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The Natural Thyroid Diet

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RET/PTC

period

positive

RET/PTC1

RET/PTC3

types

<lOyears

40/61 (66%)

23%

60%

13%

>10 years

60/130 (46%)

65%

23%

5%

* Modified from Rabes et al. (2000} (48) with permission.

* Modified from Rabes et al. (2000} (48) with permission.

population, being reported at 52-87% (49-51). Exposure to ionizing radiation not only results in a higher prevalence in RET/PTC, but also promotes the fusion of RET to the unusual rearrangement partners, since seven out of eight novel RET/PTC types have been detected in tumors associated with radiation exposure. The novel types comprised up to 13% of all rearrangements found in post-Chernobyl tumors (48) (Table 2).

The fact that post-Chernobyl tumors arising shortly after exposure had mostly RET/PTC3 and those after a longer latent period harbored predominantly RET/PTC 1 suggests at least two possibilities. First, radiation may be more effective in inducing RET/PTC3, whereas other factors (still unknown), responsible for the majority of thyroid cancers that are not associated with radiation exposure, lead mostly to RET/PTC1. Second, it is conceivable that radiation is equally efficient in generation of both types, but tumors initiated by RET/PTC3 have a higher growth rate and manifest several years earlier. The latter possibility is supported by the animal experiments demonstrating that RET/PTC3 transgenic mice develop more malignant phenotype and metastatic disease as compared to RET/PTC1 animals (52, 53), and by recent in-vitro functional studies showing that RET/PTC3 is a more potent activator of MAPK signaling pathway and more efficient in promoting proliferation of cultured thyroid cells than RET/PTC 1 (54).

The occurrence of RET/PTC has been observed after high-dose irradiation of human undifferentiated thyroid carcinoma cells (55) and of fetal human thyroid tissues transplanted into SCID mice (56, 57). In both studies, the rearrangements were detected by RT-PCR as soon as 2 days after exposure. In addition, fetal thyroid cells revealed both RET/PTC 1 and RET/PTC3 7 days after irradiation, while only RET/PTC 1 persisted and was detectable 2 months later (57). The effective dose of radiation in both studies was high (50 Gy) and lethal for dividing cells, and the cells irradiated in the study by Ito and colleagues (55) were already highly transformed and hence more susceptible to develop secondary genetic defects. Nevertheless, these observations suggest that radiation exposure may lead to the generation of RET/PTC rearrangements in thyroid cells.

Potential mechanisms of RET/PTC generation after radiation exposure

The high prevalence of RET/PTC in post-Chernobyl children and in populations affected by other types of environmental and therapeutic exposures as well as the in-vitro studies provide strong evidence for the association between ionizing radiation and RET/PTC rearrangement. The role of radiation appears particularly important for the RET/PTC3 type, since its high prevalence was uniquely associated with papillary carcinomas developed shortly after the Chernobyl accident. The mechanisms of RET/PTC generation in thyroid cells after radiation have been a subject of extensive study over the last years.

Analysis of the breakpoint sites revealed no long-sequence homology between the DNA regions involved in RET/PTC1 fusion in sporadic tumors (58) and in RET/PTC3 in post-Chernobyl carcinomas (59, 60), establishing that these rearrangements result from illegitimate rather than homologous recombination. When the breakpoint sites within the RET and ELE1 genes were mapped and analyzed in 12 post-Chernobyl tumors with RET/PTC3, it appeared that in each tumor the relative position of a breakpoint in the RET gene corresponded to the location of a breakpoint in the ELE1 gene (60). Specifically, after aligning the genes in opposite orientations, the breakpoints were locatedjust across from each other in 5 (42%) tumors (Figure 3, A), while in other tumors they could be aligned by sliding one gene with respect to another (Figure 3, B, C). Similar pattern could be deducted from another study where the breakpoints in 22 post-Chernobyl tumors with RET/PTC3 were characterized (61). Such predilection for the breakpoint site in one gene to correspond to the breakpoint site located within the certain region of the other gene suggests the presence of a stable spatial relationship between these two chromosomal loci within the nucleus (Figure 3).

If the interaction between these loci exists, it should involve folding of chromosome 10 where both RET and ELE1 genes reside separated by a linear distance of ~18 Mb. This is conceivable since one of the levels of DNA packaging involves chromatin arrangement into loops of different size attached to a chromosomal backbone (62). Therefore, although two chromosomal loci are located at a considerable linear distance from each other, they may be closely spaced in the interphase nucleus because of their location at specific areas of chromosomal loop(s).

Indeed, it has been recently demonstrated that chromosomal regions containing the RET and H4 genes are non-randomly positioned with respect to each other in the interphase nuclei of normal thyroid follicular cells (63). Utilizing fluorescence in situ hybridization, two-dimensional distances between RET and H4 were measured in

"lOObp

1670bp
1843bp

1843bp

1843bp

Figure 3. Alignment of the breakpoint sites involved in RET/PTC3 in post-Chernobyl tumors demonstrating three patterns of correspondence between the position of breakpoints in each tumor (A, B, C). Modified from Nikiforov et al. (1999) (60) with permission.

the interphase cells and compared with a theoretical Rayleigh model that describes a distribution of distances between two points of linear polymers that fold in a random manner. Previous studies have shown that interphase distances between random loci on the same chromosome conform to the Rayleigh distribution (62, 64). Indeed, in the control experiment, distances between the RET and D10S539 loci, the latter located on chromosome 10q between RET and H4 and is not known to participate in the rearrangement, were found to follow the Rayleigh distribution (Figure 4) (63). As for the RET and H4 distances, they showed a strong deviation from the Rayleigh model, primarily due to the loci either juxtaposed or closer than expected, indicating a non-random manner of RET and H4 interaction. In addition, as many as 35% of primary cultured thyroid cells had at least one pair of RET and H4 genes juxtaposed. These data suggest that generation of RET/PTC rearrangements in thyroid cells may be in part due to the structural organization of chromosome 10, resulting in non-random interaction and spatial approximation of these potentially recombinogenic DNA loci (Figure 4).

It remains unclear, however, whether RET/PTC is a direct consequence of DNA breaks induced by ionizing radiation, or it forms indirectly, after DNA damage has

Figure 4. Distribution of interphase distances between RET and D10S539 (A) and RET and H4 (B) in cultured normal thyroid cells as compared with the theoretical Rayleigh distribution (solid line). Dark bars indicate the RET - H4 distances that were in excess over the number expected based on the Rayleigh distribution. From Nikiforova et al. (2000) (63).

Interphase distances (pm) Interphase distances (jim)

Figure 4. Distribution of interphase distances between RET and D10S539 (A) and RET and H4 (B) in cultured normal thyroid cells as compared with the theoretical Rayleigh distribution (solid line). Dark bars indicate the RET - H4 distances that were in excess over the number expected based on the Rayleigh distribution. From Nikiforova et al. (2000) (63).

been repaired. Important information in this respect can be obtained by mapping and characterization the breakpoint sites involved in the fusion. Thus, if the rearrangement occurred indirectly (as a result of activation or disruption of the recombination machinery), the breakpoints are expected be located within recombinase signal sequences at both participating loci, have similarity in nucleotide composition, or cluster at certain specific hypersensitive DNA regions. However, as it had been convincingly demonstrated in post-Chernobyl tumors with RET/PTC3, the breakpoints within and surrounding ELE1 intron 5 and RET intron 11 were distributed randomly, with no breakpoints occurring at exactly the same base or within an identical sequence in any of 41 tumors reported in three different series (59-61). The breakpoints exhibited no particular nucleotide sequence or composition. In one study, no evidence of AT-rich regions, fragile sites, recombination-specific signal elements, or other target DNA sites (i.e. chi-like motifs, heptamer/nonamer signal sequences) implicated in illegitimate recombination in mammalian cells was found (60). These results favor the direct induction of RET/PTC as a result of random double-strand DNA breaks, rather than disruption of the recombination machinery. However, another study proposed an alternative mechanisms and found at least one topoisomerase I site exactly at or in close proximity to all breakpoints, suggesting the role of DNA breaks induced by this enzyme in the generation of RET/PTC3 (61).

PAX8-PPARfl REARRANGEMENT

Recently, a fusion has been identified in follicular thyroid carcinomas with cytogenetically detectable translocation t(2;3)(q13;p25) (65). This rearrangement leads to an in-frame fusion of the PAX8 gene, which encodes a paired domain transcription factor, with the peroxisome proliferator-activated receptor (PPAR) y gene. The structure and biological properties of the fusion gene are discussed in Chapter 4. In the original report, PAX8-PPARy was detected in 5 out of 8 (63%) follicular carcinomas (65), whereas in the larger follow-up series, the prevalence was 26-35% (66, 67). However, when this alteration was studied separately in follicular carcinomas from patients without a history ofradiation and in those exposed to radiation, PAX8-PPARy fusion was found in 5 out of 12 (42%) sporadic tumors and in all three (100%) tumors associated with radiation (68). Despite the small number of follicular carcinomas in the latter group, this finding points towards the possible association between radiation exposure and rearrangement. Although papillary carcinoma is by far the most common type of thyroid tumors associated with radiation, an increased risk of follicular carcinoma development has also been documented in these populations (69). Therefore, it is likely that radiation exposure may promote the development of follicular tumors through the generation of PAX8-PPARy rearrangement in a similar way it involves in the initiation of papillary thyroid carcinogenesis via RET/PTC rearrangement.

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