Abstract
Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APH-induced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication.
Highlights
The oncogene RET is involved in recurrent chromosomal rearrangements found in thyroid and more recently in lung cancer [1,2,3,4]
It rearranges with various genes in a class of rearrangements known as RET/PTC rearrangements, which are known to be carcinogenic for thyroid cells and result in development of papillary thyroid carcinoma (PTC) [5]
We analyzed the initial events of APH-induced common fragile site breakage within the RET oncogene
Summary
The oncogene RET is involved in recurrent chromosomal rearrangements found in thyroid and more recently in lung cancer [1,2,3,4]. In thyroid cells, it rearranges with various genes in a class of rearrangements known as RET/PTC rearrangements, which are known to be carcinogenic for thyroid cells and result in development of papillary thyroid carcinoma (PTC) [5]. We found that the formation of RET/PTC1 rearrangements can be induced in human thyroid cells through treatment with fragile site-inducing chemicals [9]. It is conceivable that exposure to chemicals that can induce fragile sites may contribute to the increasing rates of thyroid cancer
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