International Journal of Radiation Oncology*Biology*Physics
Biology ContributionsRadiosensitivity of human normal and tumoral thyroid cells using fluorescence in situ hybridization and clonogenic survival assay☆
Introduction
The aim of this study was to evaluate rapidly and precisely the radiosensitivity of human thyroid cells using of fluorescence in situ hybridization (FISH) and to compare the results to the well-established, but cumbersome technique of cell survival. At the Gustave Roussy Institute, differentiated thyroid carcinomas are treated by surgery followed by one or more 131I ingestions of 3.7 GBq (100 mCi) to eradicate thyroid remnants or metastases 1, 2. A dose of 3.7 GBq of iodine delivers a nonnegligible whole-body dose of 0.5 Gy, as estimated by FISH, based on the yield of stable anomalies 3, 4. Thus, assessing radiation-induced damages in thyroid tissue before treatment may be helpful in managing treatment by radioiodine. Challeton et al.(5) have shown that the radiosensitivity of different, cultured thyroid tumor tissue can be measured by clonogenic assay. Some studies based on double strand breaks and their reparation have indicated that low clonogenic survival can be linked to a misrepair of these breaks 6, 7, 8, 9. Because this lack of repair after ionizing radiation can lead to anomalies such as dicentrics and translocations, we chose to compare two methods: the clonogenic assay and the FISH technique. The clonogenic assay is, at present, the only way to estimate the radiosensitivity of a cell population. However, this technique is very time consuming. In 1986, Pinkel established a new technique based on cytogenetics that evaluates directly the radiation impact on chromosomes by measuring reciprocal translocations 10, 11. Some authors have concluded that a high level of radiation-induced reciprocal translocations in permanent cell lines is linked to a higher radiosensitivity. Nevertheless, this technique is not yet used as a predictive test because a clear correlation between the clonogenic assay and FISH technique has not been established 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. If reciprocal translocations could be shown to correspond to clonogenic survival, we would be able to estimate rapidly and reliably, after surgery, the radiosensitivity of thyroid tumors, and evaluate more precisely the iodine activity to be administered to a given patient for a given pathology.
Section snippets
Cells
Normal and tumoral thyroid cells were obtained from patients with thyroid tumor followed at the Gustave Roussy Institute. During surgery (after thyroidectomy), the thyroid or the lobo-isthmectomy was evaluated extemporaneously by the histology department. Following this, a piece of tumoral and peritumoral normal thyroid tissue was sent to the laboratory, where the tissue samples were immediately cut into small pieces and agitated in a 0.05% trypsin/0.029% EDTA solution for 2 h at 37°C.
Results
The immunohistochemistry tests confirmed the follicular epithelial origin for each cell type and patient. Only a few fibroblasts remained in our thyroid cells due to the low subculture and the special culture medium without proline (23). Nevertheless, if in a clonogenic assay, a fibroblast clone was present, it could be clearly identified and was discounted 25, 40. The ploidy of our cells for chromosome 4 was 2, and not modified in higher subcultures. Figure 1 shows the results of our FISH test
Discussion
Until now, the clonogenic assay has been regarded as a reference in the evaluation of radiosensitivity. We submitted primary cultures of normal and tumoral thyroid cells to 60Co irradiation, and employed the FISH technique to estimate the yield of reciprocal translocations in order to compare it to the clonogenic assay done at the same time. The results obtained by clonogenic assay indicated three different groups of cellular survival (depending on the pathology). Normal cells appeared to be
Acknowledgements
The authors are indebted to Ms. Ingrid Küchenthal for linguistic revision of the manuscript, and wish to thank the nurses, secretaries, and everyone who lent a helping hand in this study.
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This work was supported by a grant from Electricité de France.