Antileukemic activity of combined epigenetic agents, DNMT inhibitors zebularine and RG108 with HDAC inhibitors, against promyelocytic leukemia HL-60 cells

DNMT inhibitors are promising new drugs for cancer therapies. In this study, we have observed the antileukemic action of two diverse DNMT inhibitors, the nucleoside agent zebularine and the non-nucleoside agent RG108, in human promyelocytic leukemia (PML) HL-60 cells. Zebularine but not RG108 caused dose- and time-dependent cell growth inhibition and induction of apoptosis. However, co-treatment with either drug at a non-toxic dose and all trans retinoic acid (RA) reinforced differentiation to granulocytes, while 24 or 48 h-pretreatment with zebularine or RG108 followed by RA alone or in the presence of HDAC inhibitors (sodium phenyl butyrate or BML-210) significantly accelerated and enhanced cell maturation to granulocytes. This occurs in parallel with the expression of a surface biomarker, CD11b, and early changes in histone H4 acetylation and histone H3K4me3 methylation. The application of both drugs to HL-60 cells in continuous or sequential fashion decreased DNMT1 expression, and induced E-cadherin promoter demethylation and reactivation at both the mRNA and the protein levels in association with the induction of granulocytic differentiation. The results confirmed the utility of zebularine and RG108 in combinations with RA and HDAC inhibitors to reinforce differentiation effects in promyelocytic leukemia.     

Cu2+ is required for pyrrolidine dithiocarbamate to inhibit histone acetylation and induce human leukemia cell apoptosis

Pyrrolidine dithiocarbamate (PDTC) has been considered as a potential anticancer drug due to its powerful apoptogenic effect towards cancer cells, where Cu(2+) plays a distinct yet undefined role. Here we report that Cu(2+) is critically needed for PDTC to inhibit histone acetylation in both human leukemia HL-60 cells and human hepatoma Hep3B cells. The inhibition of histone acetylation mainly resulted from the increase of intracellular Cu(2+), but was not due to the inhibition of NF-kappaB activity by PDTC-Cu(2+) since the combinations of Cu(2+) with SN50, MG132 (two known NF-kappaB inhibitors), or bathocuproine disulfonate (BCS, a specific Cu(2+) chelator that does not cross the plasma membrane), did not lead to obvious inhibition of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cells exposed to PDTC-Cu(2+) showed a comparable decrease in histone acetylation levels in HL-60 cells in the absence or presence of the HDAC inhibitors, trichostatin A (TSA) or sodium butyrate (NaBu); the inhibition rates were about 45, 44 and 43%, respectively. PDTC-Cu(2+) had no effect on the activity of HDAC in vitro, but significantly inhibited the HAT activity both in HL-60 cells and in a cell-free in vitro system. PDTC-Cu(2+) also induced HL-60 cell apoptosis, and treating cells with TSA, NaBu or BCS significantly attenuated the apoptosis induced by PDTC-Cu(2+). Collectively, these results showed that inhibition of histone acetylation represents a distinct mechanism for the cytotoxicity of PDTC in the presence of Cu(2+), where HAT is its possible molecular target.     

Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation

Osteogenesis is a complex process associated with dramatic changes in gene expression. To elucidate whether modifications in chromatin structure are involved in osteoblast differentiation, we examined the expression levels of histone deacetylases (HDACs) and the degree of histone acetylation at the promoter regions of osteogenic genes. During osteogenesis, total HDAC enzymatic activity was decreased with significant reduction in HDAC1 expression. Consistently, recruitment of HDAC1 to the promoters of osteoblast marker genes, including osterix and osteocalcin, was down-regulated, whereas histone H3 and H4 were hyperacetylated at those promoters during osteoblast differentiation. Moreover, suppression of HDAC activity with a HDAC inhibitor, sodium butyrate, accelerated osteogenesis by inducing osteoblast marker genes including osteopontin and alkaline phosphatase. Consistently, knockdown of HDAC1 by the short interference RNA system stimulated osteoblast differentiation. Taken together, these data propose that down-regulation of HDAC1 is an important process for osteogenesis.     

Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.     

Histone acetylation and reactive oxygen species are involved in the preprophase arrest induced by sodium butyrate in maize roots

Histone acetylation plays a critical role in controlling chromatin structure, and reactive oxygen species (ROS) are involved in cell cycle progression. To study the relationship between histone acetylation and cell cycle progression in plants, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor that can cause a significant increase in histone acetylation in both mammal and plant genomes, was applied to treat maize seedlings. The results showed that NaB had significant inhibition effects on different root zones at the tissue level and caused cell cycle arrest at preprophase in the root meristem zones. This effect was accompanied by a dramatic increase in the total level of acetylated lysine 9 on histone H3 (H3K9ac) and acetylated lysine 5 on histone H4 (H4K5ac). The exposure of maize roots in NaB led to a continuous rise of intracellular ROS concentration, accompanied by a higher electrolyte leakage ratio and malondialdehyde (MDA) relative value. The NaB-treated group displayed negative results in both TdT-mediated dUTP nick end labelling (TUNEL) and γ-H2AX immunostaining assays. The expression of topoisomerase genes was reduced after treatment with NaB. These results suggested that NaB increased the levels of H3K9ac and H4K5ac and could cause preprophase arrest accompanied with ROS formation leading to the inhibition of DNA topoisomerase.     

Histone deacetylase inhibition results in a common metabolic profile associated with HT29 differentiation

Cell differentiation is an orderly process that begins with modifications in gene expression. This process is regulated by the acetylation state of histones. Removal of the acetyl groups of histones by specific enzymes (histone deacetylases, HDAC) usually downregulates expression of genes that can cause cells to differentiate, and pharmacological inhibitors of these enzymes have been shown to induce differentiation in several colon cancer cell lines. Butyrate at high (mM) concentration is both a precursor for acetyl-CoA and a known HDAC inhibitor that induces cell differentiation in colon cells. The dual role of butyrate raises the question whether its effects on HT29 cell differentiation are due to butyrate metabolism or to its HDAC inhibitor activity. To distinguish between these two possibilities, we used a tracer-based metabolomics approach to compare the metabolic changes induced by two different types of HDAC inhibitors (butyrate and the non-metabolic agent trichostatin A) and those induced by other acetyl-CoA precursors that do not inhibit HDAC (caprylic and capric acids). [1,2-¹³C₂]-d-glucose was used as a tracer and its redistribution among metabolic intermediates was measured to estimate the contribution of glycolysis, the pentose phosphate pathway and the Krebs cycle to the metabolic profile of HT29 cells under the different treatments. The results demonstrate that both HDAC inhibitors (trichostatin A and butyrate) induce a common metabolic profile that is associated with histone deacetylase inhibition and differentiation of HT29 cells whereas the metabolic effects of acetyl-CoA precursors are different from those of butyrate. The experimental findings support the concept of crosstalk between metabolic and cell signalling events, and provide an experimental approach for the rational design of new combined therapies that exploit the potential synergism between metabolic adaptation and cell differentiation processes through modification of HDAC activity.     

Acetylation of core histones in response to HDAC inhibitors is diminished in mitotic HeLa cells

Histone acetylation is a key modification that regulates chromatin accessibility. Here we show that treatment with butyrate or other histone deacetylase (HDAC) inhibitors does not induce histone hyperacetylation in metaphase-arrested HeLa cells. When compared to similarly treated interphase cells, acetylation levels are significantly decreased in all four core histones and at all individual sites examined. However, the extent of the decrease varies, ranging from only slight reduction at H3K23 and H4K12 to no acetylation at H3K27 and barely detectable acetylation at H4K16. Our results show that the bulk effect is not due to increased or butyrate-insensitive HDAC activity, though these factors may play a role with some individual sites. We conclude that the lack of histone acetylation during mitosis is primarily due to changes in histone acetyltransferases (HATs) or changes in chromatin. The effects of protein phosphatase inhibitors on histone acetylation in cell lysates suggest that the reduced ability of histones to become acetylated in mitotic cells depends on protein phosphorylation.     

Transient exposure to sodium butyrate after germinal vesicle breakdown improves meiosis but not developmental competence in pig oocytes

Oocyte maturation is a complex process during which epigenetic modifications are dramatically changed, especially histone acetylation and phosphorylation. We have investigated the effects of NaBu (sodium butyrate), a natural HDAC (histone deacetylase) inhibitor, on porcine oocyte maturation at different stages and subsequent embryonic development to improve IVF (in vitro fertilization) and embryo production. COCs (cumulus oocyte complexes) were cultured, IVM (in vitro maturation) supplemented with 1 mM NaBu before or after GVBD [GV (germinal vesicle) breakdown] during maturation. NaBu delayed oocyte meiosis in the GV and GVBD stages in an exposure-dependent manner. However, the short treatment with 1 mM NaBu after GVBD significantly improved the meiotic competence. No positive effects of NaBu on GSH levels and subsequent embryonic development following IVF were seen. Transient exposure to NaBu after GVBD improves meiotic competence, but not subsequently, probably by having an effect on histone acetylation during oocyte maturation.     

Histone deacetylase inhibitors induce apoptosis in peripheral blood lymphocytes along with histone H4 acetylation and the expression of the linker histone variant, H1 degrees

The results of this study show that H1 degrees can be induced by sodium butyrate and trichostatin A in peripheral blood lymphocytes, a cell system which does not normally express this linker histone variant. Moreover, this induced expression was found to be correlated in a dose-dependent manner with the concomitant induction of apoptosis and increased levels of histone H4 acetylation. Sodium butyrate and trichostatin A, both inhibitors of histone deacetylases, are known to induce terminal differentiation and at the same time the induction of the linker histone variant, H1 degrees, in a number of tissue/cell systems. Moreover, aside from induced expression by histone deacetylase inhibitors, H1 degrees gene expression has also been tightly associated with the process of terminal differentiation in many physiological tissue/cell systems. The concomitant induction of H1 degrees expression along with apoptosis and histone acetylation in the same cell system has not been previously reported. Histone acetylation is known to be involved in chromatin remodelling events. Such events also occur during apoptosis. The association of H1 degrees gene expression with apoptosis, and not with differentiation in these cells, leads to more general implications as to a potential functional role of H1 degrees during chromatin remodelling.