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. 2017 Jan 24;8(4):6319-6329.
doi: 10.18632/oncotarget.14062.

Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells

Jianyun Zhao  1   2   3 , Chengzhi Xie  4   5 , Holly Edwards  4   5 , Guan Wang  1 , Jeffrey W Taub  5   2   3 , Yubin Ge  1   4   5   2
Affiliations

Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells

Jianyun Zhao et al. Oncotarget. .

Abstract

Resistance to chemotherapy and a high relapse rate highlight the importance of finding new therapeutic options for the treatment of acute myeloid leukemia (AML). Histone deacetylase (HDAC) inhibitors (HDACIs) are a promising class of drugs for the treatment of AML. HDACIs have limited single-agent clinical activities, but when combined with conventional or investigational drugs they have demonstrated favorable outcomes. Previous studies have shown that decreasing expression of important DNA damage repair proteins enhances standard chemotherapy drugs. In our recent studies, the pan-HDACI panobinostat has been shown to enhance conventional chemotherapy drugs cytarabine and daunorubicin in AML cells by decreasing the expression of BRCA1, CHK1, and RAD51. In this study, we utilized class- and isoform-specific HDACIs and shRNA knockdown of individual HDACs to determine which HDACs are responsible for decreased expression of BRCA1, CHK1, and RAD51 following pan-HDACI treatment in AML cells. We found that inhibition of both HDAC1 and HDAC2 was necessary to decrease the expression of BRCA1, CHK1, and RAD51, enhance cytarabine- or daunorubicin-induced DNA damage and apoptosis, and abrogate cytarabine- or daunorubicin-induced cell cycle checkpoint activation in AML cells. These findings may aid in the development of rationally designed drug combinations for the treatment of AML.

Keywords: BRCA1; CHK1; HDAC; RAD51; acute myeloid leukemia.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Inhibition of Class II HDACs has no impact on the expression of BRCA1, CHK1, and RAD51 in AML cells
(A and B) THP-1 and OCI-AML3 cells were treated with MC1568 for 48 h, and then whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. (C amd D) THP-1 and OCI-AML3 cells were treated with Tubastatin A for 48 h, and then whole cell lysates were subjected to Western blotting and probed with the indicated antibodies.
Figure 2
Figure 2. Inhibition of HDACs 1, 2, and 3 decreases the protein and transcript levels of BRCA1, CHK1, and RAD51, and induces apoptosis in AML cell lines
(A) THP-1 cells were treated with variable concentrations of MGCD0103 for 48 h. Protein extracts were subjected to immunoprecipitation with antibodies against class I HDACs and then class I HDAC activities were measured, as described in the Materials and Methods. (B) THP-1 cells were treated with MGCD0103 for 48 h. Then total RNAs were isolated and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. (C) THP-1 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (D) OCI-AML3 cells were treated with MGCD0103 for 48 h, then total RNAs were isolated from treated cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. (E) OCI-AML3 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. (F) THP-1 and OCI-AML3 cells were treated with MGCD0103 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. *indicates p < 0.05, **indicates p < 0.01, and ***indicates p < 0.001 (panels A, B, D & F).
Figure 3
Figure 3. MGCD0103 cooperates with ara-C or DNR in inducing apoptosis and abrogates S and/or G2/M cell cycle checkpoint activation induced by ara-C or DNR in AML cells
(A and C) THP-1 and OCI-AML3 cells were treated with MGCD0103 and ara-C or DNR, alone or in combination, for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. ***indicates p < 0.001. (B and D) Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (E and F) THP-1 and OCI-AML3 cells were treated for 48 h with MGCD0103 and ara-C or DNR, alone or in combination, then fixed with ethanol and stained with PI for cell cycle analysis.
Figure 4
Figure 4. HDACs 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in AML cells
(A and C) THP-1 cells were infected with HDAC1 (THP-1/HDAC1), HDAC2 (THP-1/HDAC2), HDAC3 (THP-1/HDAC3), or NTC control (THP-1/NTC) shRNA lentivirus overnight, then washed and incubated for 48 h prior to adding puromycin to the culture medium. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. These Western blots were previously published [15]. (B) Total RNAs were isolated from the transfected cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. (D and E) THP-1/HDAC1 and THP-1/HDAC2 cells were treated with RGFP966 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies.
Figure 5
Figure 5. FK228 decreases expression of BRCA1, CHK1, and RAD51 by inhibiting HDAC1 and HDAC2 in AML cells
(A and B) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (C) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. **indicates p < 0.01 and ***indicates p < 0.001. (D and E) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h. Total RNAs were isolated from treated cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. **indicates p < 0.01 and ***indicates p < 0.001.
Figure 6
Figure 6. FK228 cooperates with ara-C or DNR in inducing apoptosis and abrogates S and/or G2/M cell cycle checkpoint activation induced by ara-C or DNR in THP-1 and OCI-AML3 AML cells
(A) THP-1 cells were treated with FK228 and ara-C or DNR, alone or in combination, for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. **indicates p < 0.01 and ***indicates p < 0.001. (B) Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (C) OCI-AML3 cells were treated with FK228 and ara-C or DNR, alone or in combination, for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. ***indicates p < 0.001. (D) Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. (E and F) THP-1 and OCI-AML3 cells were treated with FK228 and ara-C or DNR, alone or in combination for 48 h and then fixed with ethanol and stained with PI for cell cycle analysis.
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