DNA breaks at fragile sites generate oncogenic RET/PTC rearrangements in human thyroid cells

Abstract

Human chromosomal fragile sites are regions of the genome that are prone to DNA breakage, and are classified as common or rare, depending on their frequency in the population. Common fragile sites frequently coincide with the location of genes involved in carcinogenic chromosomal translocations, suggesting their role in cancer formation. However, there has been no direct evidence linking breakage at fragile sites to the formation of a cancer-specific translocation. Here, we studied the involvement of fragile sites in the formation of RET/PTC rearrangements, which are frequently found in papillary thyroid carcinoma (PTC). These rearrangements are commonly associated with radiation exposure; however, most of the tumors found in adults are not linked to radiation. In this study, we provide structural and biochemical evidence that the RET, CCDC6 and NCOA4 genes participating in two major types of RET/PTC rearrangements, are located in common fragile sites FRA10C and FRA10G, and undergo DNA breakage after exposure to fragile site-inducing chemicals. Moreover, exposure of human thyroid cells to these chemicals results in the formation of cancer-specific RET/PTC rearrangements. These results provide the direct evidence for the involvement of chromosomal fragile sites in the generation of cancer-specific rearrangements in human cells.

Figure 1

FISH on metaphase chromosomes of HTori-3 cells after treatment with fragile site-inducing chemicals. (a) Negative control without treatment showing smooth chromosomes with intact RET (red) signal. (b) Exposure to APH resulting in irregular chromosome contours and one RET signal (red) showing split in the signal whereas four other RET signal are intact. Centromeric probe for chromosome 10 is labeled in green. (c) Exposure to BrdU+2-AP resulting in the disruption of CCDC6 (green) while NCOA4 is intact (red). (d) Exposure to APH+2-AP+BrdU resulting in split in RET (red).

Figure 2

LM-PCR detection of breaks formed in HTori-3 cells after treatment with APH. LM-PCR detection of DNA breaks formed in HTori-3 cells at intron 11 of RET (a), the fragile site FRA3B (c), and the non-fragile 12p12.3 region (d) after treatment with APH. The same reaction was carried out as in (a) for intron 11 of RET, but using DNA from cells without APH treatment (b). Last lane of each gel is a 100 bp molecular weight ladder. Bands below 100bp correspond to primer dimers. Asterisks mark DNA fragments which were sequenced, and results are shown in Figure 3.

Figure 3

Location of breakpoints within intron 11 of RET induced by treatment with APH. (a) DNA samples from lanes 1, and 3–6 in Figure 2a (marked with asterisks) were sequenced, and six breakpoints are identified and indicated by black arrowheads. The locations of known breakpoints found in tumors containing RET/PTC rearrangements are indicated by grey arrowheads (Bongarzone et al., 1997; Klugbauer et al., 2001). The grey arrow corresponds to the RET-7 primer with a dual biotin label (grey circles), which is annealed to exon 12 of the RET gene. The black solid and dashed arrows correspond to the RET-R1b and RET-R1 nested PCR primers, respectively. The sequence of intron 11 is italicized. (b) The distance of each induced breakpoint from the 5′ end of the RET-R1b primer and the nearest patient tumor breakpoint was listed.

Figure 4

Detection of RET/PTC rearrangements in HT-ori3 cells after treatment with fragile site-inducing chemicals. (a) Detection of RET/PTC rearrangements in representative RT-PCR experiment after exposure to APH+2-AP+BrdU. PC, Positive control. (b) Number of rearrangement events detected in untreated cells and cells exposed to APH+2-AP+BrdU. Five independent experiments were carried out for each treatment, and, each experiment analyzed 10⁶ cells.