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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 is associated with reduced activation of IR-inducible genes. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and total RNA was collected after the indicated time points. The mRNA levels of p21, GADD45α, GADD153, and TP53inp1 were determined by real-time RT-PCR. (B) Whole cell lysates of control or IR (4 Gy)-treated Hint1 MEFs were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. Error bars represent standard deviation from triplicate experiments. *, P < 0.05 when compared with Hint1 +/+ MEFs at each time point; §, P < 0.05 when compared with Hint1 +/− MEFs.
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fig4: Loss of HINT1 is associated with reduced activation of IR-inducible genes. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and total RNA was collected after the indicated time points. The mRNA levels of p21, GADD45α, GADD153, and TP53inp1 were determined by real-time RT-PCR. (B) Whole cell lysates of control or IR (4 Gy)-treated Hint1 MEFs were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. Error bars represent standard deviation from triplicate experiments. *, P < 0.05 when compared with Hint1 +/+ MEFs at each time point; §, P < 0.05 when compared with Hint1 +/− MEFs.

Mentions: To identify genes that are differentially regulated in response to DSB in Hint1 MEFs we used Affymetrix GeneChip technology to analyze the gene expression profiles of these cells after IR. We found that the up-regulation of several DNA damage-inducible genes in response to IR was reduced in Hint1 +/− and −/− MEFs in response to IR (unpublished data). These genes include p21WAF1/CIP1, GADD45α, GADD153, and p53-inducible protein 1 (TP53inp1). To confirm these results, the mRNA levels of these genes were measured by real-time RT-PCR, using GAPDH as an internal control. We found that when compared with the Hint1 +/+ cells, there was a statistically significant (P < 0.05) decrease in activation of all four genes in Hint1 −/− MEFs at 48 h after treatment with 4 Gy of γ radiation (Fig. 4 A). In Hint1 +/− MEFs, there was also a significant decrease in induction of the expression of p21, GADD45α, and GADD153, but not TP53inp1 (Fig. 4 A). We also confirmed the real-time RT-PCR results on p21 by immunoblots of protein levels of p21 in untreated and 4-Gy γ radiation–treated Hint1 MEFs. The expression levels of p21 were similar in the three genotypes of untreated cells. 48 h after IR there was a marked increase in the protein level of p21 in Hint1 +/+ MEFs. However, induction of the p21 protein was reduced in Hint1 +/− cells and further reduced in Hint1 −/− cells (Fig. 4 B).


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 is associated with reduced activation of IR-inducible genes. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and total RNA was collected after the indicated time points. The mRNA levels of p21, GADD45α, GADD153, and TP53inp1 were determined by real-time RT-PCR. (B) Whole cell lysates of control or IR (4 Gy)-treated Hint1 MEFs were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. Error bars represent standard deviation from triplicate experiments. *, P < 0.05 when compared with Hint1 +/+ MEFs at each time point; §, P < 0.05 when compared with Hint1 +/− MEFs.
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Related In: Results  -  Collection

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fig4: Loss of HINT1 is associated with reduced activation of IR-inducible genes. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and total RNA was collected after the indicated time points. The mRNA levels of p21, GADD45α, GADD153, and TP53inp1 were determined by real-time RT-PCR. (B) Whole cell lysates of control or IR (4 Gy)-treated Hint1 MEFs were collected for immunoblot analysis of the level of the p21 protein. Actin was used as a loading control. Error bars represent standard deviation from triplicate experiments. *, P < 0.05 when compared with Hint1 +/+ MEFs at each time point; §, P < 0.05 when compared with Hint1 +/− MEFs.
Mentions: To identify genes that are differentially regulated in response to DSB in Hint1 MEFs we used Affymetrix GeneChip technology to analyze the gene expression profiles of these cells after IR. We found that the up-regulation of several DNA damage-inducible genes in response to IR was reduced in Hint1 +/− and −/− MEFs in response to IR (unpublished data). These genes include p21WAF1/CIP1, GADD45α, GADD153, and p53-inducible protein 1 (TP53inp1). To confirm these results, the mRNA levels of these genes were measured by real-time RT-PCR, using GAPDH as an internal control. We found that when compared with the Hint1 +/+ cells, there was a statistically significant (P < 0.05) decrease in activation of all four genes in Hint1 −/− MEFs at 48 h after treatment with 4 Gy of γ radiation (Fig. 4 A). In Hint1 +/− MEFs, there was also a significant decrease in induction of the expression of p21, GADD45α, and GADD153, but not TP53inp1 (Fig. 4 A). We also confirmed the real-time RT-PCR results on p21 by immunoblots of protein levels of p21 in untreated and 4-Gy γ radiation–treated Hint1 MEFs. The expression levels of p21 were similar in the three genotypes of untreated cells. 48 h after IR there was a marked increase in the protein level of p21 in Hint1 +/+ MEFs. However, induction of the p21 protein was reduced in Hint1 +/− cells and further reduced in Hint1 −/− cells (Fig. 4 B).

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