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TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1.

Zou LH, Shang ZF, Tan W, Liu XD, Xu QZ, Song M, Wang Y, Guan H, Zhang SM, Yu L, Zhong CG, Zhou PK - Oncotarget (2015)

Bottom Line: The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis.Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs.Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs.

View Article: PubMed Central - PubMed

Affiliation: School of Public Heath, Central South University, Changsha, Hunan Province 410078, P. R. China.

ABSTRACT
TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.

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Upregulation of TNKS1BP1 by ionizing radiation (IR) and its effect on the radiosensitivity of cells(A) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in HeLa cells at given times after exposed to 4 Gy of γ-rays. (B) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in MCF7, HepG2 and L02 cells at 4 h after different doses of γ-rays. (C) Increased TNKS1BP1 protein levels in both cytoplasm and nuclei in HeLa cells at 4 h after 4 Gy of γ-rays. (D) Quantitative real-time RT-PCR analysis showing the increased mRNA expression of TNKS1BP1 in HeLa cells at 2 h after 4 Gy of γ-rays. (E) Depletion of TNKS1BP1 expression in HeLa cells mediated by specific shRNA. (F) Depletion of TNKS1BP1 expression in HepG2 cells mediated by specific shRNA. (G) TNKS1BP1 depleted HeLa-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HeLa-NC cells. (H) TNKS1BP1 depleted HepG2-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HepG2-NC cells.
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Figure 1: Upregulation of TNKS1BP1 by ionizing radiation (IR) and its effect on the radiosensitivity of cells(A) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in HeLa cells at given times after exposed to 4 Gy of γ-rays. (B) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in MCF7, HepG2 and L02 cells at 4 h after different doses of γ-rays. (C) Increased TNKS1BP1 protein levels in both cytoplasm and nuclei in HeLa cells at 4 h after 4 Gy of γ-rays. (D) Quantitative real-time RT-PCR analysis showing the increased mRNA expression of TNKS1BP1 in HeLa cells at 2 h after 4 Gy of γ-rays. (E) Depletion of TNKS1BP1 expression in HeLa cells mediated by specific shRNA. (F) Depletion of TNKS1BP1 expression in HepG2 cells mediated by specific shRNA. (G) TNKS1BP1 depleted HeLa-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HeLa-NC cells. (H) TNKS1BP1 depleted HepG2-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HepG2-NC cells.

Mentions: We firstly investigated whether TNKS1BP1 was involved in the IR-induced DNA damage response. Growing HeLa cells were irradiated with 4 Gy γ-rays, and harvested at different time points post irradiation. Immunoblotting analysis revealed that the expression level of TNKS1BP1 increased at least as early as 2 h after irradiation. The inducible expression of TNKS1BP1 by ionizing irradiation reached a peak level at about 4 – 8 h (Figure 1A). To test whether the IR-induced expression occurred in different cell lines, we gave human breast cancer MCF-7 cells, human hepatocellular cancer HepG2 cells and human normal liver L02 cells different doses of γ-rays. Levels of TNKS1BP1 protein in cell lysates were detected by immunoblotting analysis at 4 h after irradiation. An increased expression of TNKS1BP1 was observed in these three cell lines after irradiation (Figure 1B). As TNKS1BP1 has been reported to localize in both cytoplasm and nucleus, we studied which part mainly contributes to IR-induced TNKS1BP1 overexpression by fractionating nucleus and cytoplasm and detecting the respective expression levels of TNKS1BP1. As shown in Figure 1C, the expression of TNKS1BP1 was increased in both the cytoplasm and nuclear fractions. We further performed a real-time PCR experiment and found that the mRNA levels of TNKS1BP1 were significantly increased in 4 Gy γ-irradiated HeLa cells (Figure 1D).


TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1.

Zou LH, Shang ZF, Tan W, Liu XD, Xu QZ, Song M, Wang Y, Guan H, Zhang SM, Yu L, Zhong CG, Zhou PK - Oncotarget (2015)

Upregulation of TNKS1BP1 by ionizing radiation (IR) and its effect on the radiosensitivity of cells(A) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in HeLa cells at given times after exposed to 4 Gy of γ-rays. (B) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in MCF7, HepG2 and L02 cells at 4 h after different doses of γ-rays. (C) Increased TNKS1BP1 protein levels in both cytoplasm and nuclei in HeLa cells at 4 h after 4 Gy of γ-rays. (D) Quantitative real-time RT-PCR analysis showing the increased mRNA expression of TNKS1BP1 in HeLa cells at 2 h after 4 Gy of γ-rays. (E) Depletion of TNKS1BP1 expression in HeLa cells mediated by specific shRNA. (F) Depletion of TNKS1BP1 expression in HepG2 cells mediated by specific shRNA. (G) TNKS1BP1 depleted HeLa-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HeLa-NC cells. (H) TNKS1BP1 depleted HepG2-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HepG2-NC cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Upregulation of TNKS1BP1 by ionizing radiation (IR) and its effect on the radiosensitivity of cells(A) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in HeLa cells at given times after exposed to 4 Gy of γ-rays. (B) Immunoblotting hybridization showing the increased expression of TNKS1BP1 protein in MCF7, HepG2 and L02 cells at 4 h after different doses of γ-rays. (C) Increased TNKS1BP1 protein levels in both cytoplasm and nuclei in HeLa cells at 4 h after 4 Gy of γ-rays. (D) Quantitative real-time RT-PCR analysis showing the increased mRNA expression of TNKS1BP1 in HeLa cells at 2 h after 4 Gy of γ-rays. (E) Depletion of TNKS1BP1 expression in HeLa cells mediated by specific shRNA. (F) Depletion of TNKS1BP1 expression in HepG2 cells mediated by specific shRNA. (G) TNKS1BP1 depleted HeLa-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HeLa-NC cells. (H) TNKS1BP1 depleted HepG2-shTNKS1BP1 cells became much more sensitive to IR as compared to the control HepG2-NC cells.
Mentions: We firstly investigated whether TNKS1BP1 was involved in the IR-induced DNA damage response. Growing HeLa cells were irradiated with 4 Gy γ-rays, and harvested at different time points post irradiation. Immunoblotting analysis revealed that the expression level of TNKS1BP1 increased at least as early as 2 h after irradiation. The inducible expression of TNKS1BP1 by ionizing irradiation reached a peak level at about 4 – 8 h (Figure 1A). To test whether the IR-induced expression occurred in different cell lines, we gave human breast cancer MCF-7 cells, human hepatocellular cancer HepG2 cells and human normal liver L02 cells different doses of γ-rays. Levels of TNKS1BP1 protein in cell lysates were detected by immunoblotting analysis at 4 h after irradiation. An increased expression of TNKS1BP1 was observed in these three cell lines after irradiation (Figure 1B). As TNKS1BP1 has been reported to localize in both cytoplasm and nucleus, we studied which part mainly contributes to IR-induced TNKS1BP1 overexpression by fractionating nucleus and cytoplasm and detecting the respective expression levels of TNKS1BP1. As shown in Figure 1C, the expression of TNKS1BP1 was increased in both the cytoplasm and nuclear fractions. We further performed a real-time PCR experiment and found that the mRNA levels of TNKS1BP1 were significantly increased in 4 Gy γ-irradiated HeLa cells (Figure 1D).

Bottom Line: The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis.Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs.Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs.

View Article: PubMed Central - PubMed

Affiliation: School of Public Heath, Central South University, Changsha, Hunan Province 410078, P. R. China.

ABSTRACT
TNKS1BP1 was originally identified as an interaction protein of tankyrase 1, which belongs to the poly(ADP-ribose) polymerase (PARP) superfamily. PARP members play important roles for example in DNA repair, telomere stability and mitosis regulation. Although the TNKS1BP1 protein was considered to be a poly(ADP-ribosyl)ation acceptor of tankyrase 1, its function is still unknown. Here we firstly identified that TNKS1BP1 was up-regulated by ionizing radiation (IR) and the depletion of TNKS1BP1 significantly sensitized cancer cells to IR. Neutral comet assay, pulsed-field gel electrophoresis, and γH2AX foci analysis indicated that TNKS1BP1 is required for the efficient repair of DNA double-strand breaks (DSB). The TNKS1BP1 protein was demonstrated to interact with DNA-dependent protein kinase (DNA-PKcs) and poly(ADP-ribose) polymerase 1 (PARP-1), by co-immunoprecipitation analysis. Moreover, TNKS1BP1 was shown to promote the association of PARP-1 and DNA-PKcs. Overexpression of TNKS1BP1 induced the autophosphorylation of DNA-PKcs/Ser2056 in a PARP-1 dependent manner, which contributed to an increased capability of DNA DSB repair. Inhibition of PARP-1 blocked the TNKS1BP1-mediated DNA-PKcs autophosphorylation and attenuated the PARylation of DNA-PKcs. TNKS1BP1 is a newly described component of the DNA DSB repair machinery, which provides much more mechanistic evidence for the rationale of developing effective anticancer measures by targeting PARP-1 and DNA-PKcs.

Show MeSH
Related in: MedlinePlus