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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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The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.
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fig2: The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.

Mentions: The results described in the previous section clearly demonstrate that HINT1 plays a role in DDR and is involved in the chromatin remodeling pathway after IR. Because IR triggers the recruitment to discrete nuclear foci of several proteins that play a role in signaling DNA damage and repair (Paull et al., 2000), it was of interest to determine whether the HINT1 protein also forms IRIF. For this purpose a FLAG-tagged Hint1 expression plasmid was constructed and transiently expressed in Hint1 −/− MEFs. These cells were either untreated or treated with a 4-Gy dose of γ radiation and fixed at the indicated times. IRIF formation was analyzed by immunofluorescent staining with specific antibodies to the FLAG tag or γ-H2AX. We found that in untreated cells, HINT1 is localized predominantly in the cytoplasm, although diffuse staining of HINT1 was also observed in the nucleus (Fig. 2 A). The cytoplasmic patterns of FLAG-Hint1 staining did not change in response to IR. However, we observed remarkable changes of FLAG-Hint1 localization in the nucleus in response to IR. There were very few foci in untreated cells, and these foci did not colocalize with γ-H2AX. At 30 min after IR, HINT1 formed distinct nuclear foci that colocalized with γ-H2AX foci (Fig. 2 A). These foci remained detectable at 3 h and disappeared at 18 h after IR. Interestingly, the kinetics of HINT1 IRIF formation correlates well with that of γ-H2AX foci (Fig. 2 B). The fact that HINT1 is recruited to IRIF provides strong evidence that HINT1 plays an important role in DDR.


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)

The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.
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fig2: The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.
Mentions: The results described in the previous section clearly demonstrate that HINT1 plays a role in DDR and is involved in the chromatin remodeling pathway after IR. Because IR triggers the recruitment to discrete nuclear foci of several proteins that play a role in signaling DNA damage and repair (Paull et al., 2000), it was of interest to determine whether the HINT1 protein also forms IRIF. For this purpose a FLAG-tagged Hint1 expression plasmid was constructed and transiently expressed in Hint1 −/− MEFs. These cells were either untreated or treated with a 4-Gy dose of γ radiation and fixed at the indicated times. IRIF formation was analyzed by immunofluorescent staining with specific antibodies to the FLAG tag or γ-H2AX. We found that in untreated cells, HINT1 is localized predominantly in the cytoplasm, although diffuse staining of HINT1 was also observed in the nucleus (Fig. 2 A). The cytoplasmic patterns of FLAG-Hint1 staining did not change in response to IR. However, we observed remarkable changes of FLAG-Hint1 localization in the nucleus in response to IR. There were very few foci in untreated cells, and these foci did not colocalize with γ-H2AX. At 30 min after IR, HINT1 formed distinct nuclear foci that colocalized with γ-H2AX foci (Fig. 2 A). These foci remained detectable at 3 h and disappeared at 18 h after IR. Interestingly, the kinetics of HINT1 IRIF formation correlates well with that of γ-H2AX foci (Fig. 2 B). The fact that HINT1 is recruited to IRIF provides strong evidence that HINT1 plays an important role in DDR.

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