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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 causes resistance to IR-induced apoptosis. SW480 cells were transfected with control shRNA or Hint1-shRNA and selected with G418, and then stable clones were pooled. (A) Whole cell lysates collected at the indicated times from untreated or IR (4 Gy)-treated pools were used for immunoblot analysis of HINT1, p-ATM-S1981, p-Chk1-S317, and p-Chk2-T68. Actin was used as a loading control. (B) Whole cell lysates of control or IR (4 Gy)-treated pools were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. (C) Control SW480 pools and SW480 pools in which HINT1 was stably knocked down by shRNA were either untreated (0 h) or treated with 4 Gy of IR. Apoptotic cells were analyzed by sub-G1 content using a flow cytometry method. A repeat experiment yielded similar results. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 when compared with the control shRNA-transfected cells at each time point.
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fig5: Loss of HINT1 causes resistance to IR-induced apoptosis. SW480 cells were transfected with control shRNA or Hint1-shRNA and selected with G418, and then stable clones were pooled. (A) Whole cell lysates collected at the indicated times from untreated or IR (4 Gy)-treated pools were used for immunoblot analysis of HINT1, p-ATM-S1981, p-Chk1-S317, and p-Chk2-T68. Actin was used as a loading control. (B) Whole cell lysates of control or IR (4 Gy)-treated pools were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. (C) Control SW480 pools and SW480 pools in which HINT1 was stably knocked down by shRNA were either untreated (0 h) or treated with 4 Gy of IR. Apoptotic cells were analyzed by sub-G1 content using a flow cytometry method. A repeat experiment yielded similar results. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 when compared with the control shRNA-transfected cells at each time point.

Mentions: Because deficient expression of HINT1 causes decreased induction of the stress-inducible genes described in Fig. 4 A, it was of interest to determine whether HINT1-deficient cells are also impaired in induction of apoptosis. However, we were not able to detect a significant increase of apoptosis even in the Hint1 +/+ MEFs after treatment with 4 Gy of γ radiation (unpublished data). Therefore, we developed stable derivatives of SW480 colon cancer cells in which HINT1 is suppressed by transfection with a Hint1–short hairpin RNA (shRNA) vector (Weiske and Huber, 2005). For comparison we also developed a control SW480 derivative that had been transfected with a control shRNA vector. Expression of the HINT1 protein was nearly undetectable in Hint1-shRNA SW480 cells. Both cell lines displayed similar growth rate. These cells also exhibited reduced activation of ATM and downstream targets such as Chk1 and Chk2 in response to IR (Fig. 5 A). The level of the p21 protein was also reduced (Fig. 5 B). After treatment with 4 Gy of γ radiation, the level of p21 was induced in the control shRNA-transfected cells, whereas the induction of p21 was markedly reduced in the Hint1-shRNA–transfected cells (Fig. 5 B). Both the control and HINT1-deficient SW480 cells displayed activation of γ-H2AX with or without IR, perhaps reflecting the combined effects of loss of HINT1 expression and their transformed state (unpublished data). Using FACS flow cytometry we quantitated the sub-G1 population as an index of apoptosis. Without IR the percentage of apoptosis in both cell types was ∼5%. At 48 h after IR this rose to ∼25% in the control cells, but only to ∼15% in the Hint1-shRNA knockdown cells (Fig. 5 C). Therefore, decreased expression of HINT1 significantly impairs the induction of apoptosis 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 causes resistance to IR-induced apoptosis. SW480 cells were transfected with control shRNA or Hint1-shRNA and selected with G418, and then stable clones were pooled. (A) Whole cell lysates collected at the indicated times from untreated or IR (4 Gy)-treated pools were used for immunoblot analysis of HINT1, p-ATM-S1981, p-Chk1-S317, and p-Chk2-T68. Actin was used as a loading control. (B) Whole cell lysates of control or IR (4 Gy)-treated pools were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. (C) Control SW480 pools and SW480 pools in which HINT1 was stably knocked down by shRNA were either untreated (0 h) or treated with 4 Gy of IR. Apoptotic cells were analyzed by sub-G1 content using a flow cytometry method. A repeat experiment yielded similar results. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 when compared with the control shRNA-transfected cells at each time point.
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fig5: Loss of HINT1 causes resistance to IR-induced apoptosis. SW480 cells were transfected with control shRNA or Hint1-shRNA and selected with G418, and then stable clones were pooled. (A) Whole cell lysates collected at the indicated times from untreated or IR (4 Gy)-treated pools were used for immunoblot analysis of HINT1, p-ATM-S1981, p-Chk1-S317, and p-Chk2-T68. Actin was used as a loading control. (B) Whole cell lysates of control or IR (4 Gy)-treated pools were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. (C) Control SW480 pools and SW480 pools in which HINT1 was stably knocked down by shRNA were either untreated (0 h) or treated with 4 Gy of IR. Apoptotic cells were analyzed by sub-G1 content using a flow cytometry method. A repeat experiment yielded similar results. Error bars represent the standard deviation from triplicate experiments. *, P < 0.05 when compared with the control shRNA-transfected cells at each time point.
Mentions: Because deficient expression of HINT1 causes decreased induction of the stress-inducible genes described in Fig. 4 A, it was of interest to determine whether HINT1-deficient cells are also impaired in induction of apoptosis. However, we were not able to detect a significant increase of apoptosis even in the Hint1 +/+ MEFs after treatment with 4 Gy of γ radiation (unpublished data). Therefore, we developed stable derivatives of SW480 colon cancer cells in which HINT1 is suppressed by transfection with a Hint1–short hairpin RNA (shRNA) vector (Weiske and Huber, 2005). For comparison we also developed a control SW480 derivative that had been transfected with a control shRNA vector. Expression of the HINT1 protein was nearly undetectable in Hint1-shRNA SW480 cells. Both cell lines displayed similar growth rate. These cells also exhibited reduced activation of ATM and downstream targets such as Chk1 and Chk2 in response to IR (Fig. 5 A). The level of the p21 protein was also reduced (Fig. 5 B). After treatment with 4 Gy of γ radiation, the level of p21 was induced in the control shRNA-transfected cells, whereas the induction of p21 was markedly reduced in the Hint1-shRNA–transfected cells (Fig. 5 B). Both the control and HINT1-deficient SW480 cells displayed activation of γ-H2AX with or without IR, perhaps reflecting the combined effects of loss of HINT1 expression and their transformed state (unpublished data). Using FACS flow cytometry we quantitated the sub-G1 population as an index of apoptosis. Without IR the percentage of apoptosis in both cell types was ∼5%. At 48 h after IR this rose to ∼25% in the control cells, but only to ∼15% in the Hint1-shRNA knockdown cells (Fig. 5 C). Therefore, decreased expression of HINT1 significantly impairs the induction of apoptosis 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