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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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Loss of HINT1 impairs the acetylation and activation of ATM. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and whole cell lysates were collected after 10 min. Immunoprecipitates were obtained using an anti-ATM antibody and resolved on SDS-PAGE. Immunoblots were then performed using either an ATM antibody or an acetyllysine antibody. (B) Hint1 +/+, +/−, and −/− MEFs were untreated (0) or treated with 4 Gy of IR. After the indicted times, cell lysates were collected and immunoblot analyses done for the levels of p-ATM-S1981 and ATM. Actin was used as an internal loading control. (C) Hint1 +/+, +/−, and −/− MEFs were untreated or treated with 4 Gy of IR and fixed after the indicated times. Cells were then stained with an antibody to p-ATM-S1981 (red) and counterstained for the nucleus with DAPI (blue). Bar, 10 μm. (D and E) Cell lysates from Hint1 MEFs untreated (0) or treated with 4 Gy of IR (D) or 100 nM bleomycin (E) were collected at the indicated times, and immunoblot analyses were done for the levels of p-Chk1-S317, p-Chk2-T68, and p-p53-S15. A repeated experiment yielded similar results.
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fig3: Loss of HINT1 impairs the acetylation and activation of ATM. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and whole cell lysates were collected after 10 min. Immunoprecipitates were obtained using an anti-ATM antibody and resolved on SDS-PAGE. Immunoblots were then performed using either an ATM antibody or an acetyllysine antibody. (B) Hint1 +/+, +/−, and −/− MEFs were untreated (0) or treated with 4 Gy of IR. After the indicted times, cell lysates were collected and immunoblot analyses done for the levels of p-ATM-S1981 and ATM. Actin was used as an internal loading control. (C) Hint1 +/+, +/−, and −/− MEFs were untreated or treated with 4 Gy of IR and fixed after the indicated times. Cells were then stained with an antibody to p-ATM-S1981 (red) and counterstained for the nucleus with DAPI (blue). Bar, 10 μm. (D and E) Cell lysates from Hint1 MEFs untreated (0) or treated with 4 Gy of IR (D) or 100 nM bleomycin (E) were collected at the indicated times, and immunoblot analyses were done for the levels of p-Chk1-S317, p-Chk2-T68, and p-p53-S15. A repeated experiment yielded similar results.

Mentions: Our previous study showed that Hint1 −/− MEFs are more resistant to the cytotoxic effects of high doses of IR (Su et al., 2003). Because cells with extensive DNA damage are normally eliminated by apoptosis (van Gent et al., 2001), this resistance to IR-induced cytotoxicity of Hint1 −/− cells suggests that these cells are defective in this response. Therefore, the ATM checkpoint pathway of DDR was examined in HINT1-deficient cells. Because ATM is recruited to IRIF (Bakkenist and Kastan, 2003; Carson et al., 2003), we first examined whether HINT1 is associated with ATM. We immunoprecipitated the HINT1 protein from lysates of FLAG-Hint1–expressing MEFs, with or without prior radiation of these cells. We found that HINT1 is associated with ATM even before radiation, and after radiation this association is increased (Fig. 2 C). The impaired acetylation of γ-H2AX in HINT1-deficient cells prompted us to further examine whether HINT1 is also required for ATM acetylation. For this purpose the ATM protein was immunoprecipitated from cell lysates of Hint1 MEFs before and after treating the cells with 4 Gy of γ radiation, and then an acetyllysine antibody was used in immunoblots to detect acetylation of ATM. We found that IR caused a marked increase in ATM acetylation in the Hint1 +/+ cells. However, in Hint1 +/− MEFs the acetylation of ATM was reduced, and in the Hint1 −/− MEFs there was no detectable acetylation of ATM (Fig. 3 A). Therefore, HINT1 is required for the acetylation of ATM induced by IR.


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)

Loss of HINT1 impairs the acetylation and activation of ATM. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and whole cell lysates were collected after 10 min. Immunoprecipitates were obtained using an anti-ATM antibody and resolved on SDS-PAGE. Immunoblots were then performed using either an ATM antibody or an acetyllysine antibody. (B) Hint1 +/+, +/−, and −/− MEFs were untreated (0) or treated with 4 Gy of IR. After the indicted times, cell lysates were collected and immunoblot analyses done for the levels of p-ATM-S1981 and ATM. Actin was used as an internal loading control. (C) Hint1 +/+, +/−, and −/− MEFs were untreated or treated with 4 Gy of IR and fixed after the indicated times. Cells were then stained with an antibody to p-ATM-S1981 (red) and counterstained for the nucleus with DAPI (blue). Bar, 10 μm. (D and E) Cell lysates from Hint1 MEFs untreated (0) or treated with 4 Gy of IR (D) or 100 nM bleomycin (E) were collected at the indicated times, and immunoblot analyses were done for the levels of p-Chk1-S317, p-Chk2-T68, and p-p53-S15. A repeated experiment yielded similar results.
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fig3: Loss of HINT1 impairs the acetylation and activation of ATM. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and whole cell lysates were collected after 10 min. Immunoprecipitates were obtained using an anti-ATM antibody and resolved on SDS-PAGE. Immunoblots were then performed using either an ATM antibody or an acetyllysine antibody. (B) Hint1 +/+, +/−, and −/− MEFs were untreated (0) or treated with 4 Gy of IR. After the indicted times, cell lysates were collected and immunoblot analyses done for the levels of p-ATM-S1981 and ATM. Actin was used as an internal loading control. (C) Hint1 +/+, +/−, and −/− MEFs were untreated or treated with 4 Gy of IR and fixed after the indicated times. Cells were then stained with an antibody to p-ATM-S1981 (red) and counterstained for the nucleus with DAPI (blue). Bar, 10 μm. (D and E) Cell lysates from Hint1 MEFs untreated (0) or treated with 4 Gy of IR (D) or 100 nM bleomycin (E) were collected at the indicated times, and immunoblot analyses were done for the levels of p-Chk1-S317, p-Chk2-T68, and p-p53-S15. A repeated experiment yielded similar results.
Mentions: Our previous study showed that Hint1 −/− MEFs are more resistant to the cytotoxic effects of high doses of IR (Su et al., 2003). Because cells with extensive DNA damage are normally eliminated by apoptosis (van Gent et al., 2001), this resistance to IR-induced cytotoxicity of Hint1 −/− cells suggests that these cells are defective in this response. Therefore, the ATM checkpoint pathway of DDR was examined in HINT1-deficient cells. Because ATM is recruited to IRIF (Bakkenist and Kastan, 2003; Carson et al., 2003), we first examined whether HINT1 is associated with ATM. We immunoprecipitated the HINT1 protein from lysates of FLAG-Hint1–expressing MEFs, with or without prior radiation of these cells. We found that HINT1 is associated with ATM even before radiation, and after radiation this association is increased (Fig. 2 C). The impaired acetylation of γ-H2AX in HINT1-deficient cells prompted us to further examine whether HINT1 is also required for ATM acetylation. For this purpose the ATM protein was immunoprecipitated from cell lysates of Hint1 MEFs before and after treating the cells with 4 Gy of γ radiation, and then an acetyllysine antibody was used in immunoblots to detect acetylation of ATM. We found that IR caused a marked increase in ATM acetylation in the Hint1 +/+ cells. However, in Hint1 +/− MEFs the acetylation of ATM was reduced, and in the Hint1 −/− MEFs there was no detectable acetylation of ATM (Fig. 3 A). Therefore, HINT1 is required for the acetylation of ATM induced by IR.

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