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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1811
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2364-2369
Copyright © 2005 by The Endocrine Society

Dose-Dependent Generation of RET/PTC in Human Thyroid Cells after in Vitro Exposure to {gamma}-Radiation: A Model of Carcinogenic Chromosomal Rearrangement Induced by Ionizing Radiation

Christy M. Caudill, Zhaowen Zhu, Raffaele Ciampi, James R. Stringer and Yuri E. Nikiforov

Departments of Pathology and Laboratory Medicine (C.M.C., Z.Z., R.C., Y.E.N.) and Molecular Genetics (J.R.S.), University of Cincinnati, Cincinnati, Ohio 45267-0529

Address all correspondence and requests for reprints to: Dr. Yuri Nikiforov, Department of Pathology, University of Cincinnati, 231 Albert Sabin Way, P.O. Box 670529, Cincinnati, Ohio 45267-0529. E-mail: Yuri.Nikiforov{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ionizing radiation is a well-known risk factor for thyroid cancer in human populations. Chromosomal rearrangements involving the RET gene, known as RET/PTC, are prevalent in thyroid papillary carcinomas from patients with radiation history. We studied the generation of RET/PTC in HTori-3 immortalized human thyroid cells exposed to a range of doses of {gamma}-radiation and harvested 2, 5–6, and 9 d later. RET/PTC1 and RET/PTC3 were detected by RT-PCR followed by Southern blotting and hybridization with internal oligonucleotide probes. No RET/PTC was found in cells harvested 2 and 5–6 d after irradiation, whereas 59 RET/PTC events were detected in cells collected 9 d after exposure. The average rate of RET/PTC induction was 0.1 x 10–6 after exposure to 0.1 Gy, 1.6 x 10–6 after 1 Gy, 3.0 x 10–6 after 5 Gy, and 0.9 x 10–6 after 10 Gy. When adjusted for cell survival, the rate after 10 Gy was comparable with those after 5 Gy. RET/PTC1 was more common than RET/PTC3 after each dose, comprising 80% of all rearrangements. In this study, we demonstrate a dose-dependent induction of RET/PTC rearrangements in human thyroid cells after exposure to 0.1–10 Gy {gamma}-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE TO IONIZING radiation is a well-established risk factor for thyroid cancer. The association was first suggested more than 50 yr ago in children who received x-ray therapy in infancy for an enlarged thymus (1). During the following decades, numerous reports have documented an increased incidence of thyroid neoplasms in children after external radiation for different benign conditions of the head, neck, and thorax (2). An increased risk of thyroid cancer has also been linked to environmental irradiation and documented in survivors of atomic bomb explosions in Japan in 1945 (3), in residents of the Marshall Islands exposed to radioactive fallout in 1954 (4), and more recently in children exposed to radiation after the Chernobyl accident (5, 6, 7). The accident released unprecedented amounts of radioactive materials into the atmosphere (8). More than 10 million people were exposed to significant levels of radiation, and the thyroid dose was 3–10 times higher in children than in adults. In the most contaminated areas of southern Belarus, estimated thyroid doses in children were among the highest, ranging between less than 0.05 and 4 Gy (8). An increase in thyroid cancer incidence in children was noted in Belarus as early as 4 yr after the accident and reached almost 30-fold (100-fold in most contaminated Gomel region) in 1995 compared with those before Chernobyl and in other countries (5, 6). The risk of thyroid cancer correlated with proximity to Chernobyl and thyroid dose and in some areas was found to be linear in the dose interval 0.07–1.2 Gy (9).

Over the last few decades, significant progress has been achieved in the understanding of the biological mechanisms of radiation carcinogenesis. It has been shown that damage to cellular DNA is primarily responsible for mutagenesis and carcinogenesis and that double-strand breaks appear to be most important in the direct generation of mutations (10, 11). Incorrect end-joining of breaks causes mutations immediately, but it can also lead to genomic instability caused by cycles of chromatid fusion and breakage. More recently, evidence has emerged that, in addition to mutations arising directly from misrepaired DNA damage, genetic consequences may be related to the persistence of genomic instability or may affect cells in proximity to those exposed to radiation (bystander effect) (12, 13).

Relatively limited information, however, is available on the specific genetic alterations underlying cell transformation after radiation exposure. In this respect, thyroid cancer in children after the Chernobyl accident represents the most thoroughly studied population. In contrast to sporadic papillary carcinomas, which commonly harbor BRAF point mutations (14, 15, 16), post-Chernobyl tumors have a high prevalence of chromosomal rearrangements involving the RET gene, known as RET/PTC. Of several types of RET/PTC described, RET/PTC1 and RET/PTC3 are the most common, comprising more than 90% of all rearrangements found in sporadic and radiation-induced tumors. RET/PTC1 is formed by RET fusion to the H4 (also known as D10S170) gene (17, 18) and RET/PTC3 by fusion to the ELE1 (RFG or ARA70) gene (19, 20). RET/PTC1 and RET/PTC3 are paracentric inversions, inv(10)(q11.2;q21) and inv(10)(q11.2), respectively, because both genes participating in each rearrangement are located on chromosome 10q (21). In contrast, RET/PTC2 and other rare forms of RET/PTC are translocations resulting from the fusion of the intracellular domain of RET to heterologous genes located on different chromosomes (reviewed in Refs. 22 and 23).

The prevalence of RET/PTC rearrangement is approximately 20–30% in sporadic thyroid papillary carcinomas and is significantly higher in tumors from patients exposed to radiation. Indeed, in children exposed to radiation after the Chernobyl accident, RET/PTC was detected in 57–87% of tumors removed 5–8 yr after exposure and in 49–65% of tumors diagnosed 7–11 yr after the accident (24, 25, 26, 27, 28, 29). A high prevalence of RET/PTC was also found in patients subjected to therapeutic x-ray irradiation for benign or malignant conditions (30, 31), providing additional evidence for the causal association between radiation exposure and generation of this rearrangement.

The occurrence of RET/PTC rearrangement has been studied in thyroid cells after in vitro radiation. RET/PTC was reported after high-dose radiation to human undifferentiated thyroid carcinoma cells (32) and to fetal human thyroid tissues transplanted into SCID mice (33, 34). In both studies, the rearrangement was detected by RT-PCR as early as 2 d after exposure to 50 Gy, a dose that is lethal for dividing cells. In addition, they employed either grafted fragments of fetal thyroid tissue, which are poorly amendable for experimental manipulations, or highly transformed, undifferentiated cells that are more susceptible to develop secondary genetic defects.

In this study, we aimed to establish and characterize a well-controlled model of induction of RET/PTC rearrangements in differentiated human thyroid cells after exposure to {gamma}-radiation. This was achieved by subjecting HTori-3 cells to a range of doses implicated in the development of thyroid cancer in human populations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell line and culture conditions

The experiments were performed on HTori-3 cells, which are human thyroid epithelial cells transfected with an origin-defective SV40 genome (35). They have been characterized as immortalized, partially transformed, differentiated cells that preserved the iodine-trapping ability and thyroglobulin (Tg) production. The cells were purchased from the European Tissue Culture Collection and grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum.

Chromosome analysis and fluorescence in situ hybridization (FISH)

Chromosomes were prepared and G-banded by standard procedures (36). Twenty-five metaphases were counted. For FISH, cells were grown directly on glass slides, fixed, and subjected to hybridization as previously described (37). A 207-bp bacterial artificial chromosome clone (RPC11-351D16, CHORI), spanning the entire RET gene region, was used as a probe. Bacterial artificial chromosome DNA was extracted and labeled with SpectrumGreen-dUTP (Vysis Inc.; Downers Grove, IL) by nick translation. Nuclei were counterstained with 4',6-diamidino-2-phenylindole. Microscopy was performed with a Leica TCS 4D confocal laser scanning fluorescence microscope with digital image capture (Leica, Wetzlar, Germany).

Expression of Tg and sodium/iodide symporter (NIS)

Expression of the Tg and NIS genes was tested by RT-PCR. Total RNA was extracted from HTori-3 cells and frozen normal thyroid tissue using Trizol reagent (Invitrogen). Three micrograms of total RNA were reverse transcribed in a volume of 20-µl reactions by using random hexamers priming and Superscript II RT (Invitrogen). The following exon-spanning primers were used: 5'-CCTCGCAGTTCAATCAGTCA-3' (Tg forward), 5-TGGCTGAAGTAGCCTGAGGT-3' (Tg reverse), 5'-CTCCCTGCTAACGACTCCAG-3' (NIS forward), and 5'-GAGGTCCCACCACAACAATC-3' (NIS reverse). For each PCR, 2 µl RT mixture was amplified in a final volume of 30 µl using 35 cycles of denaturation (94 C for 40 sec), annealing (59 C, 1 min for NIS; 57 C, 1 min for Tg), and extension (72 C for 1 min). cDNA from normal thyroid tissue was used as a positive control for gene expression. PCR products were electrophoresed in a 1.5% agarose gel and visualized by ethidium bromide staining. The expected size of the PCR products was 207 bp for NIS and 303 bp for Tg. Amplification of a 247-bp sequence of the 3-phosphoglycerate kinase (PGK) gene was achieved as described elsewhere (38) and served as a control for the quantity and quality of the extracted RNA.

Cell irradiation

In most experiments, 1.5–1.8 x 106 cells were plated in a 75-cm2 flask and 16 h later exposed to a single dose of {gamma}-irradiation from a cesium-137 source at a dose rate of 1.7 Gy/min. A larger number of cells (up to 4 x 106) was used for higher doses to compensate for cell killing. Cells were exposed to 0, 0.1, 1, 5, and 10 Gy {gamma}-irradiation, and at least four replicate plates were used per each dose. Four hours after irradiation, cells were split into six 12-well plates at a density of approximately 3 x 104 cells per well, grown for 2–9 d, and harvested. To sustain growth for 9 d, cells were transferred to 75-cm2 flasks 4–5 d after seeding into 12-well plates.

Detection of RET/PTC rearrangements

RNA was extracted from each well or flask using a Trisol reagent (Invitrogen). Then, mRNA was purified using the Oligotex mRNA minikit (QIAGEN, Valencia, CA). RT-PCR was performed using a Superscript first strand synthesis system kit and random hexamer priming (QIAGEN). PCR was performed to detect RET/PTC1 and RET/PTC3 rearrangement simultaneously in one tube, using the following oligonucleotide primers: 5'-CAAGAGAACAAGGTGCTGAAG-3' (RET/PTC1 forward), 5'-CGGTATTGTAGCTGTCCCTTTC-3' (RET/PTC3 forward), and 5'-GCAGGTCTCGAAGCTCACTC-3' (common reverse). PCR was carried out for 35 cycles. As a positive control and to test the sensitivity of the detection method, serial dilutions of the RET/PTC1-positive TPC-1 cells (39) and primary cultured papillary carcinoma cells from the RET/PTC3 positive tumor in nonirradiated HTori-3 cells were used. Ten microliters of each PCR product was electrophoresed in a 1.5% agarose gel, transferred to the nylon membrane, and hybridized with 32P-labeled oligonucleotide probes specific for RET/PTC1 (5'-CGTTACCATCGAGGATCCAAA-3') and RET/PTC3 (5'-GAACAGTCAGGAGGTCCAA-3') for 16 h at 42 C, washed, and exposed for autoradiography for 2–16 h. Evidence of RET/PTC rearrangement in the cells from a given flask was scored as one RET/PTC event.

Statistical analysis

The differences between the rate of RET/PTC induction after various doses were analyzed with one-way ANOVA, followed by t test for individual comparisons. The difference was considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The originally established HTori-3 cell line is known to possess multiple numerical and structural chromosomal abnormalities but to preserve thyroid differentiation qualities (35). We analyzed the HTori-3 cells growing in the lab by partial karyotyping and FISH with the RET probe to assure that they contained chromosomes 10 and the RET gene loci. Indeed, three intact copies of chromosome 10 were present in each cell, each containing a single, undisrupted RET locus (Fig. 1Go, left). The RT-PCR analysis revealed that these cells expressed Tg and NIS and, therefore, preserved the most specialized functions of differentiated thyroid cells (Fig. 1Go, right).



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  		FIG. 1. Characterization of HTori-3 cells used in the study. Left, FISH with RET probe (red) showing a metaphase spread (left) and an interphase cell (right) with three discrete signals. Right, RT-PCR analysis demonstrating the expression of thyroid differentiation genes Tg and NIS and control gene PGK in HTori-3 and normal thyroid cells. L 123 bp, 123-bp ladder; NC, negative control.

 
Survival of HTori-3 cells was evaluated after radiation doses ranging from 0–50 Gy. The number of surviving cells was calculated by counting detached cells and by trypan blue viability staining. The experiment was carried out in duplicate; the average cell survival was 93.4% after 0 Gy, 91.7% after 0.1 Gy, 90.7% after 1 Gy, 84% after 5 Gy, 22% after 10 Gy, and 0 after 50 Gy. Because 50 Gy radiation resulted in 100% cell death, a range of doses between 0 and 10 Gy was selected for further experiments.

Next, sensitivity of the detection of RET/PTC1 and RET/PTC3 rearrangements by RT-PCR and hybridization with specific probes were tested. Nonirradiated HTori-3 cells were mixed with variable numbers of cells carrying RET/PTC1 and RET/PTC3 rearrangement. The detection limit was one positive cell in 105 negative cells for both types of rearrangement (Fig. 2Go).



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  		FIG. 2. Detection of RET/PTC1 and RET/PTC3 rearrangements in serial dilutions of cell positive for the rearrangement in nonirradiated HT-ori3 cells negative for the rearrangement. The detection limit is 1:105 for both types of the rearrangement.

 
To assure that a cell with the rearrangement would be detected, the 2 x 106 cells in an irradiated flask were divided among 72 culture vessels 4 h postirradiation. Therefore, each well received no more than 3 x 104 cells, and if a well contained only one cell with RET/PTC, it would constitute 1 part in 3 x 104, a fraction within the limit of detection.

To determine the optimal time interval between irradiation and cell harvesting, cells were irradiated with 5 Gy and collected after either 0, 2, 5–6, or 9 d, which corresponded to zero, one, three, and five cell divisions, respectively. RET/PTC rearrangements were detected only in cells grown for 9 d after irradiation. Based on these data, the time interval of 9 d was selected for the main experiment.

In the main experiment, 16 flasks received no radiation (0 Gy), and four to five replicate flasks were subjected to a single dose of 0.1, 1, 5, and 10 Gy {gamma}-radiation. No RET/PTC rearrangement was detected after 0 Gy, indicating an extremely low level of spontaneous generation of RET/PTC in this human cell line. All other doses resulted in the generation of RET/PTC, with 59 events totally identified (Table 1Go). Representative RT-PCR blots are shown in Fig. 3Go. The number of RET/PTC events increased with dose over the range 0.1–5 Gy, with the highest number observed after 5 Gy radiation. A dose of 10 Gy resulted in a smaller absolute number of rearrangements detected, although when adjusted for cell survival, the rate of RET/PTC per survived cells was comparable with those at 5 Gy (Fig. 4Go). Statistical analysis for multiple comparisons revealed a significant variation in the mean rate of RET/PTC induction after different doses when calculated based on the number of irradiated cells (P = 0.002) and surviving cells (P = 0.02). Individual comparisons showed that the rate of RET/PTC in surviving cells was significantly different between 0 and 0.1 Gy (P = 0.009) and 0.1 and 1 Gy (P = 0.0004) but not between 1 and 5 Gy (P = 0.06). At all doses, RET/PTC1 was more common than RET/PTC3, with the ratio between the two types being at least 2:1.


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  		TABLE 1. Number of RET/PTC events and rate of RET/PTC per irradiated cells and surviving cells

 


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  		FIG. 3. Detection of RET/PTC rearrangements in representative experiments after 0, 1, and 5 Gy irradiation. PC, Positive control.

 


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  		FIG. 4. Upper panel, Cell survival; lower panel, rate of RET/PTC generation per irradiated cells (solid line) and surviving cells (dotted line) after 0–10 Gy irradiation. Each point represents the mean of four to five individual experiments (±SD).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report here the induction of RET/PTC rearrangements in differentiated human thyroid cells after in vitro exposure to {gamma}-radiation. RET/PTC was detected after 0.1 Gy, the smallest dose applied in this study, and the rate of detection increased proportionally from 0.1–1 Gy. A dose of 5 Gy produced the greatest number of rearrangements per surviving cell, but this number was roughly half that expected from the response at 1 Gy. Importantly, these doses were comparable with those received by the individuals who subsequently developed thyroid cancer in various human populations. Indeed, thyroid doses in the areas surrounding the Chernobyl accident site were in the range of 0.05–4 Gy (8). Thyroid cancer patients previously exposed to therapeutic or environmental radiation received between 0.09–12.5 Gy (40). In addition, in this study, the frequency of RET/PTC did not grow further when the dose was increased from 5–10 Gy. Therefore, the rate of RET/PTC in surviving cells reached plateau approximately at 5 Gy and then remained constant up to 10 Gy. This, again, parallels the human populations, where thyroid cancer risk associated with radiation exposure has a linear dose response for doses <10 Gy, whereas very high doses pose a low risk (40, 41).

These findings provide additional evidence for a direct link between RET/PTC rearrangement and radiation exposure. Moreover, the similarity in radiation doses and patterns of dose response, and the fact that the experiment employed human cells that preserved differentiated thyroid cell functions suggest that this in vitro model may serve as a valuable tool for studying the mechanisms of RET/PTC generation and radiation-induced carcinogenesis in human populations. The limitations of this experimental system include the usage of cultures cells which are immortalized and have significantly higher proliferation rate and the fact that the cells have three copies of the RET gene, which is expected to increase the probability of RET/PTC rearrangement compared with the diploid human cells.

In this study, RET/PTC rearrangement was identified only in cells grown for 9 d after irradiation, which corresponded to approximately five population doublings. There are two possible explanations for this phenomenon: RET/PTC was generated in a cell immediately after irradiation, but either chimeric mRNA was not expressed during the initial period or the detection method failed to detect the rearrangement in a single cell, and several cell divisions were necessary to generate a number of cells/mRNA copies sufficient for the detection; or RET/PTC was not a result of direct DNA damage and formed later, after several cell divisions. Although the experimental design used in this study could not answer this question, we will attempt to address it in the future.

For all doses used in this study, RET/PTC1 was significantly more common than RET/PTC3. This predominance of RET/PTC1 is similar to previous observations in normal human thyroid tissues transplanted in SCID mice and exposed to 50-Gy x-rays (34) and recapitulates the findings in tumors from patients subjected to therapeutic external irradiation (30, 31). However, it contradicts the experience with post-Chernobyl thyroid tumors, where RET/PTC3 was the dominant type of rearrangement in tumors developed less than 10 yr after exposure, whereas those manifested after a longer latency had predominantly RET/PTC1 (29). The most plausible explanation for this discrepancy is that, despite a smaller number of RET/PTC3 induced by radiation, this rearrangement confers thyroid cell with higher proliferative activity and tumorigenic potential, so that RET/PTC3-positive tumors require less time to reach clinically significant size and, therefore, manifest earlier than those arising through the RET/PTC1 mechanism. This possibility is supported by some experimental data. Indeed, a difference in the effects of RET/PTC1 and RET/PTC3 activation in vitro was observed in PC Cl3 rat thyroid cells transfected with both oncogenes (42). The cells expressing RET/PTC3 had significantly higher proliferation rate and approximately 3-fold higher levels of phosphorylation of the downstream target, mitogen-activated protein kinase. Other evidence comes from studies of transgenic mice. Although thyroid-specific expression of RET/PTC1 led to the development of slowly progressing and virtually nonmetastatic thyroid cancers (43, 44, 45), mice expressing RET/PTC3 developed aggressive and metastatic thyroid tumors (46). Alternatively, the reverse ratio of RET/PTC1 and RET/PTC3 may reflect the difference in radiation sources, i.e. the internal exposure to I-131 and other radioiodines after Chernobyl and external {gamma}- or x-ray irradiation in the experiments and in patients exposed to therapeutic radiation. These possibilities can be tested using the in vitro model established in this study. In addition, these experiments showed for the first time the induction of RET/PTC after doses as low as 0.1 Gy and, therefore, will allow to study the effects of low-dose ionizing radiation. Finally, because this experimental system employs cultured cells that are easy to manipulate on the genetic level, it may help to explore other important issues related to radiation carcinogenesis, particularly the role of specific genes in predisposition to chromosomal rearrangements and, therefore, radiation-induced cancer.

In summary, in this study, we demonstrate a dose-dependent generation of RET/PTC rearrangements in human thyroid cells after exposure to 0.1–10 Gy {gamma}-radiation. This provides additional evidence for a direct link between this genetic event and radiation exposure and offers a powerful experimental system for studying radiation-induced carcinogenesis in the thyroid gland.


   Acknowledgments
 
The authors are grateful to John Little for his valuable comments.


   Footnotes
 
This work was supported by National Institutes of Health Grant R01 CA88041.

First Published Online January 25, 2005

Abbreviations: FISH, Fluorescence in situ hybridization; NIS, sodium/iodide symporter; Tg, thyroglobulin.

Received September 13, 2004.

Accepted January 14, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Duffy Jr BJ, Fitzgerald PJ 1950 Cancer of the thyroid in children: a report of 28 cases. J Clin Endocrinol Metab 31:1296–1308
  2. Winship T, Rosvoll RV 1970 Cancer of the thyroid in children. Proc Natl Cancer Conf 6:677–681[Medline]
  3. Nagataki S, Shibata Y, Inoue S, Yokoyama N, Izumi M, Shimaoka K 1994 Thyroid diseases among atomic bomb survivors in Nagasaki. JAMA 272:364–370[Abstract/Free Full Text]
  4. Cronkite EP, Bond VP, Conard RA 1995 Medical effects of exposure of human beings to fallout radiation from a thermonuclear explosion. Stem Cells 13(Suppl 1):49–57
  5. Kazakov VS, Demidchik EP, Astakhova LN 1992 Thyroid cancer after Chernobyl. Nature 359:21[Medline]
  6. Williams D 2002 Cancer after nuclear fallout: lessons from the Chernobyl accident. Nat Rev Cancer 2:543–549[CrossRef][Medline]
  7. Tuttle RM, Becker DV 2000 The Chernobyl accident and its consequences: update at the millennium. Semin Nucl Med 30:133–140[CrossRef][Medline]
  8. 2000 UNSCEAR 2000 Report Vol. 2. Annex J. New York and Geneva: United Nations
  9. Jacob P, Goulko G, Heidenreich WF, Likhtarev I, Kairo I, Tronko ND, Bogdanova TI, Kenigsberg J, Buglova E, Drozdovitch V, Golovneva A, Demidchik EP, Balonov M, Zvonova I, Beral V 1998 Thyroid cancer risk to children calculated. Nature 392:31–32[CrossRef][Medline]
  10. Goodhead DT 1994 Initial events in the cellular effects of ionizing radiations: clustered damage in DNA. Int J Radiat Biol 65:7–17[Medline]
  11. Ward JF 1995 Radiation mutagenesis: the initial DNA lesions responsible. Radiat Res 142:362–368[Medline]
  12. Smith LE, Nagar S, Kim GJ, Morgan WF 2003 Radiation-induced genomic instability: radiation quality and dose response. Health Phys 85:23–29[CrossRef][Medline]
  13. Little JB 2003 Genomic instability and radiation. J Radiol Prot 23:173–181[CrossRef][Medline]
  14. Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA 2003 High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 63:1454–1457[Abstract/Free Full Text]
  15. Cohen Y, Xing M, Mambo E, Guo Z, Wu G, Trink B, Beller U, Westra WH, Ladenson PW, Sidransky D 2003 BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 95:625–627[Abstract/Free Full Text]
  16. Nikiforova MN, Ciampi R, Salvatore G, Santoro M, Gandhi M, Knauf JA, Thomas GA, Jeremiah S, Bogdanova TI, Tronko MD, Fagin JA, Nikiforov YE 2004 Low prevalence of BRAF mutations in radiation-induced thyroid tumors in contrast to sporadic papillary carcinomas. Cancer Lett 209:1–6[CrossRef][Medline]
  17. Santoro M, Dathan NA, Berlingieri MT, Bongarzone I, Paulin C, Grieco M, Pierotti MA, Vecchio G, Fusco A 1994 Molecular characterization of RET/PTC3; a novel rearranged version of the RETproto-oncogene in a human thyroid papillary carcinoma. Oncogene 9:509–516[Medline]
  18. Bongarzone I, Butti MG, Coronelli S, Borrello MG, Santoro M, Mondellini P, Pilotti S, Fusco A, Della Porta G, Pierotti MA 1994 Frequent activation of ret protooncogene by fusion with a new activating gene in papillary thyroid carcinomas. Cancer Res 54:2979–2985[Abstract/Free Full Text]
  19. Pierotti MA, Santoro M, Jenkins RB, Sozzi G, Bongarzone I, Grieco M, Monzini N, Miozzo M, Herrmann MA, Fusco A, Hay ID, Della Porta G, Vecchio G 1992 Characterization of an inversion on the long arm of chromosome 10 juxtaposing D10S170 and RET and creating the oncogenic sequence RET/PTC. Proc Natl Acad Sci USA 89:1616–1620[Abstract/Free Full Text]
  20. Minoletti F, Butti MG, Coronelli S, Miozzo M, Sozzi G, Pilotti S, Tunnacliffe A, Pierotti MA, Bongarzone I 1994 The two genes generating RET/PTC3 are localized in chromosomal band 10q11.2. Genes Chromosomes Cancer 11:51–57[Medline]
  21. Bongarzone I, Monzini N, Borrello MG, Carcano C, Ferraresi G, Arighi E, Mondellini P, Della Porta G, Pierotti MA 1993 Molecular characterization of a thyroid tumor-specific transforming sequence formed by the fusion of ret tyrosine kinase and the regulatory subunit RI {alpha} of cyclic AMP-dependent protein kinase A. Mol Cell Biol 13:358–366[Abstract/Free Full Text]
  22. Nikiforov YE 2002 RET/PTC rearrangement in thyroid tumors. Endocr Pathol 13:3–16[CrossRef][Medline]
  23. Jhiang SM 2000 The RET proto-oncogene in human cancers. Oncogene 19:5590–5597[CrossRef][Medline]
  24. Ito T, Seyama T, Iwamoto KS, Mizuno T, Tronko ND, Komissarenko IV, Cherstovoy ED, Satow Y, Takeichi N, Dohi K, Nakamura N, Akiyama M 1994 Activated RET oncogene in thyroid cancers of children from areas contaminated by Chernobyl accident. Lancet 344:259
  25. Fugazzola L, Pilotti S, Pinchera A, Vorontsova TV, Mondellini P, Bongarzone I, Greco A, Astakhova L, Butti MG, Demidchik EP 1995 Oncogenic rearrangements of the RET proto-oncogene in papillary thyroid carcinomas from children exposed to the Chernobyl nuclear accident. Cancer Res 55:5617–5620[Abstract/Free Full Text]
  26. Klugbauer S, Lengfelder E, Demidchik EP, Rabes HM 1995 High prevalence of RET rearrangement in thyroid tumors of children from Belarus after the Chernobyl reactor accident. Oncogene 11:2459–2467[Medline]
  27. Nikiforov YE, Rowland JM, Bove KE, Monforte-Munoz H, Fagin JA 1997 Distinct pattern of ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children. Cancer Res 57:1690–1694[Abstract/Free Full Text]
  28. Smida J, Salassidis K, Hieber L, Zitzelsberger H, Kellerer AM, Demidchik EP, Negele T, Spelsberg F, Lengfelder E, Werner M, Bauchinger M 1999 Distinct frequency of ret rearrangements in papillary thyroid carcinomas of children and adults from Belarus. Int J Cancer 80:32–38[CrossRef][Medline]
  29. Rabes HM, Demidchik EP, Sidorow JD, Lengfelder E, Beimfohr C, Hoelzel D, Klugbauer S 2000 Pattern of radiation-induced RET and NTRK1 rearrangements in 191 post-Chernobyl papillary thyroid carcinomas: biological, phenotypic, and clinical implications. Clin Cancer Res 6:1093–1103[Abstract/Free Full Text]
  30. Bounacer A, Wicker R, Caillou B, Cailleux AF, Sarasin A, Schlumberger M, Suarez HG 1997 High prevalence of activating ret proto-oncogene rearrangements, in thyroid tumors from patients who had received external radiation. Oncogene 15:1263–1273[CrossRef][Medline]
  31. Collins BJ, Chiappetta G, Schneider AB, Santoro M, Pentimalli F, Fogelfeld L, Gierlowski T, Shore-Freedman E, Jaffe G, Fusco A 2002 RET expression in papillary thyroid cancer from patients irradiated in childhood for benign conditions. J Clin Endocrinol Metab 87:3941–3946[Abstract/Free Full Text]
  32. Ito T, Seyama T, Iwamoto KS, Hayashi T, Mizuno T, Tsuyama N, Dohi K, Nakamura N, Akiyama M 1993 In vitro irradiation is able to cause RET oncogene rearrangement. Cancer Res 53:2940–2943[Abstract/Free Full Text]
  33. Mizuno T, Kyoizumi S, Suzuki T, Iwamoto KS, Seyama T 1997 Continued expression of a tissue specific activated oncogene in the early steps of radiation-induced human thyroid carcinogenesis. Oncogene 15:1455–1460[CrossRef][Medline]
  34. Mizuno T, Iwamoto KS, Kyoizumi S, Nagamura H, Shinohara T, Koyama K, Seyama T, Hamatani K 2000 Preferential induction of RET/PTC1 rearrangement by X-ray irradiation. Oncogene 19:438–443[CrossRef][Medline]
  35. Lemoine NR, Mayall ES, Jones T, Sheer D, McDermid S, Kendall-Taylor P, Wynford-Thomas D 1989 Characterisation of human thyroid epithelial cells immortalised in vitro by simian virus 40 DNA transfection. Br J Cancer 60:897–903[Medline]
  36. Gustashaw KM 1997 Chromosome stains. In: Spurbeck J, ed. The AGT Cytogenetics Laboratory Manual. 3rd ed. Philadelphia: Lippincott-Raven; 259–324
  37. Nikiforova MN, Stringer JR, Blough R, Medvedovic M, Fagin JA, Nikiforov YE2000 Proximity of chromosomal loci that participate in radiation-induced rearrangements in human cells. Science 290:138–141
  38. Argani P, Zakowski MF, Klimstra DS, Rosai J, Ladanyi M 1998 Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases. Mod Pathol 11:65–71[Medline]
  39. Ishizaka Y, Ushijima T, Sugimura T, Nagao M 1990 cDNA cloning and characterization of ret activated in a human papillary thyroid carcinoma cell line. Biochem Biophys Res Commun 168:402–408[CrossRef][Medline]
  40. Ron E, Lubin JH, Shore RE, Mabuchi K, Modan B, Pottern LM, Schneider AB, Tucker MA, Boice Jr JD 1995 Thyroid cancer after exposure to external radiation: a pooled analysis of seven studies. Radiat Res 141:259–277[Medline]
  41. Boice Jr JD 1998 Radiation and thyroid cancer: what more can be learned? Acta Oncol 37:321–324[CrossRef][Medline]
  42. Basolo F, Giannini R, Monaco C, Melillo RM, Carlomagno F, Pancrazi M, Salvatore G, Chiappetta G, Pacini F, Elisei R, Miccoli P, Pinchera A, Fusco A, Santoro M 2002 Potent mitogenicity of the RET/PTC3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma. Am J Pathol 160:247–254[Abstract/Free Full Text]
  43. Santoro M, Chiappetta G, Cerrato A, Salvatore D, Zhang L, Manzo G, Picone A, Portella G, Santelli G, Vecchio G, Fusco A 1996 Development of thyroid papillary carcinomas secondary to tissue-specific expression of the RET/PTC1 oncogene in transgenic mice. Oncogene 12:1821–1826[Medline]
  44. Jhiang SM, Sagartz JE, Tong Q, Parker-Thornburg J, Capen CC, Cho JY, Xing S, Ledent C 1996 Targeted expression of the ret/PTC1 oncogene induces papillary thyroid carcinomas. Endocrinology 137:375–378[Abstract]
  45. Sagartz JE, Jhiang SM, Tong Q, Capen CC 1997 Thyroid-stimulating hormone promotes growth of thyroid carcinomas in transgenic mice with targeted expression of the ret/PTC1 oncogene. Lab Invest 76:307–318[Medline]
  46. Powell Jr DJ, Russell J, Nibu K, Li G, Rhee E, Liao M, Goldstein M, Keane WM, Santoro M, Fusco A, Rothstein JL 1998 The RET/PTC3 oncogene: metastatic solid-type papillary carcinomas in murine thyroids. Cancer Res 58:5523–5528[Abstract/Free Full Text]



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Z. Zhu, R. Ciampi, M. N. Nikiforova, M. Gandhi, and Y. E. Nikiforov
Prevalence of RET/PTC Rearrangements in Thyroid Papillary Carcinomas: Effects of the Detection Methods and Genetic Heterogeneity
J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3603 - 3610.
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