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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

Inhibition or knockdown of Rad51 also leads to persistent γ-H2AXmRNA expression of HRR components in tumors and normal mucosa evaluated in biopsy material from human specimens using microarray hybridization (A) Rad51 and (B) BRCA2. (C) SW480 cells were depleted of Rad51 using siRNA for 16 h and then treated with NCS for 24 h. Whole cell extracts were immunoblotted followed by detection by the indicated antibodies. (D) SW480 cells were treated with B02 and/or ZVAD for 24 h followed by NCS for 24 h. Whole cell extracts were immunoblotted, followed by detection by the indicated antibodies. β-actin was used as a loading control.
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Figure 5: Inhibition or knockdown of Rad51 also leads to persistent γ-H2AXmRNA expression of HRR components in tumors and normal mucosa evaluated in biopsy material from human specimens using microarray hybridization (A) Rad51 and (B) BRCA2. (C) SW480 cells were depleted of Rad51 using siRNA for 16 h and then treated with NCS for 24 h. Whole cell extracts were immunoblotted followed by detection by the indicated antibodies. (D) SW480 cells were treated with B02 and/or ZVAD for 24 h followed by NCS for 24 h. Whole cell extracts were immunoblotted, followed by detection by the indicated antibodies. β-actin was used as a loading control.

Mentions: Among the proteins involved in homologous recombination repair, Rad51 and BRCA2 represent is a key modulators. As a first step to assess their importance in CRC, we determined the RNA levels corresponding to these genes by microarray hybridization of 181 CRC biopsy samples in direct comparison with 215 normal mucosa biopsies. And indeed, both Rad51 and BRCA2 expression levels were upregulated in the tumors to a highly significant extent (Figure 5A-5B; refer Supplemental Table S1 for raw data and patient characteristics). No significant relation was observed between the tumor grade, and expression of Rad51 or BRCA2 (Supplemental Figure S5). The mRNA expression levels of Rad51 and BRCA2 were highest in S phase of the cell cycle (Supplemental Figure S6). Since many tumors contain a high proportion of dividing cells, S-phase-associated expression may partially explain why Rad51 and BRCA2 levels are high in tumors. In any case, HR mediators are enhanced in CRCs and may thus support CRC progression. This prompted us to test whether direct HR inhibitors may sensitize CRC cells towards DSBs, as 5-FU does. To this end, we firstly depleted Rad51, a principal mediator of HR, and found that this strongly augmented the levels of γ-H2AX 24 h after NCS treatment (Figure 5C). Next, we employed the newly developed Rad51 inhibitor B02 [27]. We treated CRC cells with B02, with or without co-treatment with NCS and analyzed the accumulation of γ-H2AX (Figure 5D). We found that cells treated with B02 and NCS showed a strong accumulation of γ-H2AX; however, cells treated with B02 alone did so too. Since it has been previously reported that apoptosis can cause accumulation of γ-H2AX, we next determined the extent of B02- induced apoptosis by detecting cleaved caspase 3. We found that treatment with B02 alone was sufficient to induce apoptosis (Figure 5). To distinguish this caspase-mediated γ-H2AX accumulation from immediate DDR, we inhibited caspase activity by ZVAD-FMK. On simultaneous treatment of cells with B02 and ZVAD-FMK, apoptosis was reduced, as seen by lack of cleaved caspase 3. We now probed for the accumulation of γ-H2AX under these new conditions and found that treatment with B02 alone did not lead to any detectable γ-H2AX accumulation. Importantly however, treatment with B02 and NCS caused massive γ-H2AX accumulation similar to the combination of 5-FU and NCS together, even when the caspases were blocked (Figure 5D). These results strongly argue that Rad51 inhibition hinders the repair of ds DNA breaks. This raises the perspective of using Rad51 inhibitors to radiosensitize rectal cancer cells, especially in the context of cancer cell resistance towards 5-FU.


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)

Inhibition or knockdown of Rad51 also leads to persistent γ-H2AXmRNA expression of HRR components in tumors and normal mucosa evaluated in biopsy material from human specimens using microarray hybridization (A) Rad51 and (B) BRCA2. (C) SW480 cells were depleted of Rad51 using siRNA for 16 h and then treated with NCS for 24 h. Whole cell extracts were immunoblotted followed by detection by the indicated antibodies. (D) SW480 cells were treated with B02 and/or ZVAD for 24 h followed by NCS for 24 h. Whole cell extracts were immunoblotted, followed by detection by the indicated antibodies. β-actin was used as a loading control.
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Related In: Results  -  Collection

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Figure 5: Inhibition or knockdown of Rad51 also leads to persistent γ-H2AXmRNA expression of HRR components in tumors and normal mucosa evaluated in biopsy material from human specimens using microarray hybridization (A) Rad51 and (B) BRCA2. (C) SW480 cells were depleted of Rad51 using siRNA for 16 h and then treated with NCS for 24 h. Whole cell extracts were immunoblotted followed by detection by the indicated antibodies. (D) SW480 cells were treated with B02 and/or ZVAD for 24 h followed by NCS for 24 h. Whole cell extracts were immunoblotted, followed by detection by the indicated antibodies. β-actin was used as a loading control.
Mentions: Among the proteins involved in homologous recombination repair, Rad51 and BRCA2 represent is a key modulators. As a first step to assess their importance in CRC, we determined the RNA levels corresponding to these genes by microarray hybridization of 181 CRC biopsy samples in direct comparison with 215 normal mucosa biopsies. And indeed, both Rad51 and BRCA2 expression levels were upregulated in the tumors to a highly significant extent (Figure 5A-5B; refer Supplemental Table S1 for raw data and patient characteristics). No significant relation was observed between the tumor grade, and expression of Rad51 or BRCA2 (Supplemental Figure S5). The mRNA expression levels of Rad51 and BRCA2 were highest in S phase of the cell cycle (Supplemental Figure S6). Since many tumors contain a high proportion of dividing cells, S-phase-associated expression may partially explain why Rad51 and BRCA2 levels are high in tumors. In any case, HR mediators are enhanced in CRCs and may thus support CRC progression. This prompted us to test whether direct HR inhibitors may sensitize CRC cells towards DSBs, as 5-FU does. To this end, we firstly depleted Rad51, a principal mediator of HR, and found that this strongly augmented the levels of γ-H2AX 24 h after NCS treatment (Figure 5C). Next, we employed the newly developed Rad51 inhibitor B02 [27]. We treated CRC cells with B02, with or without co-treatment with NCS and analyzed the accumulation of γ-H2AX (Figure 5D). We found that cells treated with B02 and NCS showed a strong accumulation of γ-H2AX; however, cells treated with B02 alone did so too. Since it has been previously reported that apoptosis can cause accumulation of γ-H2AX, we next determined the extent of B02- induced apoptosis by detecting cleaved caspase 3. We found that treatment with B02 alone was sufficient to induce apoptosis (Figure 5). To distinguish this caspase-mediated γ-H2AX accumulation from immediate DDR, we inhibited caspase activity by ZVAD-FMK. On simultaneous treatment of cells with B02 and ZVAD-FMK, apoptosis was reduced, as seen by lack of cleaved caspase 3. We now probed for the accumulation of γ-H2AX under these new conditions and found that treatment with B02 alone did not lead to any detectable γ-H2AX accumulation. Importantly however, treatment with B02 and NCS caused massive γ-H2AX accumulation similar to the combination of 5-FU and NCS together, even when the caspases were blocked (Figure 5D). These results strongly argue that Rad51 inhibition hinders the repair of ds DNA breaks. This raises the perspective of using Rad51 inhibitors to radiosensitize rectal cancer cells, especially in the context of cancer cell resistance towards 5-FU.

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