Studies on the mechanism of action of hexamethylene bisacetamide, a potent inducer of erythroleukemic differentiation

Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up by the cell; an intracellular concentration equal to the extracellular concentration is achieved by 6–8 h. Commitment to differentiation is not detected until at least 10 h after equilibration. Both uptake and commitment to differentiate are concentration and temperature dependent. The majority of the compound is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in both cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, but acetylation of the diamine greatly increases inducing activity.     

1,6-Diaminohexane contributes to the hexamethylene bisacetamide-induced erythroid differentiation pathway by stimulating Ca2+ release from inositol 1,4,5-trisphosphate-sensitive stores and promoting Ca2+ influx

Hexamethylene bisacetamide (HMBA) stimulates Ca(2+) signals in murine erythroleukemia (MEL) cells serving as an important component of the HMBA-induced pathway that promotes differentiation to the erythroid phenotype. We observed that 1,6-diaminohexane (DAH) triggered a more rapid and robust increase in MEL cell Ca(2+) levels compared to HMBA and the monodeacetylated N-acetyl-1,6-diaminohexane (NADAH), and that polyamine deacetylase inhibition completely abolished the ability of HMBA and NADAH to induce Ca(2+) signals in MEL cells. Our work indicates that DAH mediates Ca(2+) signal propagation via its ability to activate inositol 1,4,5-trisphosphate (IP(3)) receptors, as we observed similar Ca(2+) release characteristics and heparin sensitivity of DAH and IP(3) in permeabilized MEL cells. Finally, we observed that the DAH-induced Ca(2+) release pathway robustly coupled to a Ca(2+) influx pathway that could be distinguished from thapsigargin-induced Ca(2+) influx by its unusual insensitivity to 2-aminoethoxydiphenyl borate.     

Hydrophobic chromatography of the HL-60 cellular fraction co-binding with hexamethylene bisacetamide

Methods of separating N-acetyl-1,6-diaminohexane (NADAH) and its immobilization to diol-silica have been developed. Hexamethylene bisacetamide (HMBA) and its metabolite NADAH are used as inducers of leukemia cell differentiation. The inducing mechanism of HMBA is still not clear. Experiments show that HMBA and NADAH undergo relatively strong hydrophobic reactions and do not readily undergo ion-exchange with the proteins of the cytosolic fraction of HL-60 cells during immobilization of NADAH; the retention time of the proteins was longer than that of the phosphatides. These results show that the adsorption of HMBA and NADAH to proteins was higher than that to phosphatides. The expected biospecific receptor binding with HMBA has not been found.     

Effect of 5′-methylthioadenosine on induction of murine erythroleukemia cell differentiation

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of proliferation and differentiation. The present study has monitored the effects of 5′-methylthioadenosine, the metabolic product of spermidine and spermine synthesis, on the appearance of a differentiated murine erythroleukemia cell phenotype. The results demonstrate that increasing concentrations of 5′-methylthioadenosine (1 × 10^?6 to 5 × 10^?4M) progressively inhibit murine erythroleukemia cell heme synthesis and hemoglobin production. The results also demonstrate that this inhibition of differentiation is not related to depletion of intracellular spermidine or cytostasis. Since 5′-methylthioadenosine is also a known inhibitor of DNA methylation, this naturally occurring nucleoside may be an intermediate involved in both murine erythroleukemia cell proliferation and differentiation.     

Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one scaffold for the development of novel chemical inhibitors for Bromodomain and Extraterminal Domain (BET) family

The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-inhibitory activity with IC₅₀ value of 33 nM and anti-proliferation potency with IC₅₀ value of 110 nM in HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion, these results suggested that compound 28 has merely potential for leukemia treatment.     

肿瘤细胞诱导分化剂NADAH在硅胶载体上的固定化及其与DNA作用的研究

1976年Reuben等[1]发现六亚甲基二乙酰胺（hexamethylenebisacetallllde,HMBA）在5×10-3mol/L浓度可诱导99％以上的Friend红白血病细胞分化．已报道HMBA在体外可引起动物多种实体瘤和白血病细胞系的分化［2］．阐明HMBA诱导肿瘤细胞分化的机制有着重要意义．HMBA去掉一个乙酸基后单乙酸己二胺（N-acetyl-diallllnohexaneNADAH）：CH3CONHCHZCHZCHZCHZCHZCHZNHZ,具有与HMBA几乎同样诱导肿瘤细胞分化的活性［3,'」,由于NADAH有一个活泼基因NHZ,为固相化研究其诱导肿瘤细胞分化机理提供了可能。通过两步合成将…     

环六亚甲基双乙酰胺对小鼠畸胎癌B7-2克隆细胞的诱导分化

近年来,有不少研究工作者报道了用化学物质诱导恶性癌细胞进行有限的分化。这类物质主要有维生素A酸(RA),丁酸钠(Na-butyrare),二甲基亚砜(DM-SO),环腺嘌呤核苷酸(c-AMP)以及环六亚甲基双乙酰胺(hexamethylene bisaceta-mide,HMBA)等,它们能使癌细胞恶性表型受到抑制,并向一定类型的体细胞分化。HMBA最早用于研究小鼠红白血病细胞的诱导分化,并为研究珠蛋白基因表达提供了很好的实验模型。随后,Jakob等     

Induction of differentiation in mouse neuroblastoma cells by hexamethylene bisacetamide

Hexamethylene bisacetamide (HMBA), a potent inducer of erythroid differentiation in murine erythroleukemia cells (1), induces differentiation in mouse neuroblastoma cells, as indicated by the extension of neurites and the development of an excitable membrane. HMBA is effective at concentrations 50-fold lower than dimethylsulfoxide (2), another inducer of differentiation in both mouse neuroblastoma and murine erythroleukemia cells.