Limits...
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 and/or NCS do not decrease the recruitment of RPA2 and Rad51(A) Confocal microscopy images of SW480 cells treated with, DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2 h (B) RPA2 foci were counted in at least 100 cells per sample per experiment (n=3), and the percentage of cells containing detectable foci were plotted as a histogram. The RPA2 foci were counted using the FociCounter software, (C) Representative IF images for Rad51 colocalized with γH2AX foci at the indicated time points after treatment with DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2, 8 and 24 h, (D) Quantification of Rad51 colocalized with γH2AX foci counted in at least 100 cells each at the indicated time points. Error bars represent the SEM of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494959&req=5

Figure 3: 5-FU and/or NCS do not decrease the recruitment of RPA2 and Rad51(A) Confocal microscopy images of SW480 cells treated with, DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2 h (B) RPA2 foci were counted in at least 100 cells per sample per experiment (n=3), and the percentage of cells containing detectable foci were plotted as a histogram. The RPA2 foci were counted using the FociCounter software, (C) Representative IF images for Rad51 colocalized with γH2AX foci at the indicated time points after treatment with DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2, 8 and 24 h, (D) Quantification of Rad51 colocalized with γH2AX foci counted in at least 100 cells each at the indicated time points. Error bars represent the SEM of two independent experiments.

Mentions: In principle, impaired factor recruitment could represent one reason for reduced HRR. One group of such factors is represented by the RPA complex. We therefore assessed the recruitment of RPA2, a subunit of the RPA complex, to see if 5-FU treatment interferes with early stages of HRR. 5-FU did not impair the recruitment of RPA2 to the intranuclear structures, as indicated by the foci formation of RPA2 detected by immunofluorescence and digital image analysis (Figure 3A and 3B). On the contrary, treatment with 5-FU and NCS together led to an increase in RPA2 foci formation 2 h post NCS treatment. The more pronounced foci formation 2 h post NCS treatment is in accordance with the extensive DNA damage induced by 5-FU in combination with NCS. At longer times post NCS treatment, i. e. 8 and 24 h, cells that were pretreated with 5-FU showed even more pronounced RPA2 foci and more discrete colocalization with γ-H2AX (Supplemental Figure S3 A-C). It can therefore be concluded that 5-FU does not impair the recruitment of RPA2. We next investigated the recruitment of Rad51, a key component of HRR, in response to treatment with 5-FU and NCS (Figure 3C and 3D, and Supplemental Figure S4). Treatment with NCS alone led to the formation of Rad51 foci that mostly colocalized with γ-H2AX foci at 2 h post treatment and increased further at 8 h post treatment before being reduced to near-background levels 24 h post treatment. In contrast, treatment with a combination of 5-FU and NCS gave rise to sustained Rad51 foci 24 h post NCS treatment. We conclude that 5-FU does not impair the recruitment of repair proteins, like RPA2 and Rad51. This suggests that 5-FU-induced HRR inhibition does not occur through interference with the early steps of 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)

5-FU and/or NCS do not decrease the recruitment of RPA2 and Rad51(A) Confocal microscopy images of SW480 cells treated with, DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2 h (B) RPA2 foci were counted in at least 100 cells per sample per experiment (n=3), and the percentage of cells containing detectable foci were plotted as a histogram. The RPA2 foci were counted using the FociCounter software, (C) Representative IF images for Rad51 colocalized with γH2AX foci at the indicated time points after treatment with DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2, 8 and 24 h, (D) Quantification of Rad51 colocalized with γH2AX foci counted in at least 100 cells each at the indicated time points. Error bars represent the SEM of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: 5-FU and/or NCS do not decrease the recruitment of RPA2 and Rad51(A) Confocal microscopy images of SW480 cells treated with, DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2 h (B) RPA2 foci were counted in at least 100 cells per sample per experiment (n=3), and the percentage of cells containing detectable foci were plotted as a histogram. The RPA2 foci were counted using the FociCounter software, (C) Representative IF images for Rad51 colocalized with γH2AX foci at the indicated time points after treatment with DMSO, 5 μM 5-FU + 100ng /ml NCS, 5 μM 5-FU, and 100 ng/ml NCS for 2, 8 and 24 h, (D) Quantification of Rad51 colocalized with γH2AX foci counted in at least 100 cells each at the indicated time points. Error bars represent the SEM of two independent experiments.
Mentions: In principle, impaired factor recruitment could represent one reason for reduced HRR. One group of such factors is represented by the RPA complex. We therefore assessed the recruitment of RPA2, a subunit of the RPA complex, to see if 5-FU treatment interferes with early stages of HRR. 5-FU did not impair the recruitment of RPA2 to the intranuclear structures, as indicated by the foci formation of RPA2 detected by immunofluorescence and digital image analysis (Figure 3A and 3B). On the contrary, treatment with 5-FU and NCS together led to an increase in RPA2 foci formation 2 h post NCS treatment. The more pronounced foci formation 2 h post NCS treatment is in accordance with the extensive DNA damage induced by 5-FU in combination with NCS. At longer times post NCS treatment, i. e. 8 and 24 h, cells that were pretreated with 5-FU showed even more pronounced RPA2 foci and more discrete colocalization with γ-H2AX (Supplemental Figure S3 A-C). It can therefore be concluded that 5-FU does not impair the recruitment of RPA2. We next investigated the recruitment of Rad51, a key component of HRR, in response to treatment with 5-FU and NCS (Figure 3C and 3D, and Supplemental Figure S4). Treatment with NCS alone led to the formation of Rad51 foci that mostly colocalized with γ-H2AX foci at 2 h post treatment and increased further at 8 h post treatment before being reduced to near-background levels 24 h post treatment. In contrast, treatment with a combination of 5-FU and NCS gave rise to sustained Rad51 foci 24 h post NCS treatment. We conclude that 5-FU does not impair the recruitment of repair proteins, like RPA2 and Rad51. This suggests that 5-FU-induced HRR inhibition does not occur through interference with the early steps of 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