(1999) 18, 6330 ± 6334 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Chromosomal breakpoint positions suggest a direct role for radiation in inducing illegitimate recombination between the ELE1 and RET genes in radiation-induced thyroid carcinomas

YE Nikiforov*,1,2, A Kosho€er1, M Nikiforova2, J Stringer3 and JA Fagin2

1Department of Pathology, University of Cincinnati College of Medicine, PO Box 670529, Cincinnati, Ohio, OH 45267-0529, USA, 2Division of Endocrinology, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, Ohio, OH 45267-0547, USA, 3Department of Molecular , University of Cincinnati College of Medicine, PO Box 670524, Cincinnati, Ohio, OH 45267-0524, USA

The RET/PTC3 rearrangement is formed by fusion of Nikiforov et al., 1997), but not in tumors from the ELE1 and RET genes, and is highly prevalent in unexposed children (Bongarzone et al., 1996; Nikifor- radiation-induced post-Chernobyl papillary thyroid car- ov et al., 1997). We have also found that this type of cinomas. We characterized the breakpoints in the ELE1 RET rearrangement is associated with a speci®c and RET genes in 12 post-Chernobyl pediatric papillary histotype of radiation-induced thyroid tumors ± the carcinomas with known RET/PTC3 rearrangement. We solid growth papillary carcinoma, that is rare in found that the breakpoints within each intron were sporadic adult or pediatric populations (Nikiforov et distributed in a relatively random fashion, except for al., 1997). On the other hand, RAS and p53 , clustering in the Alu regions of ELE1. None of the prevalent in sporadic thyroid , are virtually breakpoints occurred at the same base or within a similar absent among post-Chernobyl tumors (Nikiforov et al., sequence. There was also no evidence of preferential 1996; Suchy et al., 1998). Thus, RET/PTC3 rearrange- cleavage in AT-rich regions or other target DNA sites ment is a major genetic event in a well-characterized implicated in illegitimate recombination in mammalian population of radiation-induced post-Chernobyl pedia- cells. Modi®cation of sequences at the cleavage sites was tric papillary carcinomas and most likely has direct minimal, typically involving a 1 ± 3 nucleotide association with radiation exposure. and/or duplication. Surprisingly, the alignment of ELE1 RET/PTC3 results from a paracentric inversion of and RET introns in opposite orientation revealed that in the long arm of 10 and is formed by each tumor the position of the break in one gene fusion of the intracellular domain of RET tyrosine corresponded to the position of the break in the other kinase receptor gene with the ELE1(RFG) gene gene. This tendency suggests that the two genes may lie (Santoro et al., 1994). Both genes are located at next to each other but point in opposite directions in the 10q11.2, with a minimum distance of 500 kb between nucleus. Such a structure would facilitate formation of them (Minoletti et al., 1994). The ELE1 gene is RET/PTC3 rearrangements because a single radiation expressed ubiquitously and drives the expression of track could produce concerted breaks in both genes, the truncated RET receptor in thyroid follicular cells. leading to inversion due to reciprocal exchange via end- The precise mechanisms of how radiation may joining. induce oncogenic mutations, such as RET/PTC3 rearrangement, and lead to papillary thyroid carcino- Keywords: RET; gene rearrangement; illegitimate ma development are unknown. One possibility is that recombination; radiation-induced; thyroid radiation directly caused the as a result of misrepaired DNA damage. It is well known that radiation produces a random pattern of primary particle tracks, that (by direct ionization and through Introduction the agency of chemical radicals) cause damage to the chromatin structure manifesting as single-strand DNA The Chernobyl nuclear power accident in April 1986 breaks, double-strand DNA breaks, base damage and produced one of the most serious environmental DNA-protein crosslinks (Ward, 1988; Kovacs et al., disasters ever recorded and created a rich paradigm 1994; Grosovsky et al., 1988). DNA strand breaks can of radiation-induced human neoplasia, o€ering re- be lethal to the , and several enzymatic mechanisms markable insights into thyroid carcinogenesis (Kaza- exist to repair them. One such repair mechanism is to kov et al., 1992; Nikiforov and Gnepp, 1994; Pacini et ligate the ends of severed (end-joining). al., 1997). We and others observed that a speci®c type Incorrectly rejoined breaks may result in chromosomal of the RET gene rearrangement, RET/PTC3, is highly aberrations such as chromosomal translocations, prevalent (50 ± 58%) in post-Chernobyl pediatric deletions, and inversions, through which an oncogene papillary carcinomas removed 5 ± 7 years after the (such as the RET/PTC3 fusion gene) can form. Indeed, accident (Klugbauer et al., 1995; Fugazzola et al., 1995; the induction of the RET gene rearrangement has been observed in human fetal thyroid tissue transplanted into SCID mice as early as 2 days following 50 Gy exposure (Mizuno et al., 1997). The direct action model *Correspondence: YE Nikiforov or radiation-induced does not explain the long Received 8 April 1999; revised 14 June 1999; accepted 14 June 1999 period of time between radiation exposure and RET rearrangements in radiation-induced thyroid tumors YE Nikiforov et al 6331 development of the disease, but this lag period would was obtained. Comparison of the nucleotide composi- be expected if the RET/PTC3 fusion is necessary, but tion of the chimeric introns in each tumor with both not sucient to cause tumor growth. The lag period contributing germline sequences allowed us to deter- would be the time required for a second genetic event to occur. Indeed, it has been shown that constitutive expression of RET/PTC1 (another form of RET rearrangement also leading to the activation of the truncated RET receptor) in well-di€erentiated thyroid PCCL3 cells was associated with loss of di€erentiated properties, but was not sucient for transformation (Santoro et al., 1993). However, malignant phenotype was obtained by cotransfection with H-RAS or K-RAS . An alternative to the direct pathway is that radiation does not act directly to produce the RET/PTC3 fusion, but instead may cause persistent genomic instability, which eventually produces one or more mutations that result in tumor progression (reviewed in Nikiforov and Fagin, 1998). Whether the gene fusion occurs early or late in the process of tumor cell evolution, two general mechan- isms for such fusions are possible. One mechanism would be for concerted breaks to form in the RET and ELE1 genes. The RET/PTC3 fusion could then be formed by end-joining. This mechanism would be expected to be reciprocal. Concerted breaks could form as the direct result of radiation damage, or due to the action of an endonuclease. The alternative pathway to gene fusion would be for breaks to occur independently and to be joined at random. This mechanism would not be expected to produce reciprocal rearrangements. To explore the potential mechanisms causing the RET/PTC3 fusion in radiation-induced tumors, we identi®ed and analysed the precise breakpoint sites in the RET and ELE1 gene in 12 pediatric post- Chernobyl papillary thyroid carcinomas. The struc- tures of these breakpoints were most consistent with the direct mechanism of induction of RET/PTC3 fusion via reciprocal exchange at a pair of double strand breaks which are in proximity to each other due to the folding of the chromosome 10.

Results

A series of PCRs with all possible combinations of primers located sequentially through intervals of 200 ± 250 bp across ELE1 intron 5 (1670 bp) and RET intron 11 (1843 bp) was performed to amplify genomic DNA from each tumor (Figure 1). The ampli®cation product containing ELE1/RET breakpoint was ob- tained in 12 of 22 tumors. The breakpoint sites within the two genes were identi®ed by sequencing of PCR products. Then, the reciprocal sequence of RET/ELE1

Figure 2 Nucleotide sequences of ELE1/RET and RET/ELE1 breakpoint sites in 12 tumors and germ-line sequences of the ELE1 and RET genes. Arrows indicate positions of the Figure 1 Strategy for breakpoint site identi®cation by PCR breakpoints. Nucleotides deleted or inserted (duplicated) are ampli®cation of genomic DNA from tumors with RET/PTC3 shown in boxes. Short- between germline using all possible combinations of the forward primers (F1 ± F7) RET and ELE1 sequences in the proximity of the breakpoint sites and the reverse primers (R1 ± R7) are indicated in italics and underlined RET rearrangements in radiation-induced thyroid tumors YE Nikiforov et al 6332 mine the precise composition of the breakpoints and C24 in RET) were close to topoisomerase II modi®cation of sequences at these sites (Figure 2). The recognition sites. location and distribution of the breakpoint sites in the Thus, the breakpoint sites in each intron appeared to RET or ELE1 genes are shown in Figure 3. be distributed in a relatively random fashion. However, Analysis of these data revealed that breakpoints in upon further analysis a certain pattern emerged. Despite the ELE1 gene were asymmetrically distributed, with the great range of the chimeric intron lengths (from 304 nine of 12 breakpoints located in the right half of the to 3240 bp), these fusion introns tended to be comprised intron. Most of this half of the intron is occupied by of equal lengths of DNA from each gene. To illustrate, two Alu sequences, and eight breakpoints fell within the 304 bp intron in the C20 fusion contained 152 bp one or the other of these two SINES. Breakpoints in from the ELE1 gene, and 152 bp from the RET gene. the RET intron 11 were distributed more evenly along Similarly, the 3240 bp C14 intron contained 1517 bp the intron, with four cases (C2, C15, C28, C24) from ELE1 and 1723 bp from the RET gene. Moreover, clustered within an 80 bp region. after alignment of the respective introns in opposite None of the RET or ELE1 breakpoints occurred at directions, the position of the breakpoint in one gene exactly the same base or within a similar sequence in showed a correspondence to the position of the any of the 12 tumors. breakpoint in the other gene in each tumor (Figure 4). There was no long-sequence homology between As shown in Figure 4A, such an arrangement aligns ®ve regions contributing to these rearrangements, indicat- of the breakpoint pairs directly across from each other. ing that they are the result of illegitimate rather than The other seven pairs of breakpoints can be aligned by . Short-sequence homology sliding one gene with respect to the other (Figure 4B and (3 ± 5 nucleotides) was found at or immediately C). Interestingly, in the two other previously reported adjacent to breakpoints in seven cases. cases of Chernobyl-related cancers where fusion Each of these inversions was formed via a fairly occurred between the ELE1 intron 5 and RET intron precise reciprocal recombination event. Modi®cation of 11 (CH10 and CH4) (Bongarzone et al., 1997), the breaks sequences at the breakpoint sites was minimal, typically that were joined to form RET/PTC3 can also be aligned involving a 1 ± 3 nucleotide deletion and/or duplication. by this procedure (Figure 4). Four cases had su€ered larger deletions of 9 ± 98 bp. Breakpoints exhibited no particular nucleotide sequence or composition. In the ELE1 intron, break- Discussion point sites were located in regions with an average AT- content of 52%, close to the AT-content of the entire The analysis of the breakpoint sites in the RET and intron, which is 61%. The same was true for the RET ELE1 genes composing RET/PTC3 rearrangement in intron, where breakpoints occurred within regions with these radiation-induced tumors suggests a direct 37% AT-content, the same as the AT-content of the mechanism of RET/PTC3 induction as a result of entire intron. Only one of 12 breakpoints in ELE1 gene random double-strand DNA breaks produced by was located within a 214 bp AT-rich region (76% AT) ionizing radiation, rather than a later occurrence of this at the 5'-end of the intron. Two of the 24 breakpoint mutation due to general destabilization of the . sites in both genes were found at or close to a poly-A This, if the RET/PTC intrachromosomal inversion arose sequence (C8 and C30 in ELE1), and one breakpoint as a result of activation of the recombination machinery was found within a run of six T residues (C11 in or errors in cleavage by enzymes that cut and join DNA ELE1). Short /pyrimidine tracts were seen at two (such as topoisomerase II), one would expect the breaks rearrangement sites (C20, C24). Other direct repeats, to be located within recombinase signal sequences at palindromes, chi-like minisatellite octamer sequences, both participating loci, to have similarity in sequences at or telomere-like repeats were not found at or close to the breakpoints, or to be clustered at certain speci®c breakpoint sites. No perfect vertebrate topoisomerase II consensus sequence occurs within either of the introns. However, if a 1 ± 3 bp mismatch is allowed, three of 24 breakpoint sites (C27 and C17 in ELE1,

Figure 3 Distribution of breakpoint sites (arrows) in the ELE1 and RET genes constituting the RET/PTC3 rearrangement in 12 Figure 4 Alignment of two introns involved in RET/PTC3 post-Chernobyl thyroid carcinomas. Case numbers (in bold) and demonstrates a spatial arrangement of breakpoints in two genes nucleotide positions of breakpoints within introns are shown in 12 of our cases (solid lines) and two cases (CH4, CH10) above arrows. (Alu-Alu repeats; T=topoisomerase II recognition reported by Bongarzone et al. (1997) (dotted lines), as break- sequences; Chi-like=Chi-like sequence, poly-A and poly- points in each tumor correspond to one of three stable patterns T=polynucleotide sequences) (A, B or C) RET rearrangements in radiation-induced thyroid tumors YE Nikiforov et al 6333 hypersensitive DNA regions such as AT-rich regions, stage of cell cycle. Schematic representation of the most Alu repeats, repeated purine/pyrimidine tracts, and other possible mechanism of RET/PTC3 formation in these chromosomal regions with increased lability (fragile) tumors is depicted in Figure 5. sites (Ehrlich et al., 1993; Stary and Sarasin, 1992; In conclusion, our ®ndings indicate that RET/PTC3 Hyrien et al., 1987). By contrast, our results indicate that rearrangements in these radiation-induced tumors are a in post-Chernobyl tumors the RET/PTC breakpoints result of illegitimate reciprocal recombination. The were distributed relatively randomly across the respec- simplest path to such events is ligation of ends that tive introns, except for clustering in the Alu sequences of were produced by double-strand DNA breaks induced one of the two contributing genes, with no breakpoints by radiation at the same time in the same place in a occurring at exactly the same base or within an identical nucleus. sequence in any of the 12 tumors. Although it is possible that cleavage within Alu regions favored recombination, the overall lack of consistency in speci®c DNA sites Materials and methods implicated in illegitimate recombination in mammalian cells suggests a direct induction of these genetic events as Patient population a result of random double-strand DNA breaks. We studied 22 post-Chernobyl papillary thyroid carcinomas Despite the random distributions of the breakpoint with RET/PTC3 rearrangements previously identi®ed by RT ± sites in each intron, the alignment of two introns in PCR (Nikiforov et al., 1997). All patients resided in the areas opposite directions disclosed a certain pattern of of Belarus contaminated as a result of the Chernobyl nuclear correspondence of ELE1 and RET breaks in each accident in April, 1986. They underwent surgery at the tumor. In addition, breakpoint sites in RET/PTC3 in Thyroid Tumor Center in Minsk, Belarus in 1991 and 1992. two previously reported post-Chernobyl cases (Bongar- There were 15 males and seven females in this group. The age of patients at the time of surgery ranged from 5 ± 18 years. zone et al., 1997) showed similar patterns of correspon- dence. Such predictable relative positioning of chromosomal breaks in two di€erent genes can occur if the chromosome is folded in a way to space these distant loci next to each other but pointed in opposite directions at the time of chromosomal breaks. In such cases, passage of a single radiation track can produce concerted double-strand DNA breaks in two chromosomal fragments closely spaced in the thyroid cell nucleus. A number of studies indicate that chromosomes and their domains occupy distinct territories in interphase nuclei, rather than being randomly dispersed throughout the nuclear volume (Lichter et al., 1988; Pinkel et al., 1988). Furthermore, chromosome arms and bands also form exclusive domains within a given territory (Cremer et al., 1996). It has also been demonstrated that nuclear localization of centromeres and other discrete chromo- somal regions is non-random, and varies between di€erent phases of the cell cycle and according to the cell type (Manuelidis, 1984; Ferguson and Ward, 1992; Gerdes et al., 1994). Moreover, it has been shown that for two random chromosomal loci the relationship between interphase distance and genomic separation was not always linear, leading to a model of chromatin arrangement in ¯exible loops of several Mbp arranged along the chromosomal backbone (Yokota et al., 1995). Thus, although two chromosomal loci are located at a considerable linear distance from each other (such as the ELE1 and RET genes), they may be closely spaced in the interphase nucleus because of their position at speci®c areas of chromosomal loop(s). The position of breakpoint sites in DNA recombina- tion is thought to be determined mostly by sequence- speci®c factors, or secondary DNA structures at the breakpoint site. Thus, two distant regions consistently involved in a rearrangement event should possess a Figure 5 Putative mechanism of RET/PTC3 rearrangement certain degree of nucleotide homology between them or based on the results of the current study. Schematic steps of RET/PTC3 formation starting from the linear arrangement of the contain recombinase-speci®c sequences at the cleavage wild-type genes in the long arm of chromosome 10 (top), to site. However, the ELE1 and RET breakpoints do not formation of chromosomal loop dextraposing the two genes display such features. Furthermore, our ®ndings suggest adjacent to each other but pointed in opposite directions, to that the preferential occurrence of this rearrangement simultaneous breaks in both genes at the same sites with reciprocal exchange via end-joining leading to inversion and may be due in large part to the structural organization of gene rearrangement as demonstrated in a linear fashion (bottom). the chromosome, resulting in the proximity of poten- Note that as a result of this reciprocal recombination the regions tially recombinogenic DNA sequences during a certain 1 and 2, internal to the target genes, are also inverted RET rearrangements in radiation-induced thyroid tumors YE Nikiforov et al 6334 Identi®cation of breakpoints ELE1 for each tumor. PCR and sequencing techniques were identical to those described above. The position and PCR-based approach was utilized to identify the precise nucleotide composition of primers for ELE1/RET and breakpoint sites of RET/PTC3 rearrangement located within RET/ELE1, as well as of the oligonucleotide probes are ELE1 intron 5 (1670 bp) and RET intron 11 (1843 bp). For available upon request. these experiments, we determined that the quality of DNA extracted from paran-embedded tissue (performed as previously reported in Nikiforov et al., 1996) was sucient Analysis of breakpoint sites and search for sequence features to amplify fragments of 400 ± 500 bp, and that the break- The sequences obtained from each tumor where compared points in RET and ELE1 may occur at any site across the with the available germline sequences of the RET intron 11 respective introns. Based on the reported sequences of RET and ELE1 intron 5 utilizing the DNAStar program intron 11 (GenBank Acc. # X77860) and ELE1 intron 5 (DNASTAR, Inc.) to identify the breakpoints sites in both (GenBank Acc. # X77859), we designed primers located genes, and to analyse possible modi®cation of nucleotide sequentially through intervals of 200 ± 250 bp across both sequence at the breakpoint sites. introns (Figure 1). A series of PCRs in a ®nal volume of AT-rich regions, Alu sequences, direct DNA repeats, 20 ml with all possible combinations of upstream and palindromes, purine/pyrimidine tracts, telomere-like repeats downstream primers were performed to amplify the genomic (TTAGGG) , chi-like octamer conserved within minisatellite DNA from each tumor as previously described (Nikiforov et n repeats (GG(A/T) GG(A/T)CG), as well as vertebrate al., 1996). Ten ml of each PCR product were loaded on topoisomerase II consensus sequences ((A/G)N(T/ agarose gels, blotted to the nylon membranes and hybridized C)NNCNNG(T/C)NG(G/T) TN(T/C)N(T/C)) were with an internal oligonucleotide probe end-labeled with 32P searched for in both introns and in the 100 bp of DNA (Nikiforov et al., 1997). Another 10 ml of the PCR products surrounding each breakpoint. were electrophoresed in an agarose gel and stained with ethidium bromide. The band of interest was excised, puri®ed, and sequenced with an automated ABI model 377 sequencer using the ABI PRISM Dye Terminator Cycle Sequencing kit (Perkin-Elmer, Foster City, CA, USA). If the initially identi®ed ampli®cation signal was larger than 500 bp or Acknowledgments was visualized only after hybridization, additional internal This work was supported by a TRAC award from Knoll primers were designed to narrow the region in order to Pharmaceuticals to Dr Nikiforov and in parts by grants facilitate sequencing. CA 50706, CA 72597 to Dr Fagin and ES 05652 to Dr After detecting the breakpoint sites in ELE1/RET we Stringer. We are grateful to Dr Fenoglio-Preiser for designed primers to obtain the reciprocal sequence of RET/ valuable comments.

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