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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 cells display increased chromosome aberrations. Hint1 MEFS were treated with or without a 4-Gy dose of IR and 18 h later were treated with cytochalasin B for 22 h. Cells were then fixed and stained with Acridine orange for the analysis of chromosomal abnormalities. (A) Representative images showing normal binuclear morphology of untreated Hint1 +/+ MEF and chromosome aberrations in untreated Hint1 −/− MEFs, including micronuclei (top right), unequal division (middle left), nuclear blebbing (middle right), chromatin bridge (bottom left), and multinucleation (bottom right). (B) Micronuclei frequency per binucleated cell was compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (C) Chromosomal abnormality frequencies of the indicated type per binucleated cell were compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (D) Telomere labeling of Hint1 +/+ and −/− MEFs. CF, centromeric fragment; CB, chromosome break; arrows, chromatid breaks. (E) Chromosomal analysis of Hint1 +/+ and −/− MEFs. *, P < 0.05 when compared with the +/+ MEFs (Fisher's exact test).
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fig7: HINT1-deficient cells display increased chromosome aberrations. Hint1 MEFS were treated with or without a 4-Gy dose of IR and 18 h later were treated with cytochalasin B for 22 h. Cells were then fixed and stained with Acridine orange for the analysis of chromosomal abnormalities. (A) Representative images showing normal binuclear morphology of untreated Hint1 +/+ MEF and chromosome aberrations in untreated Hint1 −/− MEFs, including micronuclei (top right), unequal division (middle left), nuclear blebbing (middle right), chromatin bridge (bottom left), and multinucleation (bottom right). (B) Micronuclei frequency per binucleated cell was compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (C) Chromosomal abnormality frequencies of the indicated type per binucleated cell were compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (D) Telomere labeling of Hint1 +/+ and −/− MEFs. CF, centromeric fragment; CB, chromosome break; arrows, chromatid breaks. (E) Chromosomal analysis of Hint1 +/+ and −/− MEFs. *, P < 0.05 when compared with the +/+ MEFs (Fisher's exact test).

Mentions: Collectively, our findings suggest that HINT1-deficient cells display defects in both DNA repair and ATM checkpoint pathways. To examine whether this is associated with genomic instability, micronuclei formation and chromosomal abnormalities were analyzed in the three genotypes of Hint1 MEFs. We first examined the frequency of abnormalities in unirradiated cells and found that the frequencies of micronuclei, unequal nuclear division, and nuclear blebbing were significantly higher in +/− and −/− cells than in +/+ cells (Fig. 7, A–C). No chromatin bridges or multinucleation were observed in the +/+ cells; however, these abnormalities were observed in both the +/− and −/− cells (Fig. 7 C). After treatment of these cells with 4 Gy of IR, there was a significant increase in the frequency of micronuclei formation in all three genotypes (Fig. 7 B). A substantial increase in micronuclei number per binucleated cell was also observed after IR treatment in the HINT1-deficient cells (Fig. 7 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)

HINT1-deficient cells display increased chromosome aberrations. Hint1 MEFS were treated with or without a 4-Gy dose of IR and 18 h later were treated with cytochalasin B for 22 h. Cells were then fixed and stained with Acridine orange for the analysis of chromosomal abnormalities. (A) Representative images showing normal binuclear morphology of untreated Hint1 +/+ MEF and chromosome aberrations in untreated Hint1 −/− MEFs, including micronuclei (top right), unequal division (middle left), nuclear blebbing (middle right), chromatin bridge (bottom left), and multinucleation (bottom right). (B) Micronuclei frequency per binucleated cell was compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (C) Chromosomal abnormality frequencies of the indicated type per binucleated cell were compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (D) Telomere labeling of Hint1 +/+ and −/− MEFs. CF, centromeric fragment; CB, chromosome break; arrows, chromatid breaks. (E) Chromosomal analysis of Hint1 +/+ and −/− MEFs. *, P < 0.05 when compared with the +/+ MEFs (Fisher's exact test).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2568022&req=5

fig7: HINT1-deficient cells display increased chromosome aberrations. Hint1 MEFS were treated with or without a 4-Gy dose of IR and 18 h later were treated with cytochalasin B for 22 h. Cells were then fixed and stained with Acridine orange for the analysis of chromosomal abnormalities. (A) Representative images showing normal binuclear morphology of untreated Hint1 +/+ MEF and chromosome aberrations in untreated Hint1 −/− MEFs, including micronuclei (top right), unequal division (middle left), nuclear blebbing (middle right), chromatin bridge (bottom left), and multinucleation (bottom right). (B) Micronuclei frequency per binucleated cell was compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (C) Chromosomal abnormality frequencies of the indicated type per binucleated cell were compared in Hint1 MEFs untreated or treated with 4 Gy of IR. (D) Telomere labeling of Hint1 +/+ and −/− MEFs. CF, centromeric fragment; CB, chromosome break; arrows, chromatid breaks. (E) Chromosomal analysis of Hint1 +/+ and −/− MEFs. *, P < 0.05 when compared with the +/+ MEFs (Fisher's exact test).
Mentions: Collectively, our findings suggest that HINT1-deficient cells display defects in both DNA repair and ATM checkpoint pathways. To examine whether this is associated with genomic instability, micronuclei formation and chromosomal abnormalities were analyzed in the three genotypes of Hint1 MEFs. We first examined the frequency of abnormalities in unirradiated cells and found that the frequencies of micronuclei, unequal nuclear division, and nuclear blebbing were significantly higher in +/− and −/− cells than in +/+ cells (Fig. 7, A–C). No chromatin bridges or multinucleation were observed in the +/+ cells; however, these abnormalities were observed in both the +/− and −/− cells (Fig. 7 C). After treatment of these cells with 4 Gy of IR, there was a significant increase in the frequency of micronuclei formation in all three genotypes (Fig. 7 B). A substantial increase in micronuclei number per binucleated cell was also observed after IR treatment in the HINT1-deficient cells (Fig. 7 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