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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

Reduced HRR is not a consequence of a shift in cell cycle distribution(A) Propidium iodide staining and flow cytometry of SW480 cells treated with DMSO, 5 μM 5-FU and/or 100 ng/ml NCS as detailed in Figure 1A. (B) Quantification of the cell cycle distribution. (C) EdU assay in SW480 cells to determine the percentage of cells in S phase. The cells were first treated as shown in Figure 1A; 2 h prior to fixation, 15 μM EdU was added to the cells. The cells were then fixed on the plate, and EdU was detected by Alexa 488 azide. Fluorescence intensity was quantified as described for γ-H2AX in Figure 1C.
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Figure 4: Reduced HRR is not a consequence of a shift in cell cycle distribution(A) Propidium iodide staining and flow cytometry of SW480 cells treated with DMSO, 5 μM 5-FU and/or 100 ng/ml NCS as detailed in Figure 1A. (B) Quantification of the cell cycle distribution. (C) EdU assay in SW480 cells to determine the percentage of cells in S phase. The cells were first treated as shown in Figure 1A; 2 h prior to fixation, 15 μM EdU was added to the cells. The cells were then fixed on the plate, and EdU was detected by Alexa 488 azide. Fluorescence intensity was quantified as described for γ-H2AX in Figure 1C.

Mentions: Double strand break repair processes are cell cycle dependent. Most of the NHEJ is restricted to the G1 phase, while HRR is active in the S and G2 phases [26]. We therefore assessed whether the reduction in homologous recombination was a consequence of a shift in the cell cycle phase distribution upon treatment with 5-FU. To this end, we performed propidium iodide staining and flow cytometry of SW480 cells (Figure 4A) and found that 5-FU actually increases the proportion of cells in S phase (Figure 4B). This was further confirmed by the detection of DNA synthesizing cells through 5-ethynyl-2′deoxyuridine (EdU) incorporation. Indeed, 5-FU treatment increased the fraction of EdU positive cells (Figure 4C) This indicated that the observed reduction in HRR upon 5-FU treatment did not occur due to arrest of the cells in an unfavorable cell cycle phase. The accumulation of cells in S phase upon 5-FU treatment is compatible with active HRR. Nonetheless, 5-FU leads to a reduction in this repair process, possibly through interference with DNA polymerization during HRR.


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)

Reduced HRR is not a consequence of a shift in cell cycle distribution(A) Propidium iodide staining and flow cytometry of SW480 cells treated with DMSO, 5 μM 5-FU and/or 100 ng/ml NCS as detailed in Figure 1A. (B) Quantification of the cell cycle distribution. (C) EdU assay in SW480 cells to determine the percentage of cells in S phase. The cells were first treated as shown in Figure 1A; 2 h prior to fixation, 15 μM EdU was added to the cells. The cells were then fixed on the plate, and EdU was detected by Alexa 488 azide. Fluorescence intensity was quantified as described for γ-H2AX in Figure 1C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Reduced HRR is not a consequence of a shift in cell cycle distribution(A) Propidium iodide staining and flow cytometry of SW480 cells treated with DMSO, 5 μM 5-FU and/or 100 ng/ml NCS as detailed in Figure 1A. (B) Quantification of the cell cycle distribution. (C) EdU assay in SW480 cells to determine the percentage of cells in S phase. The cells were first treated as shown in Figure 1A; 2 h prior to fixation, 15 μM EdU was added to the cells. The cells were then fixed on the plate, and EdU was detected by Alexa 488 azide. Fluorescence intensity was quantified as described for γ-H2AX in Figure 1C.
Mentions: Double strand break repair processes are cell cycle dependent. Most of the NHEJ is restricted to the G1 phase, while HRR is active in the S and G2 phases [26]. We therefore assessed whether the reduction in homologous recombination was a consequence of a shift in the cell cycle phase distribution upon treatment with 5-FU. To this end, we performed propidium iodide staining and flow cytometry of SW480 cells (Figure 4A) and found that 5-FU actually increases the proportion of cells in S phase (Figure 4B). This was further confirmed by the detection of DNA synthesizing cells through 5-ethynyl-2′deoxyuridine (EdU) incorporation. Indeed, 5-FU treatment increased the fraction of EdU positive cells (Figure 4C) This indicated that the observed reduction in HRR upon 5-FU treatment did not occur due to arrest of the cells in an unfavorable cell cycle phase. The accumulation of cells in S phase upon 5-FU treatment is compatible with active HRR. Nonetheless, 5-FU leads to a reduction in this repair process, possibly through interference with DNA polymerization during HRR.

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