DNA topoisomerases participate in fragility of the oncogene RET

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.

Figure 1. LM-PCR detection of DNA breaks within intron 11 of RET.

DNA breaks formed in intron 11 of RET were detected by LM-PCR without treatment (A) or following 24-hour treatment with 0.4 µM APH alone (B) or in combination with the DNA topoisomerase I and II catalytic inhibitors 6 nM betulinic acid (C) or 3 µM merbarone (D). Representative gels are shown. Each lane represents a separate PCR reaction using DNA from approximately 1300 cells. The first lane of each gel is a 100-bp molecular weight ladder.

Figure 2. Location of APH-induced DNA breakpoints within intron 11 of RET detected by LM-PCR.

(A) The location of 144 APH-induced DNA breakpoints isolated within intron 11 of RET by LM-PCR were determined by DNA sequencing (arrowheads). DNA breaks identified on the strand shown by the sequence are indicated by black arrowheads, and on the complementary strand by grey arrowheads. Open arrowheads indicate the locations of known patient breakpoints observed in PTC tumors containing RET/PTC rearrangements [,–50]. The location of BanI and XbaI digestion sites within intron 11 are labeled. RET primer sets (see Table S1) are indicated by arrows. Lines with circles are dual biotin-labeled primers followed by two nested primers. The dashed black lines represent primer set 1, dashed grey lines primer set 2, solid black lines primer set 3, and solid grey lines primer set 4. The sequence of intron 11 is displayed along with the flanking exon 10 and 11 sequences, shown in italics. (B) The distribution of APH-induced DNA breakpoints within intron 11 are depicted as a smooth curve fit of the percentage of breakpoints (y axis) located every 50 bp of intron 11 in a 5’ to 3’ direction (x axis).

Figure 3. Comparison of 144 APH-induced DNA breakpoints to predicted DNA topoisomerase I and II cleavage sites.

(A) Topoisomerase I cleavage sites within RET intron 11 were predicted based on the consensus [5’-(A/T/G) (C/G/A) (A/T) (T/C)-3’] immediately upstream of the cleavage site [51], compared to APH-induced DNA breakpoints, and represented as the distance in bp from each APH breakpoint to the closest predicted cleavage site (x axis). A positive distance refers to the closest topoisomase I cleavage site being downstream of the APH breakpoint, and a negative distance being upstream. The percentage of all APH-induced breakpoints is displayed on the y axis. (B) Topoisomerase IIα cleavage sites were predicted using the consensus sequence [5’-(no A) (no T) (A/no C) (-) (C/no A) (-) (-) (-) (-) (no T) (-) (T/no G) (C/ no A) (-)-3’] [52], where breakage occurs between the nucleotides five and six. The locations of APH-induced breakpoints were compared to the predicted sites and represented in the same manner as in (A).

Figure 4. Comparison of CPT 11- and VP-16- induced topoisomerase I and II cleavage to predicted cleavage sites.

Topoisomerase I and II DNA cleavage was induced by treatment of HTori-3 cells with 10 µM CPT-11 or VP-16, respectively, for 1.5 hours. The location of CPT 11- (n=22) or VP-16- (n=21) induced DNA cleavage within RET intron 11 was detected using LM-PCR and RET primer set 1 and compared to either topoisomerase I (CPT 11) or II (VP-16) predicted cleavage sites. A positive distance indicates the predicted cleavage site is downstream of the drug-induced site, and a negative distance indicates the predicted cleavage site is upstream. The percentage of all drug-induced breakpoints is represented on the y axis.

Figure 5. Location of 144 APH-induced RET intron 11 breakpoints on predicted DNA secondary structures.

(A) A representative predicted DNA secondary structure is shown, corresponding to the RET gene nucleotides 43,610,735 to 43,611,034 (hg37.2), with the locations of APH-induced breakpoints indicated by arrows. The program Mfold was used to predict potential DNA secondary structures within the RET intron 11 sequence, by analyzing 300-nt fragments one at a time with a 150-nt shift increment on both DNA strands, and selecting the most energetically favorable structure for each fragment. The location of each APH-induced DNA breakpoint on the predicted secondary structures was analyzed. (B) The percentage of the APH-induced RET breakpoints is shown for each DNA secondary structural features recognized by the DNA topoisomerases I and II.

Figure 6. Effects of DNA topoisomerase catalytic inhibitors on the APH-induced common fragile site breakage.

(A) HTori-3 cells were treated with 0.4 µM APH in combination with the topoisomerase I and II catalytic inhibitors, 6 nM betulinic acid (BA) or 3 µM merbarone, for 24 hours. LM-PCR was used to detect DNA breaks within RET intron 11 using RET primer set 1. The frequency of DNA breakage within RET intron 11 following 0.4 µM APH treatment combined with 6 nM betulinic acid or 3 µM merbarone significantly decreases compared to APH treatment alone (*P ≤ 3.34E-3), to levels similar to untreated cells. (B) The frequency of DNA breakage within FHIT intron 4, located within the APH-induced common fragile site FRA3B, shows a significant increase with 0.4 µM APH treatment. As with RET intron 11, the rate of APH-induced DNA breakage within FHIT intron 4 significantly decreased when combined with BA or merbarone (*P ≤ 7.21E-6). All data were averaged from 5–7 replicated experiments. All statistical analyses were performed using a two-tailed Student’s T-test. Error bars indicate standard deviations.

Figure 7. Rate of APH-induced DNA breakage in combination with CPT 11 treatment.

Co-treatment of HTori-3 cells with APH and CPT 11 significantly increased the level of DNA breakage within RET intron 11 and FRA3B relative to APH treatment alone (*P ≤ 5.76E-4). This breakage was significantly greater than in the non-fragile 12p12.3 region ( ^# P ≤ 2.59E-5). The level of DNA breakage for each treatment was measured using LM-PCR and averaged over at least three independent experiments. Significance was calculated using a two-tailed Student’s T-test. Error bars represent standard deviations.