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5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair.

Srinivas US, Dyczkowski J, Beißbarth T, Gaedcke J, Mansour WY, Borgmann K, Dobbelstein M - Oncotarget (2015)

Bottom Line: However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci.Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa.Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Oncology, University Medical Center Göttingen, Germany.

ABSTRACT
Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

No MeSH data available.


Related in: MedlinePlus

5-FU reduces the homologous recombination repair(A) DRGFP assay to assess the HRR. The normalized GFP intensity, as determined by flow cytometry upon transfection of reporter plasmids, to assess the activity of HRR upon drug treatment. GFP positive cells were measured using flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells. (B) Schematic representation of the DRGFP assay. The plasmid contains two non-functional, truncated GFP cassettes. The cassette at the 5′ end has an I-SceI restriction endonuclease cleavage site. The synthesis of I-SceI in the cell by the virtue of a closed circular transfected expression plasmid produces a double strand break in the GFP cassette as shown. If the cell uses the GFP cassette at the 3′ end (iGFP) to repair the double strand break using HRR a functional GFP cassette is generated. (C) NHEJ assay in analogy to A. GFP positive cells were measured using a flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells (D) Schematic representation of the NHEJ assay. The NHEJ plasmid contains two exons that together encode GFP, separated by an adenovirus derived (AD) exon. The Hind III restriction endonuclease sites are present on either side of the AD exon, as depicted in the figure. The plasmid was first digested with Hind III in vitro and then transfected in to HeLa cells. If the cell repairs the damage using NHEJ, the splice donors and acceptor sites are so placed that a functional GFP transcript is produced. Results from at least three independent experiments are shown with columns representing the standard error of the mean.
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Figure 2: 5-FU reduces the homologous recombination repair(A) DRGFP assay to assess the HRR. The normalized GFP intensity, as determined by flow cytometry upon transfection of reporter plasmids, to assess the activity of HRR upon drug treatment. GFP positive cells were measured using flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells. (B) Schematic representation of the DRGFP assay. The plasmid contains two non-functional, truncated GFP cassettes. The cassette at the 5′ end has an I-SceI restriction endonuclease cleavage site. The synthesis of I-SceI in the cell by the virtue of a closed circular transfected expression plasmid produces a double strand break in the GFP cassette as shown. If the cell uses the GFP cassette at the 3′ end (iGFP) to repair the double strand break using HRR a functional GFP cassette is generated. (C) NHEJ assay in analogy to A. GFP positive cells were measured using a flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells (D) Schematic representation of the NHEJ assay. The NHEJ plasmid contains two exons that together encode GFP, separated by an adenovirus derived (AD) exon. The Hind III restriction endonuclease sites are present on either side of the AD exon, as depicted in the figure. The plasmid was first digested with Hind III in vitro and then transfected in to HeLa cells. If the cell repairs the damage using NHEJ, the splice donors and acceptor sites are so placed that a functional GFP transcript is produced. Results from at least three independent experiments are shown with columns representing the standard error of the mean.

Mentions: NCS, like γ-radiation, primarily induces double strand DNA breaks. The persistent accumulation of γ-H2AX suggested that 5-FU might interfere with at least one of the principal repair mechanisms for DSBs, HRR or NHEJ. To assess this directly, we carried out plasmid based reporter assays. These assays are based on the repair of a GFP-encoding DNA upon transfection of reporter plasmids [24, 25]. Strikingly, we found that 5-FU reduces homologous recombination repair (Figure 2A-2B). In contrast, we did not observe any significant change in NHEJ efficacy upon treatment with either 5-FU or NCS (Figure 2C-D). We conclude that 5-FU treatment reduces the efficiency by that a cell carries out HRR, whereas the mechanisms of NHEJ remain largely unaffected.


5-Fluorouracil sensitizes colorectal tumor cells towards double stranded DNA breaks by interfering with homologous recombination repair.

Srinivas US, Dyczkowski J, Beißbarth T, Gaedcke J, Mansour WY, Borgmann K, Dobbelstein M - Oncotarget (2015)

5-FU reduces the homologous recombination repair(A) DRGFP assay to assess the HRR. The normalized GFP intensity, as determined by flow cytometry upon transfection of reporter plasmids, to assess the activity of HRR upon drug treatment. GFP positive cells were measured using flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells. (B) Schematic representation of the DRGFP assay. The plasmid contains two non-functional, truncated GFP cassettes. The cassette at the 5′ end has an I-SceI restriction endonuclease cleavage site. The synthesis of I-SceI in the cell by the virtue of a closed circular transfected expression plasmid produces a double strand break in the GFP cassette as shown. If the cell uses the GFP cassette at the 3′ end (iGFP) to repair the double strand break using HRR a functional GFP cassette is generated. (C) NHEJ assay in analogy to A. GFP positive cells were measured using a flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells (D) Schematic representation of the NHEJ assay. The NHEJ plasmid contains two exons that together encode GFP, separated by an adenovirus derived (AD) exon. The Hind III restriction endonuclease sites are present on either side of the AD exon, as depicted in the figure. The plasmid was first digested with Hind III in vitro and then transfected in to HeLa cells. If the cell repairs the damage using NHEJ, the splice donors and acceptor sites are so placed that a functional GFP transcript is produced. Results from at least three independent experiments are shown with columns representing the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494959&req=5

Figure 2: 5-FU reduces the homologous recombination repair(A) DRGFP assay to assess the HRR. The normalized GFP intensity, as determined by flow cytometry upon transfection of reporter plasmids, to assess the activity of HRR upon drug treatment. GFP positive cells were measured using flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells. (B) Schematic representation of the DRGFP assay. The plasmid contains two non-functional, truncated GFP cassettes. The cassette at the 5′ end has an I-SceI restriction endonuclease cleavage site. The synthesis of I-SceI in the cell by the virtue of a closed circular transfected expression plasmid produces a double strand break in the GFP cassette as shown. If the cell uses the GFP cassette at the 3′ end (iGFP) to repair the double strand break using HRR a functional GFP cassette is generated. (C) NHEJ assay in analogy to A. GFP positive cells were measured using a flow cytometer and normalized to the control. Note that these assays require extreme transfection efficiencies and were therefore performed only in HeLa cells (D) Schematic representation of the NHEJ assay. The NHEJ plasmid contains two exons that together encode GFP, separated by an adenovirus derived (AD) exon. The Hind III restriction endonuclease sites are present on either side of the AD exon, as depicted in the figure. The plasmid was first digested with Hind III in vitro and then transfected in to HeLa cells. If the cell repairs the damage using NHEJ, the splice donors and acceptor sites are so placed that a functional GFP transcript is produced. Results from at least three independent experiments are shown with columns representing the standard error of the mean.
Mentions: NCS, like γ-radiation, primarily induces double strand DNA breaks. The persistent accumulation of γ-H2AX suggested that 5-FU might interfere with at least one of the principal repair mechanisms for DSBs, HRR or NHEJ. To assess this directly, we carried out plasmid based reporter assays. These assays are based on the repair of a GFP-encoding DNA upon transfection of reporter plasmids [24, 25]. Strikingly, we found that 5-FU reduces homologous recombination repair (Figure 2A-2B). In contrast, we did not observe any significant change in NHEJ efficacy upon treatment with either 5-FU or NCS (Figure 2C-D). We conclude that 5-FU treatment reduces the efficiency by that a cell carries out HRR, whereas the mechanisms of NHEJ remain largely unaffected.

Bottom Line: However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci.Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa.Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Oncology, University Medical Center Göttingen, Germany.

ABSTRACT
Malignant tumors of the rectum are treated by neoadjuvant radiochemotherapy. This involves a combination of 5-fluorouracil (5-FU) and double stranded DNA-break (DSB)-inducing radiotherapy. Here we explored how 5-FU cooperates with DSB-induction to achieve sustainable DNA damage in colorectal cancer (CRC) cells. After DSB induction by neocarzinostatin, phosphorylated histone 2AX (γ-H2AX) rapidly accumulated but then largely vanished within a few hours. In contrast, when CRC cells were pre-treated with 5-FU, gammaH2AX remained for at least 24 hours. GFP-reporter assays revealed that 5-FU decreases the efficiency of homologous recombination (HR) repair. However, 5-FU did not prevent the initial steps of HR repair, such as the accumulation of RPA and Rad51 at nuclear foci. Thus, we propose that 5-FU interferes with the continuation of HR repair, e. g. the synthesis of new DNA strands. Two key mediators of HR, Rad51 and BRCA2, were found upregulated in CRC biopsies as compared to normal mucosa. Inhibition of HR by targeting Rad51 enhanced DNA damage upon DSB-inducing treatment, outlining an alternative way of enhancing therapeutic efficacy. Taken together, our results strongly suggest that interfering with HR represents a key mechanism to enhance the efficacy when treating CRC with DNA-damaging therapy.

No MeSH data available.


Related in: MedlinePlus