A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases

Abstract

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity in vitro. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.

Figure 1

Effect of HPCs on histone acetylation in MEL cells. (A) MEL cells were cultured with TSA (75 nM), HMBA (5 mM), and SAHA (2.5 μM) for the indicated times. Histones were extracted from the treated cells and were analyzed on an AUT gel. (B) MEL cells were cultured for 6 h with no addition (lane 1), 5 mM HMBA (lane 2), 0.3 mM EMBA (lane 3), 30 μM SBHA (lane 4), 2.5 μM SAHA (lane 5), and 4.0 μM CBHA (lane 6). Histones were isolated and analyzed on an AUT gel. The degree of histone acetylation of histone H4 is indicated as follows: Ac0, nonacetylated H4; Ac1, monoacetylated H4; Ac2, diacetylated H4; Ac3, triacetylated H4; Ac4, tetraacetylated H4.

Figure 2

Characterization of SAHA-resistant cell clones, SAHA R-4 and SAHA R-6. (A) Effect of HPCs and TSA on commitment to terminal differentiation. Cells were cultured with no addition (CTL), 5 mM HMBA, 3 μM SAHA, or TSA (12.5 ng/ml), as indicated, and commitment assays performed after 48 h of culture. After a 5-day growth period in methylcellulose, cells were scored as committed to terminal differentiation if they formed small colonies (<32 cells) and were expressing hemoglobin as determined by benzidine staining. (B) Effect of SAHA on histone acetylation in DS19 and SAHA R-4 cells. Cells were cultured in 0, 0.5, 1.0, and 2.5 μM SAHA for 2 h. Histones were isolated and analyzed on an AUT gel as indicated.

Figure 3

Effect of HPCs on histone acetylation in T24 and ARP-1 cells. MEL cells were cultured for 6 h with no addition (lane 1) or 4 μM 3-Cl-UCHA (lane 3). T24 cells were cultured for 6 h with no addition (lane 1), 10 mM HMBA (lane 2), 3 μM 3-Cl-UCHA (lane 3), or TSA (500 ng/ml; lane 4). ARP-1 cells were cultured for 6 h with no addition (lane 1), 5 mM HMBA (lane 2), 1 μM 3-Cl-UCHA (lane 3), or TSA (20 ng/ml; lane 4). The histones were extracted from the treated cells and then analyzed on an AUT gel.

Figure 4

Inhibition of HDAC1 and HDAC3 by HPCs. (A) Cellular extracts from Jurkat cells (5 × 10⁷ cells) were subjected to immunoprecipitation with a polyclonal antiserum against HDAC1 and HDAC3 in the presence of a 100-fold excess of HDAC3 peptide (residues 413–428) or HDAC1 peptide (residues 467–482). As an additional control, the corresponding preimmune serum was used. Immunoprecipitated complexes were tested for HDAC activity by measuring the release of [³H]acetate from an acetylated amino-terminal H4 peptide (in cpm), in the absence or presence of 400 nM TSA. (B) Cellular extracts from Jurkat cells were subjected to immunoprecipitation with polyclonal antiserum against HDAC1 (♦) or HDAC3 (□) and tested for HDAC activity in the absence or the presence of increasing concentration of the following inhibitors: HMBA, 0.005, 0.05, and 0.5 mM; EMBA, 0.005, 0.05, and 0.5 mM; SAHA, 0.001, 0.01, 0.1, 1, and 10 μM; CBHA or SBHA, 0.001, 0.01, 0.1, 0.33, 1, 10, and 100 μM.