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Cancer Research 68, 8986, November 1, 2008. doi: 10.1158/0008-5472.CAN-08-1032
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Enhanced Sensitivity of the RET Proto-Oncogene to Ionizing Radiation In vitro

Claudia Béu Volpato1, Minerva Martínez-Alfaro2, Raffaella Corvi4, Coralie Gabus2, Sylvie Sauvaigo5, Pietro Ferrari3, Elena Bonora6, Alessandro De Grandi1 and Giovanni Romeo6

1 Institute of Genetic Medicine, European Academy, Bolzano, Italy; 2 Genetics Cancer Susceptibility Unit and 3 Nutrition and Cancer Unit, IARC, Lyon, France; 4 European Center for the Validation of Alternative Methods, Institute for Health and Consumer Protection, JRC of the European Commission, Ispra, Italy; 5 Laboratoire des Lésions des Acides Nucléiques, CEA Grenoble, France; and 6 Unità di Genetica Medica, Dipartimento di Scienze Ginecologiche, Ostetriche, Pediatriche, Policlinico S. Orsola-Malpighi, Bologna, Italy

Requests for reprints: Giovanni Romeo, Dipartimento di Scienze Ginecologiche, Ostetriche, Pediatriche, Cattedra di Genetica Medica, Padiglione 11. Policlinico S. Orsola-Malpighi, Via Massarenti 9, 40138 Bologna, Italy. Phone: 39-051-306474; Fax: 39-0516364004; E-mail: romeo{at}eurogene.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Exposure to ionizing radiation is a well-known risk factor for a number of human cancers, including leukemia and thyroid cancer. It has been known for a long time that exposure of cells to radiation results in extensive DNA damage; however, a small number of studies have tried to explain the mechanisms of radiation-induced carcinogenesis. The high prevalence of RET/PTC rearrangements in patients who have received external radiation, and the evidence of in vitro induction of RET rearrangements in human cells, suggest an enhanced sensitivity of the RET genomic region to damage by ionizing radiation. To assess whether RET is indeed more sensitive to radiations than other genomic regions, we used a COMET assay coupled with fluorescence in situ hybridization, which allows the measurement of DNA fragmentation in defined genomic regions of single cells. We compared the initial DNA damage of the genomic regions of RET, CXCL12/SDF1, ABL, MYC, PLA2G2A, p53, and JAK2 induced by ionizing radiation in both a lymphoblastoid and a fetal thyroid cell line. In both cell lines, RET fragmentation was significantly higher than in other genomic regions. Moreover, a differential distribution of signals within the COMET was associated with a higher percentage of RET fragments in the tail. RET was more susceptible to fragmentation in the thyroid-derived cells than in lymphoblasts. This enhanced susceptibility of RET to ionizing radiation suggests the possibility of using it as a radiation exposure marker. [Cancer Res 2008;68(21):8986–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The most common molecular alteration in papillary thyroid carcinoma (PTC) involves the proto-oncogene RET (REarranged during Transfection), which maps to the long arm of chromosome 10 at band q11.2. These rearrangements (inversions or translocations), which are found exclusively in thyroid tumors, always lead to the juxtaposition of its intracellular tyrosine kinase (TK) domain to the 5' portion of different donor genes constitutively expressed in the thyroid (1). PTC represents the main example of radiation-associated cancer, whose incidence has increased up to 100-fold in children and adolescents from certain districts of Belarus and Ukraine surrounding Chernobyl during a 10-year period (2–4). Several rearranged forms of RET have been described in PTCs (5–12), most of which have been detected in radiation associated tumors. PTC patients that have been exposed to ionizing radiations present a very high frequency of RET/PTC rearrangements compared with nonexposed patients. This group of patients includes not only children exposed to the radioactive fallout but also individuals who developed the tumor after therapeutic or accidental ionizing radiation exposure (13, 14). PTC1 and PTC3 rearrangements have also been induced in vitro and in vivo by irradiation of tumor cell lines and fetal thyroid tissue that had previously been transplanted into severe combined immunodeficient (SCID) mice (15–17). Several studies suggest that different kinds of radiation (UVA and ionizing radiation) induce a nonrandom distribution of the DNA lesions across the whole genome (18, 19).

A study on the evaluation of chromosome rearrangement frequency in peripheral lymphocytes of children exposed to the Chernobyl fallout suggested a preferential involvement of chromosome 10 in comparison with chromosome 1 and chromosome 3 (20). Low levels of ionizing radiation also generated a significantly greater extent of chromosome 10 translocations in human lymphocytes in vitro (21). Unfortunately, the exact breakpoint location was not precisely investigated in these two studies. The association between the formation of RET rearrangements and radiation exposure could therefore be explained by a higher sensitivity of the genomic RET region to breakage (21).

The extensive characterization of the breakpoints within the RET gene, which nearly exclusively occur within intron 11, has shown that they are distributed in a relatively random fashion. None of the breakpoints occurred at the same base or within a similar sequence, and there is no evidence of preferential cleavage in AT-rich regions or within Alu elements (22, 23). Two-color fluorescence in situ hybridization (FISH) and three-dimensional microscopy have been used to map the positions of the RET and the H4 loci within normal interphase nuclei. Those experiments suggested that the spatial contiguity of the two genes might provide a structural basis for generation of RET/PTC1 rearrangement by allowing a single radiation track to produce a double strand break in each gene at the same site in the nucleus (24).

To date, no study has been performed to quantitatively investigate the induced RET breakages after ionizing radiation exposure and the possibility of using it as radiation damage indicator. For this reason, in the present study, we wished to assess whether RET is indeed more sensitive to radiation than other genomic regions, and to investigate whether it can be used as a marker of ionizing radiation exposure. To this end, we established a procedure that combines the COMET assay (single cell gel electrophoresis) and FISH. The COMET assay allows the measurement of overall DNA damage in a single cell by using an electric field to separate damaged DNA from undamaged DNA. FISH is able to detect the specific fragmentation of genes in cells exposed to ionizing radiations. In the present work, we analyzed the initial DNA fragmentation observed in irradiated cells in culture soon after radiation exposure (time 0) before DNA repair occurs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell lines. Experiments were performed using two cell lines: TAD2 (fetal thyroid cell line) obtained by transfection of fetal human thyroid cells with SV40 (kind gift from Dr. Davies, Mount Sinai School of Medicine, New York; ref. 25) and a lymphoblastoid cell line established from lymphocytes of a healthy person. The cell lines were cultured in RPMI and in Dulbecco's medium, respectively, with 10% FCS and 1% penicillin/streptomycin.

Establishment of the working irradiation dose. Initially the COMET-FISH assay was performed on cells exposed to increasing ionizing radiation doses (2, 3, 4, 5, 6, and 7 Gy) to establish the best irradiation dose to be used in our study (data not shown). The working dose chosen was 3 Gy, a dose that produced a discrete breakage of the genomic regions studied without generating too many fragments. A high ionizing radiation dose was avoided because this might have caused heavy DNA damage and random breakage of genomic sequences. Moreover, a previous epidemiologic study had shown that the risk for PTC is higher at low radiation dose (0.5 Gy,) but decreases at high radiation dose (20 Gy; ref. 26).

Irradiation. The cells were irradiated with Cs137 on ice. All cultures were tested for viability using trypan blue exclusion staining. They showed 95% viable cells before irradiation and 90% immediately after irradiation.

COMET assay. Experiments were performed using a modification of the alkaline COMET-assay protocol initially described by Singh (27). Frosted microscope slides were covered with 200 µL of 0.1% agarose in PBS. After polymerization of the gel, 2 x 105 cells were resuspended in 10 µL PBS and 75 µL 0.5% low-melting-point agarose and were added to each slide. The slides were initially placed (15 slides from irradiated and 5 slides from nonirradiated cells) in cold fresh lyses buffer (2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris-HCl, and 1% Triton x-100) for 1 h and subsequently in a horizontal gel electrophoresis unit filled with chilled electrophoresis buffer (300 mmol/L NaOH and 1 mmol/L Na2EDTA) for 30 min. Electrophoresis was conducted in a chamber (20 x 25 cm) for 60 min at 14 V. Slides were then drained, neutralized, and dried with alcohol. The comets were counterstained with 4,6-diamidino-2 phenylindole dihydrochloride (DAPI). DNA damage was analyzed using the Europe Komet 3.1 software (Kinetic Imaging) that gave us a measure of the tail moment and Metafer Comet-FISH (Metasystem from Zeiss-Germany).

Probes. The digoxigenin-labeled DNA probe for p53 (Oncor) was used according to the manufacturer's instructions. A mixture of four overlapping cosmids R1, E5, B6, and F2 covering ~100 kb of the RET genomic region (28), three BACs, respectively, for ABL1 (clone dJ 1146L15; refs. 29, 30), CXCL12/SDF1 (clone RP11 20J15; ref. 31) and MYC (clone dJ 968N11; refs. 32, 33), and two PACs, respectively, for PLA2G2A (clone B15251; ref. 34) and JAK2 (clone RP11 39k24; ref. 35) were used as probes. All the BACs and PACs sizes were between 100 to 180 kb.

These probes were labeled with biotin-16-dUTP (Roche Diagnostics) by nick translation (36). Labeled probes were mixed with a 10-fold excess of Cot-1 human DNA (Roche Diagnostics) and Salmon Sperm DNA in 10 µL hybridization buffer (50% Dextran sulfate 4x SSC) + 10 µL formamide, denatured at 95°C for 5 min, and prehybridized at 37°C for 30 min.

FISH. The slides were immersed in 0.5 mol/L NaOH for 30 min at room temperature and subsequently dehydrated in an ethanol series (70%, 80%, and 100%). Denatured probes were added to the slides at a concentration of 20 ng/µL for cosmids and 27 ng/µL for PACs. The slides were then covered with plastic coverslips and incubated in a humidified chamber at 37°C for 12 h. After hybridization, the coverslips were carefully removed and the slides were washed (2x SSC, 25% formamide) for 5 min. The slides hybridized with biotin- and digoxigenin-labeled probes were incubated for 30 min at 37°C with FITC-conjugated avidin (Vector Laboratories) and rhodamine-conjugated antidigoxigenin, Fab fragment (Roche Diagnostics), respectively. Finally, they were washed with 4x SSC containing 0.1% tween. Nuclei were counterstained with DAPI and mounted in Vectashield (Vector Laboratories).

COMET-FISH analysis. The DNA damage was analyzed using the Komet 3.1 software (Kinetic Imaging) that gave us a measure of the tail moment (% DNA x Tail Length; refs. 37, 38). Subsequently, analysis of the fluorescence hybridization image was performed using an Axioplan 1 Carl Zeiss microscope and the IPlaps Vysis capture software. The number of signals was counted in each comet and their distribution within the comet (head or tail) was analyzed by measuring the distance of each spot from the center of the head and the radius of the nucleus. Analysis was performed on at three independent samples with at least 50 cells in each experiment. Each sample had comparable average tail moments. Irradiated and nonirradiated (control cells) samples were comparable in each experiment. The data were collected in a double-blind manner to avoid bias. Images were saved under a code that was independent of cell type or experimental procedure, and analyzed without knowledge of the sample type.

Statistical analysis. Statistical regression models were used to assess differences of signal distributions in the two cell lines with each probe.

A Poisson regression model was used to assess the difference of gene fragmentation, by cell lines, using the total number of signals in the comet as dependent variable. In a second step, the number of signals in the tail was used as dependent variable in a Poisson regression model, and the total number of signals in the comet was used as the "offset" variable. This analysis was aimed at detecting any statistically significant difference in the mean number of signals in the tail. Finally, a logistic model, with a dichotomous dependent variable (cells with signals in tail and cells without signals in tail), was used to compare cells hybridized with different probes. In each of the three models, the use of nested effects (comets within cell lines) allowed the simultaneous comparison of these quantities within cell lines (between comets) and between cell lines. Statistical analyses were performed using the SAS software (39).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
A fragmentation assay, which combines the COMET assay and the FISH analysis, was established with the aim of studying the RET sensitivity to ionizing radiation exposure, and to elucidate whether this radiosensitivity is cell type specific. This study was performed in two different cell lines: a normal lymphoblastoid cell line and a fetal thyroid cell line (TAD2). The cells were first exposed to ionizing radiation, electrophoresed in agarose, and the resulting comets were subsequently hybridized with the different genomic probes.

COMET analysis. The experiments were always conducted in parallel on irradiated and nonirradiated cells. Nonirradiated cells very rarely showed a tail and a signal on it, whereas cells irradiated with 3 Gy always presented a tail, which could vary in size and could contain several signals. This was confirmed by the measure of tail moment (% DNA x tail length), which is directly proportional to DNA damage and indeed is found to be much higher in irradiated cells (39, 40). The analysis of DNA damage in the two cell lines, before and after irradiation, showed also that the tail moment was in general more elevated in lymphoblastoid cells (Table 1 ). These different values might well be due to the distinct chromatin structure of the two cell lines, which may affect the accessibility of DNA to DAPI counterstain and therefore the fluorescence patterns (41). To assess the assay reproducibility of the analysis, the experiments were repeated on three different series of comets. For the subsequent FISH analysis, it was necessary to use a series of comets that showed a similar tail moment to be sure that the general DNA damage was comparable between the different experiments.


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  		Table 1. Range of tail moment in non irradiated and irradiated cells used for comet

 
COMET-FISH analysis. The comets were hybridized with probes encompassing the genomic region of RET (10q11); CXCL12/SDF1 (10q11.1), ABL (9q24), MYC (8q24.1), PLA2G2A (1p35), p53 (17p13), and JAK2 (9p24). The probe P53 was used as control because it had been previously shown that this DNA sequence was almost stable upon radiation exposure (42). ABL probe was used to compare RET fragility because it belongs to the TK family like RET, it is often translocated in leukemia, and it is localized in a telomeric region (28, 29), whereas RET maps in the 10q pericentromeric region.

The genomic regions of PLA2G2A, p53, and JAK2 were used as comparison against RET breakage susceptibility because PLA2G2A (33) and p53 (43) are localized in chromosomal regions often deleted in cancer, whereas JAK2 is rearranged in acute leukemia (44) and amplified in Hodgkin cells (45). CXCL12/SDF1 and MYC were used to compare radiation breakages in centromeric and telomeric regions, respectively. CXCL12/SDF1, from now on defined as SDF, map on chromosome 10 centromeric to RET at distance of 12 MB (30). MYC is localized in the telomeric region of chromosome 8 and can be used to investigate the radiation breakages for this chromosomal region (31, 32).

The hybridization experiments led to the detection of DNA fragmentation as discrete spots within the comet. In the case of no fragmentation of the genomic site analyzed, the comet showed two spots in the head, corresponding to the two homologous copies of the sequence. If the genomic region of interest was fragmented, several spots could be observed, which were localized either in the head or in the tail of the comet (Fig. 1 ). When nonirradiated control cells were hybridized, two labeled sites were usually located in the head, showing the typical hybridization pattern known from interphase FISH on standard microscope slides. In this case, the hybridization pattern was similar for the different genes analyzed (Fig. 1A). In contrast, many of the irradiated cells showed gene fragmentation that seemed to be specific for each genomic region (Fig. 1B). Data on fragmentation level are shown in Tables 2 to 4 . RET fragmentation was higher than fragmentation of the other genes in both cell lines. A differential distribution of signals within the comet was also observed, accompanied by a higher percentage of RET fragments present in the tail compared with the percentage seen in other genomic regions (Table 3). Because it has been hypothesized that large DNA fragments do not migrate to the tails, the presence of RET fragments in the tail would indicate that the RET region was cut into smaller fragments (18).


Figure 1
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  		Figure 1. Representative pictures of TAD2 cells after COMET-FISH. A, nonirradiated comets cell hybridized with RET (a) and PLA2G2A (b). B, irradiated comets cell hybridized with RET (a), p53 (b), PLA2G2A (c), and JAK2 (d). Comets were counterstained with DAPI; p53 was detected with rhodamine-conjugated antidigoxigenin (B-b) and the other probes with FITC-conjugated avidin (A, a and b; B, a, c, and d). Nonirradiated cells are almost rounding, whereas irradiated cells show an evident tail. Irradiated comets show gene fragmentation with the exception of the comet hybridized with JAK2.

 

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  		Table 2. Mean number of signal obtained by COMET hybridization with different genomic regions and mutual comparison within and between cell lines

 

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  		Table 3. Percentage of signals in the tail and mutual comparison with and between cell lines

 

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  		Table 4. Percentage of cells with at least one signals in the tail and reciprocal comparisons with and between cell lines

 
Statistical analysis. The regression models were used to evaluate the degree of radiation sensitivity of the different genomic regions in the two cell lines. The comparative statistical analysis was performed considering each series separately and considering the two series together. The results were analogous in the two cases. Results from the same experiment performed on different series, concerning the total number of fragments, were always concordant, clearly demonstrating the experimental reproducibility. Our results, therefore, did not need to be corrected for the probe size because all the probes used have the same size order of magnitude (RET, 105 kb; ABL, ~150 kb; MYC, ~180 kb; PLA2G2A, ~120 kb; p53, ~100 kb; JAK2, 100 kb; SDF, 184 kb; ref. 18).

We compared (a) the total number of fragments, (b) the distribution of signals within the comet, and (c) the number of cells with at least one signal in the tail (indicating how much the genomic regions were fragmented). This last comparison has been used by others (18, 46) to evaluate the damage of the genomic regions resulting from genotoxic agents. Tables 2 to 4 shows the comparison of the signals among different genomic regions in the two cells lines. Considering the mean number of signals by comet and the percentage of signals in the tail, ABL and MYC show approximately the same level of RET radiosensitivity, whereas Pla2G2A, p53, and Jak2 are in general less radiosensitive (Tables 2A and 3A). Almost the same distribution of signals is exhibited in TAD2 (Table 2B). In general, no significant fragmentation differences were observed when the other genes ABL, MYC, SDF1, PLA2G2A, p53, and JAK2 were compared with each other in both cell lines.

As it is possible infer from Table 3, ABL is more fragmented in lymphoblasts than in TAD2. The MYC and p53 genomic region showed a discrete fragmentation in TAD2 cells compared with lymphoblasts but less than RET (Table 2). RET fragmentation was generally, significantly higher than the other genomic regions.

Moreover, the analysis of the fragment distribution showed a number of spots in the tail that were more relevant in the case of RET (Table 3). RET was significantly more fragmented in thyroid cells than in lymphoblasts, despite the higher tail moment found in the lymphoblasts (Tables 3 and 4). A significantly higher number of cells with at least one signal in the tail were observed generally in the case of RET (Table 4).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
To our knowledge, this is the first report on the quantitative comparison of the sensitivity of different genomic regions to initial damage caused by ionizing radiation in cell lines.

Our results clearly establish that the RET genomic region is more sensitive than other regions to ionizing radiation at a dose of 3 Gy in both cell lines for all analyzed parameters. It shows more breakages and more fragmentation than the other genomic regions. In irradiated cells, there is a higher percentage of signals in the tails of the TAD2 cell line than in the lymphoblast cell line. Furthermore, the RET genomic region exhibits a higher initial damage than any other genomic region in the TAD2 cells. This fact is suggestive of the possibility of more frequent DNA damages involving RET in the thyroid than in other tissues and could be of importance in the induction of RET rearrangements in this tissue.

It is possible to group the analyzed genomic regions into four categories corresponding to the grade of radiosensitivity: (1) RET, (2) ABL, MYC, (3) SDF, (4) Pla2G2A, p53, and Jak2, where number 1 is the most sensitive. Groups 2 and 3 were used to evaluate radiation breakage in different chromosomal locations. Because the radiosensitivity of RET could be dependent on the cell type, our results should be replicated in principle in other types of human cell lines, such as primary thyrocytes and lymphocytes. In particular, one could argue that due to a diverse level of differentiation, the fetal derivation of TAD2 might cause a different susceptibility to the ionizing radiations with respect to an adult-derived thyroid cell line. However, previously published observations stand against this argument because irradiated mouse fetal cells when transplanted into SCID mice do not behave differently from adult tumor cell lines (15, 16). In addition, our present results obtained in vitro are in agreement the results we previously obtained in vivo in C57B1/6 and CBA/J mice (47).

As a general conclusion, it can be said that our in vitro and in vivo observations clearly show, as a proof of principle, the RET region differential susceptibility to ionizing radiations. It remains highly interesting to verify in as many as possible other cellular systems the applicability of our results obtained in the TAD2 cell line, which, however, is widely used in the literature as a model thyroid cellular system.

ABL and MYC (group 2) are telomeric on chromosomes 9 and 8, respectively, and are in regions with a comparable gene density. SDF is more centromeric than RET on chromosome 10. These three metacentric chromosomes have approximately the same size. ABL is often translocated in leukemia and belongs to the TK gene family as does RET. The sensitivity to ionizing radiations of the ABL genomic region is similar to that of RET in lymphoblasts, but sensitivity of TAD2 is significantly less. The fact that TAD2 cell line is derived from fetal thyroid cells, not involved in leukemia, could be an explanation for our observations. MYC exhibits a discrete sensitivity to ionizing radiation, whereas SDF is slightly less sensitive than MYC in both cell lines. These observations indicate that SDF significant differences could depend on the chromosomal position. This is supported by previous research that showed that the induced number of chromatin strand breaks does not correlate with the chromosome size, and that there was an inverse correlation between the density of active genes and the sensitivity toward UV-A radiation (18).

Group 4 is the least sensitive to ionizing radiation in the lymphoblastoid and fetal thyroid cell lines used. Pla2G2A, p53, Jak2 (group 4), and also SDF (group 3) are involved in carcinogenesis pathways but this is not correlated with higher radiosensitivity. Our results indicate that RET is not the only radiosensitive genomic region, but it is always the most prone to breakage upon radiation exposure.

The chromosomal localization and the involvement in carcinogenesis of RET does not seem to influence sensitivity of RET to ionizing radiation in our experiments. The elevated initial fragmentation of RET clearly shows that damage by ionizing radiation is not distributed randomly over the whole genome. This issue has been reviewed by Johnson (19). A prevalence of breakpoints was found at heterochromatic regions and/or telomeres, and a clustering of breakpoints was observed near the centromeres of many chromosomes. In our study, the most damaged genomic region includes the RET proto-oncogene.

Radiation-induced breakpoints of chromosome 1 have been mapped to G-light bands (48). This observation could not be confirmed by our study. Among the genes analyzed, RET and JAK2 are contained in regions of the genome that have a moderate gene density, whereas PLA2G2A and p53 are localized within gene-rich regions. This could reflects the high frequency of RET rearrangements found in PTC of children exposed to the Chernobyl fallout (49) and of adult patients who received external radiation (13). Moreover, Mizuno and colleagues (16) have examined the capacity of ionizing radiations to induce the formation of RET chimeric genes in human tissue transplanted in SCID mice after 50 Gy exposure. They showed the induction of several types of rearrangements and the persistence of the PTC1 rearrangement throughout a 2-month period, which indicates that the cells containing this type of rearrangement have a selective growth advantage during the early phases of thyroid carcinogenesis. Our data infer that RET radiosensitivity in vitro is similar to that reported in vivo by our group using mouse lymphocytes (47).

Furthermore, RET is more susceptible to fragmentation in TAD2 cells than in lymphoblasts, although across the genome, we observed more DNA damage in lymphoblasts than in TAD2, suggesting the possibility that the RET genomic region is more radiosensitive in thyroid cells. This observation might be explained by a different chromatin condensation on chromosome 10q11.2 in various cell types or by a differential expression of RET in these cells. A high frequency of RET rearrangements has also been observed in microcarcinomas of patients who have not been exposed to radiation (50), suggesting that the RET sequence carries an intrinsic fragility. Another explanation for the enhanced rearrangement frequency of a defined chromosomal region could be a differential DNA repair of the DNA lesions. It has in fact been hypothesized that the chromatin of gene-rich regions with a high transcriptional activity is more accessible to the repair apparatus (38). These data again suggest that chromosome fragility might be due more to structural differences of the chromatin, than to functional differences. In conclusion, in our experimental model, the RET genomic region exhibits an enhanced sensitivity to ionizing radiation compared with other regions of the genome.


   Disclosure of Potential Conflicts of Interest
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 Abstract
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 Materials and Methods
 Results
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 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Grant QLG1-CT-2001-01646-"The RET Proto-Oncogene" and by grant LSHC-CT-2006-037530 "HERMIONE" from the EU, by FIRB grant for PRIME-"Proggetto Integrato Malattie Ereditarie," and by funds from the Carisbo Foundation (Bologna; G. Romeo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Davies, Mount Sinai School of Medicine, New York, for kindly providing the TAD2 cell line, Suzanne Pauly for her excellent technical assistance, and Dr. Andrew Moore for helpful comments on the manuscript.


   Footnotes
 
Note: C.B. Volpato and M. Martínez-Alfaro contributed equally to this work.

M. Martínez-Alfaro is recipient of a CONACYT-Mexico fellowship.

Received 3/19/08. Revised 7/25/08. Accepted 8/11/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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M. D. Johnson, P. L. Moots, Z. Zhuang, and R. J. Weil
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