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The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage.

Li H, Balajee AS, Su T, Cen B, Hei TK, Weinstein IB - J. Cell Biol. (2008)

Bottom Line: HINT1 deficiency does not affect the formation of gamma-H2AX foci; however, it impairs the removal of gamma-H2AX foci after DNA damage and this is associated with impaired acetylation of gamma-H2AX.HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR.HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA.

ABSTRACT
Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)-induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with gamma-H2AX and ATM. HINT1 deficiency does not affect the formation of gamma-H2AX foci; however, it impairs the removal of gamma-H2AX foci after DNA damage and this is associated with impaired acetylation of gamma-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both gamma-H2AX and ATM.

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HINT1-deficient MEFs display retarded DNA rejoining. (A and B) Analysis of time course kinetics of DNA rejoining by alkaline comet assay. Hint1 MEFs were irradiated with 4 Gy of γ radiation on ice and postincubated for the indicated recovery times. Comet slides were prepared, lysed in an alkaline buffer, and electrophoresed. Comet images were captured using a fluorescent microscope, and the tail moment was analyzed in 100 randomly chosen comets using comet analysis software. Representative comet images and the histogram of cell number versus tail moment (A) and the mean of tail moments observed at different times are shown (B). *, P < 0.005 compared with Hint1 +/+ MEFs (nonparametric Mann-Whitney test). A repeat experiment gave similar results. (C) PFGE analysis of DNA DSB repair in Hint1 MEFs. Hint1 MEFs were irradiated with 40 Gy of γ radiation on ice and postincubated for the indicated recovery times. Agarose plugs were prepared, cells were lysed by proteinase K, and genomic DNA was electrophoresed. The percentage of DSB unrepaired were calculated and DNA-PKcs −/− MEFs were used for comparison. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 compared with Hint1 +/+ MEFs (Student's t test).
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fig6: HINT1-deficient MEFs display retarded DNA rejoining. (A and B) Analysis of time course kinetics of DNA rejoining by alkaline comet assay. Hint1 MEFs were irradiated with 4 Gy of γ radiation on ice and postincubated for the indicated recovery times. Comet slides were prepared, lysed in an alkaline buffer, and electrophoresed. Comet images were captured using a fluorescent microscope, and the tail moment was analyzed in 100 randomly chosen comets using comet analysis software. Representative comet images and the histogram of cell number versus tail moment (A) and the mean of tail moments observed at different times are shown (B). *, P < 0.005 compared with Hint1 +/+ MEFs (nonparametric Mann-Whitney test). A repeat experiment gave similar results. (C) PFGE analysis of DNA DSB repair in Hint1 MEFs. Hint1 MEFs were irradiated with 40 Gy of γ radiation on ice and postincubated for the indicated recovery times. Agarose plugs were prepared, cells were lysed by proteinase K, and genomic DNA was electrophoresed. The percentage of DSB unrepaired were calculated and DNA-PKcs −/− MEFs were used for comparison. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 compared with Hint1 +/+ MEFs (Student's t test).

Mentions: To further address whether the spontaneous and prolonged γ-H2AX foci staining in the HINT1-deficient MEFs was caused by an inability to remove H2AX after DNA breaks had been repaired or by the presence of unrepaired DNA breaks, we compared DNA-rejoining capabilities of Hint1 +/+ and −/− MEFs. We first performed alkaline comet assays because this method is sensitive enough to detect low levels of DNA breaks. Hint1 MEFs were either mock treated or irradiated with a 4-Gy dose of γ radiation on ice, and then allowed to recover at 37°C for 0 min, 1 h, or 8 h. The tail moment was analyzed in 100 randomly chosen comets of the three genotypes of cells. Consistent with the spontaneous γ-H2AX foci staining, the unirradiated Hint1 −/− MEFs exhibited an increased tail moment compared with the Hint1 +/+ MEFs (Fig. 6, A and B). The Hint1 +/+ MEFs rejoined most of the DNA breaks after 1 h. However, the DNA breaks in the Hint1 −/− MEFs remained unrepaired at 8 h after exposure to IR (Fig. 6, A and B). Therefore, HINT1 deficiency also causes retarded DNA repair of spontaneous and IR-induced DNA strand breaks.


The HINT1 tumor suppressor regulates both gamma-H2AX and ATM in response to DNA damage.

Li H, Balajee AS, Su T, Cen B, Hei TK, Weinstein IB - J. Cell Biol. (2008)

HINT1-deficient MEFs display retarded DNA rejoining. (A and B) Analysis of time course kinetics of DNA rejoining by alkaline comet assay. Hint1 MEFs were irradiated with 4 Gy of γ radiation on ice and postincubated for the indicated recovery times. Comet slides were prepared, lysed in an alkaline buffer, and electrophoresed. Comet images were captured using a fluorescent microscope, and the tail moment was analyzed in 100 randomly chosen comets using comet analysis software. Representative comet images and the histogram of cell number versus tail moment (A) and the mean of tail moments observed at different times are shown (B). *, P < 0.005 compared with Hint1 +/+ MEFs (nonparametric Mann-Whitney test). A repeat experiment gave similar results. (C) PFGE analysis of DNA DSB repair in Hint1 MEFs. Hint1 MEFs were irradiated with 40 Gy of γ radiation on ice and postincubated for the indicated recovery times. Agarose plugs were prepared, cells were lysed by proteinase K, and genomic DNA was electrophoresed. The percentage of DSB unrepaired were calculated and DNA-PKcs −/− MEFs were used for comparison. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 compared with Hint1 +/+ MEFs (Student's t test).
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fig6: HINT1-deficient MEFs display retarded DNA rejoining. (A and B) Analysis of time course kinetics of DNA rejoining by alkaline comet assay. Hint1 MEFs were irradiated with 4 Gy of γ radiation on ice and postincubated for the indicated recovery times. Comet slides were prepared, lysed in an alkaline buffer, and electrophoresed. Comet images were captured using a fluorescent microscope, and the tail moment was analyzed in 100 randomly chosen comets using comet analysis software. Representative comet images and the histogram of cell number versus tail moment (A) and the mean of tail moments observed at different times are shown (B). *, P < 0.005 compared with Hint1 +/+ MEFs (nonparametric Mann-Whitney test). A repeat experiment gave similar results. (C) PFGE analysis of DNA DSB repair in Hint1 MEFs. Hint1 MEFs were irradiated with 40 Gy of γ radiation on ice and postincubated for the indicated recovery times. Agarose plugs were prepared, cells were lysed by proteinase K, and genomic DNA was electrophoresed. The percentage of DSB unrepaired were calculated and DNA-PKcs −/− MEFs were used for comparison. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 compared with Hint1 +/+ MEFs (Student's t test).
Mentions: To further address whether the spontaneous and prolonged γ-H2AX foci staining in the HINT1-deficient MEFs was caused by an inability to remove H2AX after DNA breaks had been repaired or by the presence of unrepaired DNA breaks, we compared DNA-rejoining capabilities of Hint1 +/+ and −/− MEFs. We first performed alkaline comet assays because this method is sensitive enough to detect low levels of DNA breaks. Hint1 MEFs were either mock treated or irradiated with a 4-Gy dose of γ radiation on ice, and then allowed to recover at 37°C for 0 min, 1 h, or 8 h. The tail moment was analyzed in 100 randomly chosen comets of the three genotypes of cells. Consistent with the spontaneous γ-H2AX foci staining, the unirradiated Hint1 −/− MEFs exhibited an increased tail moment compared with the Hint1 +/+ MEFs (Fig. 6, A and B). The Hint1 +/+ MEFs rejoined most of the DNA breaks after 1 h. However, the DNA breaks in the Hint1 −/− MEFs remained unrepaired at 8 h after exposure to IR (Fig. 6, A and B). Therefore, HINT1 deficiency also causes retarded DNA repair of spontaneous and IR-induced DNA strand breaks.

Bottom Line: HINT1 deficiency does not affect the formation of gamma-H2AX foci; however, it impairs the removal of gamma-H2AX foci after DNA damage and this is associated with impaired acetylation of gamma-H2AX.HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR.HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities.

View Article: PubMed Central - PubMed

Affiliation: Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA.

ABSTRACT
Hint1 is a haploinsufficient tumor suppressor gene and the underlying molecular mechanisms for its tumor suppressor function are unknown. In this study we demonstrate that HINT1 participates in ionizing radiation (IR)-induced DNA damage responses. In response to IR, HINT1 is recruited to IR-induced foci (IRIF) and associates with gamma-H2AX and ATM. HINT1 deficiency does not affect the formation of gamma-H2AX foci; however, it impairs the removal of gamma-H2AX foci after DNA damage and this is associated with impaired acetylation of gamma-H2AX. HINT1 deficiency also impairs acetylation of ATM and activation of ATM and its downstream effectors, and retards DNA repair, in response to IR. HINT1-deficient cells exhibit resistance to IR-induced apoptosis and several types of chromosomal abnormalities. Our findings suggest that the tumor suppressor function of HINT1 is caused by, at least in part, its normal role in enhancing cellular responses to DNA damage by regulating the functions of both gamma-H2AX and ATM.

Show MeSH
Related in: MedlinePlus