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…     

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.     

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.     

Determination of hexamethylene bisacetamide, an antineoplastic compound, in mouse and human plasma by LC-MS/MS

Hexamethylene bisacetamide (HMBA) is a polar compound which has recently been discovered to have antineoplastic activity by up-regulating the expression of an endogenous antiproliferative breast cancer protein, HEXIM1 (hexamethylene bisacetamide inducible protein 1) in vivo. HMBA has been shown in the past to induce terminal differentiation in multiple leukemia types at a concentration of 2-5mM, but its phase I and II clinical trials were largely unsuccessful due to serious side effects (notably, thrombocytopenia) with dose escalation. In this work, a sensitive and simple LC-MS/MS method for direct determination of HMBA in mouse and human plasma is described. Plasma samples were prepared by deproteinization with acetonitrile. Separation was achieved on a Waters Atlantis(?) T3 (2.1 mm × 50 mm, 3 μm) column with retention times of 2.2 and 3.7 min for HMBA and 7MBA (internal standard), respectively. The quantitation was carried out by tandem mass spectrometry using positive MRM mode. The linear range of the method was 0.500-100 ng/mL in both mouse and human plasma with injection volume of 5 μL. This method has been validated in accordance with the US Food and Drug Administration (FDA) guidelines for bioanalytical method development and applied to the determination of HMBA concentrations in FVB mice over time after a single dose of HMBA in saline (0.9% NaCl) at 10mg/kg.     

Hexamethylene Bisacetamide (HMBA) simultaneously targets AKT and MAPK pathway and represses NF-κB activity: Implications for cancer therapy

Hexamethylene Bisacetamide (HMBA) is a hybrid polar compound originally developed as a differentiation inducing agent. We show in this study that HMBA can inhibit activation of several NF-κB target genes in both lung and breast cancer cell lines. Furthermore, consistent with its ability to inhibit NF-κB function, HMBA can also sensitize cells to apoptosis. We show that HMBA mediates inhibition of the Akt and ERK/MAPK cascade, both of which are critical for cell survival and proliferation and are well known regulators of NF-κB activation. We also show that PTEN negative breast cancer cells which have hyper activation of the PI3K/Akt pathway show increased sensitivity to growth inhibitory effects of combination of HMBA and TNFα. Furthermore, HMBA can decrease the kinase activity of the IKK complex leading to defective phosphorylation of IκBα and Ser536 of p65. This study gives mechanistic insight into the mechanism of action of HMBA, provides the rationale for the potential use of HMBA in combination with various existing kinase inhibitors in combination therapy and also suggests useful biomarkers for monitoring tumor response to HMBA.     

Hexamethylene bisacetamide leads to reduced helper virus-free HSV-1 amplicon expression titers via suppression of ICP0

The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.     

Conversion of differentiation inducer resistance to differentiation inducer sensitivity in erythroleukemia cells.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line R1, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (less than 10 ng of VC per ml). Four independently derived VC-resistant cell lines from HMBA-resistant R1 cells, designated R1[VCR]a to R1[VCR]d, acquired sensitivity to HMBA and the accelerated kinetics of commitment that are characteristic of VC-resistant MELC derived from the parental DS19-SC9 cells. The calcium channel blocker verapamil suppresses the VC resistance of R1[VCR] cells but does not alter the accelerated response to HMBA. In R1[VCR] cells there was no detectable increase in the level of the 140-kilodalton P-glycoprotein. Transient inhibition of protein synthesis during the latent period delays inducer-mediated commitment of VC-sensitive DS19-SC9 MELC but does not alter the accelerated commitment kinetics of R1[VCR]a cells. Previously, we have reported evidence that protein kinase C beta (PKC beta) plays a role in HMBA-induced MELC differentiation and that compared with DS19-SC9 cells, R1 cells have a relatively low level and R1[VCR]a cells have a high level of PKC beta. These findings suggest that (i) acquisition of VC resistance overcomes the block acquired by R1 cells to HMBA-mediated differentiation; (ii) the accelerated kinetics of HMBA-induced commitment of VC-resistant MELC is not dependent on the verapamil-sensitive transport channel that is responsible, at least in part, for resistance to VC; (iii) in VC-resistant MELC, there is constitutive expression or accumulation of a protein required for HMBA-induced differentiation; and (iv) an elevated level of PKC beta activity may play a role in the altered response of R1[VCR] and other VC-resistant MELC to HMBA.     

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.     

Kinetics of histone H10 accumulation and commitment to differentiation in murine erythroleukemia cells

The accumulation of histone H10 (also denoted IP 25) in murine erythroleukemia cells, induced to differentiate with hexamethylene bis-acetamide, was shown to precede by 15-20 h the appearance in the culture of cells irreversibly committed to differentiate. In addition the rates of accumulation of H10 and of committed cells vary in a similar manner with the HMBA concentration. Flow microfluorimetric analysis demonstrated that the accumulation of H10 did not occur simultaneously in all the cells. This accumulation of histone H10 was initiated first in cell in the G2 phase of the cell cycle and subsequently in the cells situated in all the phases of the cell cycle.     

Amiloride in differentiation and commitment of Friend erythroleukemia cells

Dimethylsulfoxide (DMSO) converts almost all of the undifferentiated murine erythroleukemia cells (MEL or Friend cells, clone 745A) in a culture to differentiated cells that contain high levels of hemoglobin and that stop growing after a limited number of cell divisions. Contrary to other reports--that amiloride strongly inhibits DMSO-induced differentiation in MEL cells--in this laboratory, inhibition by amiloride, tested with DMSO over a range of concentrations in two kinds of media and at various cell densities, was found to be only weak or absent. Similarly, amiloride did not inhibit induction by N,N'-hexamethylene bis-acetamide (HMBA). As expected from previous findings with other cell systems, amiloride inhibited protein synthesis and cell multiplication.     

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.     

Glucose deprivation results in the induction of glucose-regulated proteins and domes in MDCK monolayers in hormonally defined serum-free medium

Madin-Darby canine kidney (MDCK) cells spontaneously form dome-like structures in vitro, a phenomenon which has been proposed to be indicative of cellular differentiation. This study indicates the existence of a correlation between the induction of domes and of glucose-regulated proteins during glucose starvation. When MDCK monolayers were glucose deprived, domes appeared very rapidly. After only 3 h of glucose deprivation domes appeared in 69% of the microscope fields. The level of expression of glucose-regulated proteins (grps) as well as domes was examined over a 6-h time interval of glucose deprivation. Both grp 76 and 97 were induced over this time interval, with grp 76 being the more readily detectable. The level of induction of grp 76 as a function of time was quantitated by means of densitometry measurements. The induction of domes was examined in parallel with the induction of grp 76. The results indicated that the induction of grp 76 and domes occurs with a similar time course. The effect of glucose deprivation on the initial rate of ouabain-sensitive Rb+ uptake was also examined. Within the first 4 h of glucose deprivation, the initial rate of ouabain-sensitive Rb+ uptake did not differ significantly in glucose-deprived and control MDCK monolayers. These observations indicate that unlike the case with other methods of dome induction (e.g., treatment with either prostaglandin E1 (PGE1) or hexamethylene bis-acetamide (HMBA] glucose deprivation does not affect the Na+K+ ATPase activity of MDCK monolayers. These observations suggest that PGE1, HMBA, and glucose deprivation affect dome formation in MDCK monolayers by means of distinct mechanisms.     

Polyamine regulation of Na+/glucose symporter expression in LLC-PK1 cells

Addition of polyamines or their analogs to newly confluent LLC-PK₁ cells resulted in down-regulation of Na⁺-glucose transport (symport) activity. Polyamines prevented the induction of this symporter by the differentiation inducer hexamethylene bisacetamide (HMBA) but did not influence induction by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Partial depletion of endogenous polyamines after addition of α -difluoromethylornithine (DFMO) resulted in a 4 to 5-fold increase in symporter expression. Symporter induction by either HMBA or DFMO was inhibited by the protein kinase inhibitor H-7 but H-7 did not affect symporter induction by IBMX. Changes in symporter activity were accompanied by changes in levels of the 75 kD symporter subunit detected by Western blot. Cultures exposed to HMBA exhibited reduced levels of ornithine decarboxylase activity. Our results suggest that induction of symporter expression by HMBA may be mediated in part by its effects on polymine metabolism, and point to parallel roles of polyamines and cyclic AMP in regulating the expression of this physiologically important renal transport system. © 1993 Wiley-Liss, Inc.     

Differential expression of protein kinase C isozymes and erythroleukemia cell differentiation

Hexamethylene bisacetamide (HMBA) and other polar/apolar chemical agents are potent inducers of erythroid differentiation in murine erythroleukemia cells (MELC), as well as other transformed cell lines. Although the mechanism of action of HMBA is not yet known, evidence has been obtained that protein kinase C (PKC) plays a role in this process. In this study we provide further evidence that establishes this relationship. MELC contain two principal PKC activities, PKC beta and PKC alpha. MELC variants, selected for resistance to vincristine (VC), which display acceleration of their rates of induced differentiation, are enriched in PKC beta activity. When MELC are exposed to HMBA there is a fall in PKC activity, largely accounted for by a decline in PKC beta. This decline in PKC activity is faster in the VC-resistant, rapidly differentiating MELC. We previously demonstrated that VC-resistant MELC are resistant to the inhibition of differentiation by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In both VC-sensitive and -resistant MELC, PMA causes rapid membrane translocation and then a decline in PKC activity, accompanied by a generation of a Ca2+- and phospholipid-independent protein kinase activity. In VC/PMA-resistant variants, this Ca2+/phospholipid-independent protein kinase activity persists considerably longer than in the VC-sensitive variants. This correlates with the resistance to PMA and provides additional evidence for a role for the Ca2+/phospholipid-independent protein kinase activity during induced differentiation.     

Involvement of monoamine oxidase and diamine oxidase in the metabolism of the cell differentiating agent hexamethylene bisacetamide (HMBA)

We have previously demonstrated a number of metabolites of hexamethylene bisacetamide (HMBA) in the urine of patients treated with HMBA. These include N-acetyl-1,6-diaminohexane (NADAH), 6-acetamidohexanoic acid (6AcHA), 1,6-diaminohexane (DAH) and 6-aminohexanoic acid (6AmHA). Because these compounds have potential roles in the dose-limiting metabolic acidosis and neurotoxicity associated with HMBA therapy, and are similar in structure to known substrates of monoamine oxidase (MAO) and diamine oxidase (DAO), we investigated the activities of these enzymes in the metabolic interconversion of HMBA metabolites. NADAH (5 mM) was incubated with MAO and aldehyde dehydrogenase. 6AcHA production was verified by gas chromatography-mass spectrometry and quantified by gas chromatography. 6AcHA production was linear for up to 4 hr. Complete inhibition of MAO activity was observed with 2 mM tranyl-cypromine or pargyline. Mouse liver microsomes, which do not contain MAO, did not convert NADAH to 6AcHA and, in control experiments, did not degrade 6AcHA. The HMBA metabolite, DAH, was a substrate for DAO, producing 3,4,5,6-tetrahydro-2H-azepine. Participation of DAO in the metabolism of HMBA implies potential interaction of HMBA and metabolites with polyamine metabolism and may represent a mechanism for HMBA's effects on cellular growth and differentiation. Metabolism of NADAH, also a differentiator, by MAO implies that concurrent use of HMBA and an MAO inhibitor may be clinically useful.