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SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells.

Wang Z, Yuan H, Roth M, Stark JM, Bhatia R, Chen WY - Oncogene (2012)

Bottom Line: The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations.SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin.These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA.

ABSTRACT
BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

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SIRT1 altered functions of DNA damage repair in CML cells(a) Flow cytometry analysis of HR and NHEJ repair after SIRT1 knockdown using stably integrated reporter constructs in KCL-22 cells. I-SceI was introduced by electroporation and DS-Red was used for transfection control. The repair rate was normalized to DS-Red. (b–d) NHEJ repair assay using inducible I-SceI expression. Gene knockdown was carried out in inducible I-SceI expressing EJ5-GFP KCL-22 cells. 48 hrs after gene knockdown, I-SceI expression was induced by doxycycline for 72 hrs, and GFP positive cells were analyzed by flow cytometry. b, effect of Ku70 and SIRT1 knockdown; c, effect of NBS1 knockdown; d, effect of RAD51 knockdown. ** indicates P<0.05.
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Figure 6: SIRT1 altered functions of DNA damage repair in CML cells(a) Flow cytometry analysis of HR and NHEJ repair after SIRT1 knockdown using stably integrated reporter constructs in KCL-22 cells. I-SceI was introduced by electroporation and DS-Red was used for transfection control. The repair rate was normalized to DS-Red. (b–d) NHEJ repair assay using inducible I-SceI expression. Gene knockdown was carried out in inducible I-SceI expressing EJ5-GFP KCL-22 cells. 48 hrs after gene knockdown, I-SceI expression was induced by doxycycline for 72 hrs, and GFP positive cells were analyzed by flow cytometry. b, effect of Ku70 and SIRT1 knockdown; c, effect of NBS1 knockdown; d, effect of RAD51 knockdown. ** indicates P<0.05.

Mentions: To further determine the role of SIRT1 in DNA damage repair in CML cells, we generated stable KCL-22 cell clones bearing a NHEJ reporter construct EJ5-GFP,44 and clones carrying a HR reporter DR-GFP.45 After introducing DNA damage on these reporters with the endonuclease I-SceI by electroporation, we found that NHEJ was more efficient than HR for DNA damage repair in KCL-22 cells, and that SIRT1 knockdown suppressed both NHEJ and HR activity with more prominent effect on NHEJ (Figure 6a).


SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells.

Wang Z, Yuan H, Roth M, Stark JM, Bhatia R, Chen WY - Oncogene (2012)

SIRT1 altered functions of DNA damage repair in CML cells(a) Flow cytometry analysis of HR and NHEJ repair after SIRT1 knockdown using stably integrated reporter constructs in KCL-22 cells. I-SceI was introduced by electroporation and DS-Red was used for transfection control. The repair rate was normalized to DS-Red. (b–d) NHEJ repair assay using inducible I-SceI expression. Gene knockdown was carried out in inducible I-SceI expressing EJ5-GFP KCL-22 cells. 48 hrs after gene knockdown, I-SceI expression was induced by doxycycline for 72 hrs, and GFP positive cells were analyzed by flow cytometry. b, effect of Ku70 and SIRT1 knockdown; c, effect of NBS1 knockdown; d, effect of RAD51 knockdown. ** indicates P<0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376246&req=5

Figure 6: SIRT1 altered functions of DNA damage repair in CML cells(a) Flow cytometry analysis of HR and NHEJ repair after SIRT1 knockdown using stably integrated reporter constructs in KCL-22 cells. I-SceI was introduced by electroporation and DS-Red was used for transfection control. The repair rate was normalized to DS-Red. (b–d) NHEJ repair assay using inducible I-SceI expression. Gene knockdown was carried out in inducible I-SceI expressing EJ5-GFP KCL-22 cells. 48 hrs after gene knockdown, I-SceI expression was induced by doxycycline for 72 hrs, and GFP positive cells were analyzed by flow cytometry. b, effect of Ku70 and SIRT1 knockdown; c, effect of NBS1 knockdown; d, effect of RAD51 knockdown. ** indicates P<0.05.
Mentions: To further determine the role of SIRT1 in DNA damage repair in CML cells, we generated stable KCL-22 cell clones bearing a NHEJ reporter construct EJ5-GFP,44 and clones carrying a HR reporter DR-GFP.45 After introducing DNA damage on these reporters with the endonuclease I-SceI by electroporation, we found that NHEJ was more efficient than HR for DNA damage repair in KCL-22 cells, and that SIRT1 knockdown suppressed both NHEJ and HR activity with more prominent effect on NHEJ (Figure 6a).

Bottom Line: The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations.SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin.These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA.

ABSTRACT
BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

Show MeSH
Related in: MedlinePlus