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Class I histone deacetylase inhibitors inhibit the retention of BRCA1 and 53BP1 at the site of DNA damage.

Fukuda T, Wu W, Okada M, Maeda I, Kojima Y, Hayami R, Miyoshi Y, Tsugawa K, Ohta T - Cancer Sci. (2015)

Bottom Line: Reflecting their effects on histone modifications, the HDAC inhibitors inhibit ionizing radiation-induced foci (IRIF) formation of BRCA1 and BARD1 as well as 53BP1 and RIF1, whereas UNC0638 suppresses IRIF formation of BRCA1 and BARD1 but not 53BP1 and RIF1.Although HDAC inhibitors suppressed HDR, they did not cooperate with the poly(ADP-ribose) polymerase inhibitor olaparib to block cancer cell growth, possibly due to simultaneous suppression of NHEJ pathway components.Collectively, these results suggest the mechanism by that HDAC inhibitors inhibit both the HDR and NHEJ pathways, whereas HKMT inhibitor inhibits only the HDR pathway; this finding may affect the chemosensitizing effects of the inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.

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Induction of H3K56ac and H4ac by the histone deacetylase (HDAC) inhibitors, but not the HKMT inhibitor at doses that do not affect the expression levels of DNA repair genes. (a, b) U2OS (a) or MCF-7 (b) cells were incubated for 24 h with DMSO (−), MS-275 (2.5 μM) or FK228 (2.5 nM) and immunoblotted with the indicated antibodies. (c) U2OS cells were incubated for 24 h with DMSO (−) or UNC0638 (3 μM) and immunoblotted with the indicated antibodies. UNC0638 did not inhibit the expression levels of DNA repair genes BRCA1, BARD1 and 53BP1 at the doses tested. (d) U2OS cells were treated with MS-275 (2.5 μM), FK228 (2.5 nM) or UNC0638 (3 μM) for 24 h. Gene expression levels for of BRCA2, ATM, RIF1 and the loading control, GAPDH, were analyzed by RT-PCR.
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fig02: Induction of H3K56ac and H4ac by the histone deacetylase (HDAC) inhibitors, but not the HKMT inhibitor at doses that do not affect the expression levels of DNA repair genes. (a, b) U2OS (a) or MCF-7 (b) cells were incubated for 24 h with DMSO (−), MS-275 (2.5 μM) or FK228 (2.5 nM) and immunoblotted with the indicated antibodies. (c) U2OS cells were incubated for 24 h with DMSO (−) or UNC0638 (3 μM) and immunoblotted with the indicated antibodies. UNC0638 did not inhibit the expression levels of DNA repair genes BRCA1, BARD1 and 53BP1 at the doses tested. (d) U2OS cells were treated with MS-275 (2.5 μM), FK228 (2.5 nM) or UNC0638 (3 μM) for 24 h. Gene expression levels for of BRCA2, ATM, RIF1 and the loading control, GAPDH, were analyzed by RT-PCR.

Mentions: Inhibition of H3K9me2 with HKMT inhibitor UNC0638 dramatically suppressed ionizing irradiation-induced foci (IRIF) formation of BRCA1 and BARD1.10 To explore whether class I HDAC inhibitors exhibit similar effects, we first analyzed the effects of MS-275 and FK228 on the H3K9 modification. A time course analysis in U2OS cells suggested that H3K9me2 was significantly reduced in response to the HDAC inhibitors by 24 h, whereas H3K9ac was markedly enhanced (Fig.1a). To avoid effects on the expression levels of DNA repair genes, we searched for an appropriate dose of the HDAC inhibitors to reduce the H3K9me2 but not affect the expression levels of the DNA repair genes during the time period. H3K9me2 was significantly reduced after treatment with 2.5 μM MS-275 and 2.5 nM FK228, accompanied by substantial upregulation of H3K9ac (Fig.1b). Although the protein expression levels of BRCA1 and BARD1 were reduced at higher doses (data not shown), they were unchanged in response to 2.5 μM MS-275 or 2.5 nM FK228 (Fig.1b). The HDAC inhibitor effects on histone modifications using the doses at concentrations that did not alter BRCA1 and BARD1 expression were reproducible in MCF-7 cells (Fig.1c). Because TSA promotes H3K56ac and H4K16ac, which inhibit the interaction between 53BP1 and H4K20me2,4,7 we tested whether MS-275 and FK228, at our trial doses, also accelerated this modification. An anti-H4ac antibody that recognizes acetylation of N-terminal lysines including H4K16ac was used, as in a previous study.4 Similar to TSA, MS-275 and FK228 effectively elevated H3K56ac and H4ac in U2OS cells (Fig.2a), as well as in MCF-7 cells (Fig.2b). Conversely, the HKMT inhibitor UNC0638 reduced H3K9me2 without enhancing H3K56ac, H4ac or H3K9ac (Fig.2c). The expression levels of other DNA repair genes, including the genes that have been reported to be reduced by HDAC inhibitors,2 RAD51, 53BP1, BRCA2, ATM and RIF1, were not affected by treatment with HDAC inhibitors or UNC0638 (Figs1b,c and 2c,d).


Class I histone deacetylase inhibitors inhibit the retention of BRCA1 and 53BP1 at the site of DNA damage.

Fukuda T, Wu W, Okada M, Maeda I, Kojima Y, Hayami R, Miyoshi Y, Tsugawa K, Ohta T - Cancer Sci. (2015)

Induction of H3K56ac and H4ac by the histone deacetylase (HDAC) inhibitors, but not the HKMT inhibitor at doses that do not affect the expression levels of DNA repair genes. (a, b) U2OS (a) or MCF-7 (b) cells were incubated for 24 h with DMSO (−), MS-275 (2.5 μM) or FK228 (2.5 nM) and immunoblotted with the indicated antibodies. (c) U2OS cells were incubated for 24 h with DMSO (−) or UNC0638 (3 μM) and immunoblotted with the indicated antibodies. UNC0638 did not inhibit the expression levels of DNA repair genes BRCA1, BARD1 and 53BP1 at the doses tested. (d) U2OS cells were treated with MS-275 (2.5 μM), FK228 (2.5 nM) or UNC0638 (3 μM) for 24 h. Gene expression levels for of BRCA2, ATM, RIF1 and the loading control, GAPDH, were analyzed by RT-PCR.
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fig02: Induction of H3K56ac and H4ac by the histone deacetylase (HDAC) inhibitors, but not the HKMT inhibitor at doses that do not affect the expression levels of DNA repair genes. (a, b) U2OS (a) or MCF-7 (b) cells were incubated for 24 h with DMSO (−), MS-275 (2.5 μM) or FK228 (2.5 nM) and immunoblotted with the indicated antibodies. (c) U2OS cells were incubated for 24 h with DMSO (−) or UNC0638 (3 μM) and immunoblotted with the indicated antibodies. UNC0638 did not inhibit the expression levels of DNA repair genes BRCA1, BARD1 and 53BP1 at the doses tested. (d) U2OS cells were treated with MS-275 (2.5 μM), FK228 (2.5 nM) or UNC0638 (3 μM) for 24 h. Gene expression levels for of BRCA2, ATM, RIF1 and the loading control, GAPDH, were analyzed by RT-PCR.
Mentions: Inhibition of H3K9me2 with HKMT inhibitor UNC0638 dramatically suppressed ionizing irradiation-induced foci (IRIF) formation of BRCA1 and BARD1.10 To explore whether class I HDAC inhibitors exhibit similar effects, we first analyzed the effects of MS-275 and FK228 on the H3K9 modification. A time course analysis in U2OS cells suggested that H3K9me2 was significantly reduced in response to the HDAC inhibitors by 24 h, whereas H3K9ac was markedly enhanced (Fig.1a). To avoid effects on the expression levels of DNA repair genes, we searched for an appropriate dose of the HDAC inhibitors to reduce the H3K9me2 but not affect the expression levels of the DNA repair genes during the time period. H3K9me2 was significantly reduced after treatment with 2.5 μM MS-275 and 2.5 nM FK228, accompanied by substantial upregulation of H3K9ac (Fig.1b). Although the protein expression levels of BRCA1 and BARD1 were reduced at higher doses (data not shown), they were unchanged in response to 2.5 μM MS-275 or 2.5 nM FK228 (Fig.1b). The HDAC inhibitor effects on histone modifications using the doses at concentrations that did not alter BRCA1 and BARD1 expression were reproducible in MCF-7 cells (Fig.1c). Because TSA promotes H3K56ac and H4K16ac, which inhibit the interaction between 53BP1 and H4K20me2,4,7 we tested whether MS-275 and FK228, at our trial doses, also accelerated this modification. An anti-H4ac antibody that recognizes acetylation of N-terminal lysines including H4K16ac was used, as in a previous study.4 Similar to TSA, MS-275 and FK228 effectively elevated H3K56ac and H4ac in U2OS cells (Fig.2a), as well as in MCF-7 cells (Fig.2b). Conversely, the HKMT inhibitor UNC0638 reduced H3K9me2 without enhancing H3K56ac, H4ac or H3K9ac (Fig.2c). The expression levels of other DNA repair genes, including the genes that have been reported to be reduced by HDAC inhibitors,2 RAD51, 53BP1, BRCA2, ATM and RIF1, were not affected by treatment with HDAC inhibitors or UNC0638 (Figs1b,c and 2c,d).

Bottom Line: Reflecting their effects on histone modifications, the HDAC inhibitors inhibit ionizing radiation-induced foci (IRIF) formation of BRCA1 and BARD1 as well as 53BP1 and RIF1, whereas UNC0638 suppresses IRIF formation of BRCA1 and BARD1 but not 53BP1 and RIF1.Although HDAC inhibitors suppressed HDR, they did not cooperate with the poly(ADP-ribose) polymerase inhibitor olaparib to block cancer cell growth, possibly due to simultaneous suppression of NHEJ pathway components.Collectively, these results suggest the mechanism by that HDAC inhibitors inhibit both the HDR and NHEJ pathways, whereas HKMT inhibitor inhibits only the HDR pathway; this finding may affect the chemosensitizing effects of the inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Translational Oncology, St. Marianna University Graduate School of Medicine, Kawasaki, Japan.

Show MeSH
Related in: MedlinePlus