeding and pus discharge via sulcus in Oriental Medicine. In control group, the cells were cultured alone with Dulbeco's Modified Eagle's Medium contained with 10% fetal bovine serum, 100U/ml penicillin, 100microgram/ml streptomycin, 0.5microgram/ml amphotericin-B. In experimental group, extracts of Drynariae Rhizoma(0.1, 1, 5, 10, 50microgram/ml) were added into the above culture condition. And then each group was characterized by examing the cell proliferation at 1, 3, 7, 14, 21, 30th day, the amount of total protein synthesis and alkaline phosphatase activity of RCV at 2,4th day and those of RBM at 3, 6th day. And also, the calcified nodule of RCV was examed at 3, 5th day in three goup, control, experimental, culture with the PDGF group. The results were as follow; 1. Both RCV and RBM cells in Drynariae Rhizoma-treated experimental group proliferated more rapidly than non-treated control group. The experimental group below 5microgram/ml Drynariae Rhizoma-treated showed more prominent cell proliferation from the 7th day to the 21st day than the control group and above 10 microgram/ml treated group in RCV. 2. Amount of total protein synthesis was more increased in Drynariae Rhizoma-treated group than in control group. In 5microgram/ml Drynariae Rhizoma-treated group showed most prominent protein synthesis of the any other exrperimental group and control group. 3. Alkaline phosphatase activity also more increased in Drynariae Rhizoma-treated group than control group.

Detection of Antibodies against Shope Fibroma Virus and Formation of Fibroma in the Korean Domestic Rabbits

In our preliminary study to find antiviral or antitumor agents from Korean natural products, we found that the Shope fibroma virus (SFV) induced fibromas reaching maximum size at 5~6 days with spontaneous disappearance at 15~20 days after SFV intracutaneous inoculation into Korean domestic rabbits. However, the sizes of fibromas of rabbits at day 5 after virus inoculation were significantly different individually. Assuming that the variation of tumor size was due to either susceptibility or the preexisting antibodies against SFV in the Korean domestic rabbits, the rabbits were checked for the antibodies against SFV by IFAT using SFV infected RKl3 cells. The antibody positive rate of normal Korean domestic rabbits was 32.8% and the sizes of the fibromas of the positive rabbits were significantly smaller than those of negative rabbits (p<0.0001). The fibroma sizes were dependent on the antibody titers of rabbits to SFV. The sizes of fibromas after inoculation of SFV into immunized rabbits were about one tenth of those by the first inoculation into normal rabbits. This is the first report on the antibody prevalence against SFV among normal Korean domestic rabbits and it suggest the existence of a wild fibroma virus or related virus in Korea.