Gene expression profiling in undifferentiated thyroid carcinoma induced by high-dose radiation

Abstract

Published gene expression studies for radiation-induced thyroid carcinogenesis have used various methodologies. In this study, we identified differential gene expression in a human thyroid epithelial cell line after exposure to high-dose γ-radiation. HTori-3 cells were exposed to 5 or 10 Gy of ionizing radiation using two dose rates (high-dose rate: 4.68 Gy/min, and low-dose rate: 40 mGy/h) and then implanted into the backs of BALB/c nude mice after 4 (10 Gy) or 5 weeks (5 Gy). Decreases in cell viability, increases in giant cell frequency, anchorage-independent growth in vitro, and tumorigenicity in vivo were observed. Particularly, the cells irradiated with 5 Gy at the high-dose rate or 10 Gy at the low-dose rate demonstrated more prominent tumorigenicity. Gene expression profiling was analyzed via microarray. Numerous genes that were significantly altered by a fold-change of >50% following irradiation were identified in each group. Gene expression analysis identified six commonly misregulated genes, including CRYAB, IL-18, ZNF845, CYP24A1, OR4N4 and VN1R4, at all doses. These genes involve apoptosis, the immune response, regulation of transcription, and receptor signaling pathways. Overall, the altered genes in high-dose rate (HDR) 5 Gy and low-dose rate (LDR) 10 Gy were more than those of LDR 5 Gy and HDR 10 Gy. Thus, we investigated genes associated with aggressive tumor development using the two dosage treatments. In this study, the identified gene expression profiles reflect the molecular response following high doses of external radiation exposure and may provide helpful information about radiation-induced thyroid tumors in the high-dose range.

Keywords: carcinogenesis; cell line; gene expression; radiation; thyroid gland.

Fig. 1.

Schematic representation of irradiation protocols. In case of low-dose-rate irradiation, HTori-3 cells were irradiated at 40 mGy/h for 125 h (5 Gy) or 250 h (10 Gy). A high-dose-rate radiation group was irradiated at 4.68 Gy/min for 64 s (5 Gy) or 128 s (10 Gy) (the solid line represents the radiation-irradiated period; the dotted line represents non-irradiated period).

Fig. 2.

Time- and dose-dependent effect of γ-radiation on HTori-3 cell viability. HTori-3 cells were exposed to various doses of γ-radiation (at 4.68 Gy/min), and cell viability was determined by MTT assay at the indicated times. The data shown represent three independent experiments, with standard error bars indicated. *P < 0.05 and **P < 0.01.

Fig. 3.

Alterations in the morphology of HTori-3 cells after radiation exposure. HTori-3 cells were exposed to low-dose-rate (40 mGy/h) or high-dose-rate (4.68 Gy/min) radiation. After 5 weeks, the cells were observed and photographed under the inverted microscope. Arrows point to multinucleated giant cells with cytoplasmic vacuoles.

Fig. 4.

Anchorage-independent cell growth in irradiated HTori-3 cells. A colony-forming assay was used to detect the anchorage-independent growth of transformed cells from the various experimental groups. HTori-3 cells were exposed to low-dose-rate (40 mGy/h) or high-dose-rate (4.68 Gy/min) radiation. After 8 weeks, cells were seeded into 6-well plates in duplicate (1 × 10³ cells per well) and incubated for 4 weeks. The resulting colonies were stained with nitroblue tetrazolium. The data shown represent three independent experiments, with standard error bars indicated. *P < 0.0001, using Student's t-test on the differences between control and irradiated groups.

Fig. 5.

Tumorigenicity of HTori-3 cells in nude mice. (A) Representative photographs of tumor formation in nude mice. Arrows indicate location of tumors. Tumor size was monitored once per week. (B) The tumor incidence rate in each group after 16 weeks: low-dose-rate (40 mGy/h) 5 Gy, 20.24%; high-dose-rate (4.68 Gy/min) 5 Gy, 24.39%; low-dose-rate 10 Gy, 33.78%; and high-dose-rate 10 Gy, 30.88%.

Fig. 6.

Representative histologic image of tumor tissues from xenografts. (A) Adipose tissue invasion, (B) necrosis, (C) mitotic structures, (D) giant cell (multinucleated) and (E) apoptotic body. Scale bars, 300 µm (A, B) and 100 µm (C–E). Insets show malignant tumor-like morphology in enlarged images.

Fig. 7.

Identification of radiosensitivity genes in γ-radiation–induced tumor cells. Six genes were commonly up- and downexpressed in tumor cells of all groups. IL-18, ZNF845 and CRYAB were upregulated in radiation-induced tumor cells compared with control, whereas CYP24A1, OR4N4 and VN1R4 were downregulated in tumor cells compared with the control.