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.