Enantioselective analysis of ketone bodies in patients with β-ketothiolase deficiency, medium-chain acyl coenzyme A dehydrogenase deficiency and ketonemic vomiting

Enantioselective multidimensional gas chromatography–mass spectrometry (enantio-MDGC–MS) is a valuable tool for the differentiation of enantiomers from complex matrices when present in trace amounts. The separation of chiral compounds provides further information on the diagnosis of diseases, and on normal and abnormal biochemical pathways. The formation of the normal urinary metabolite 3-hydroxy-2-methylbutanoic acid (HMBA), excreted in abnormally high amounts in β-ketothiolase deficiency, is not absolutely clarified. Metabolic pathways involving this metabolite are isoleucine catabolism, as well as presumably β-oxidation of fatty acids and ketogenesis. The latter two pathways are distinguishable in their enantioselectivity. Enantioselective analysis gives further information on interfering metabolic pathways and the selectivity of the enzyme(s) forming HMBA. Different ratios of the stereoisomers of HMBA in control urine samples and patients with β-ketothiolase deficiency were detected. Analogous to HMBA urinary 3-hydroxybutanoic acid (HBA) was investigated in several diseases. The formation of HBA and HMBA is expected to result from the same or similar metabolic pathways. Differences in the enantiomeric ratio of HMBA may originate from the enantioselectivity of different enzyme systems.     

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.     

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.     

Relative resistance to 1,25-dihydroxyvitamin D3 in a keratinocyte model of tumor progression.

We have examined the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on mitogen-stimulated growth and on c-myc proto-oncogene expression in a keratinocyte model of tumor progression. A dose-dependent inhibition of cell growth by 1,25-(OH)2D3 was demonstrated in both established (HPK1A) and malignant (HPK1A-ras) cells. However, this inhibition was observed with the addition of 1,25-(OH)2D3 at a higher concentration in HPK1A-ras cells than in HPK1A cells. Cell cycle analysis revealed a blockage of the normal progression of the cell cycle from G0 to S phase in the presence of 1,25-(OH)2D3. A higher concentration of 1,25-(OH)2D3 was required in HPK1A-ras cells to overcome the mitogen-stimulated progression into S phase, when compared with HPK1A cells. Analysis of c-myc messenger RNA revealed a strong inhibition of its expression at early time points with higher concentrations of 1,25-(OH)2D3 being required to obtain an inhibition in HPK1A-ras cells similar to that obtained in HPK1A cells. 1,25-(OH)2D3 receptor characterization by sucrose gradient analysis and equilibrium binding demonstrated the presence of a single 3.7 S protein with similar receptor numbers and affinity in both cell lines. These observations therefore demonstrate that an alteration of the growth inhibitory response to 1,25-(OH)2D3 occurs when keratinocytes acquire the malignant phenotype and suggest that the alteration lies beyond the interaction of the ligand with its receptor. In addition, relative resistance to 1,25-(OH)2D3 was also observed in the expression of the cell-cycle associated oncogene c-myc. These studies may therefore have important implications in vivo in the development and growth of epithelial cell cancers.     

The modulation of growth by HMBA in PKC overproducing HT29 colon cancer cells.

To examine whether protein kinase C (PKC) plays a role in mediating growth inhibitory effects of hexamethylene bisacetamide (HMBA) we compared a control H29 colon cancer cell line to a derivative, HT29-PKC7, that overexpresses high levels of PKC beta 1. We found that although HMBA markedly inhibited the growth of the control cells, no inhibition was seen with the HT29-PKC7 cells. On the other hand the tumor promoter 12-0-tetradecanoyl-phorbol-13 acetate inhibited the growth of HT29-PKC7 cells, but no inhibition was seen with the control cells. Maximum inhibition of the growth of both cell lines was obtained by combined treatment with HMBA and TPA. These results may be relevant to the use of HMBA in combination with other agents in the therapy of specific cancers.     

Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin

Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.     

Suppression of Cell-cycle Progression during the S Phase of Rat Fibroblasts by 3-Deoxyglucosone,a Maillard Reaction Intermediate

3-Deoxyglucosone (3DG), the main intermediate compound in the Maillard reaction of proteins with glucose, suppressed the proliferation of various cell lines by inhibition of DNA synthesis. We investigated the mechanism of the suppression of cell proliferation from the standpoint of the progression of cell cycle. When 3DG was added to the culture of 3Y1 cells, rat fibroblasts, growing in exponential phase, the addition of 300 or 600μg/ml of 3DG increased the numbers of the cells apparently arrested at the G1 or G2/M phase, respectively. We observed that 3DG specifically inhibited the time-dependent progression during the S phase of a synchronous culture released from the early S phase in 3Y1 cells. 3DG influenced the cells released from the GO phase but not the GO-arrested cells. When an intracellular concentration of reduced glutathione (GSH) in 3Y1 cells was decreased by using a GSH synthetase inhibitor, the inhibition of [³H]thymidine incorporation by 3DG was enhanced. Therefore, we assumed that the cells proliferating actively, in which the intracellular GSH concentrations have been reported to be lower, were more susceptible to the inhibitory effects of 3DG on the cell-cycle progression during the S phase.     

Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive Drug Metabolites Acting on TRPV1 in the Brain

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT₃ or 5-HT_1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.     

Modification of transformation of C3H/10T1/2 cells by a differentiation-inducing substance

Transformation of C3H/10T1/2 cells was induced by 3-methylcholanthrene. Treatment with hexamethylene bisacetamide (HMBA), a differentiation inducing and poly (ADP-ribose)-synthesis modifying substance, influences expression of multilayered foci in a treatment schedule-dependent manner. Inhibition of transformation occurred only if HMBA was present after the genotoxic damage. After HMBA treatment most of transformed cells showed an end-differentiation like form.     

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…     

A comparison of time-lapse cinemicrography and flow cytometry for the study of accelerated cell-cycle transit

Abstract. Two methods for the study of cell-cycle progression, time-lapse cinemicrography (TLCM) and flow cytometry (FCM), were compared for their ability to measure the shortening of cell-cycle transit time induced by temporary inhibition of DNA synthesis. DNA synthesis was reversibly inhibited by aphidicolin (APH) in synchronized HeLa cells obtained by mitotic collection. TLCM directly measured intermitotic time intervals and thereby directly obtained the cell-cycle transit time distribution. In contrast, FCM measured time dependent changes in the fractions of cells in the cell-cycle phases from which the distribution of cells traversing a cell-cycle boundary, such as that between G₁ and S phase, was determined. Nevertheless, both methods provided equivalent measures of the cell-cycle transit time and its dispersion. However, TLCM apeared to provide a better measure of skewness of the transit time distribution than did FCM. Further, both methods were able to detect changes in the cell cycle transit on the order of 1 h or less. The TLCM data showed a greater precision (due to a larger number of data points) than that from FCM. However, FCM was able to directly measure changes in the transit of G₁ phase whereas TLCM would require two different experiments to make a similar determination. The results obtained in this study show that FCM can replace TLCM to study most aspects of cell-cycle progression.     

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.     

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.     

Deletion of histone deacetylase 3 reveals critical roles in S phase progression and DNA damage control

Histone deacetylases (HDACs) are enzymes that modify key residues in histones to regulate chromatin architecture, and they play a vital role in cell survival, cell-cycle progression, and tumorigenesis. To understand the function of Hdac3, a critical component of the N-CoR/SMRT repression complex, a conditional allele of Hdac3 was engineered. Cre-recombinase-mediated inactivation of Hdac3 led to a delay in cell-cycle progression, cell-cycle-dependent DNA damage, and apoptosis in mouse embryonic fibroblasts (MEFs). While no overt defects in mitosis were observed in Hdac3-/- MEFs, including normal H3Ser10 phosphorylation, DNA damage was observed in Hdac3-/- interphase cells, which appears to be associated with defective DNA double-strand break repair. Moreover, we noted that Hdac3-/- MEFs were protected from DNA damage when quiescent, which may provide a mechanistic basis for the action of HDAC inhibitors on cycling tumor cells.     

Bioactivation of 7-hydroxymethyl-12- methyibenz[a]anthracene by rat liver bile acid sulfotransferase I

The bioactivation of 7-hydroxy-methyl-12-methylbenz[a]anthracene (HMBA) to an electrophilic sulfuric acid ester metabolite has been shown to be catalyzed by rat liver bile acid sulfotransferase I (BAST I). The sulfation and activation of HMBA by BAST I was determined by the ability of sulfated HMBA to form DNA ad-ducts. The BAST I was also shown to react with rabbit anti-human dehydroepiandrosterone sulfotransferase antisera and to represent a major form of hydroxysteroid/bile acid sulfotransferase in female rat liver cytosol. Higher levels of BAST I activity and immunoreactivity as well as HMBA-DNA adduct formation were detected in female rat liver cytosol than in male rat liver cytosol. The bioactivation of HMBA by pure BAST I was dependent on the presence of 3′-phosphoadenosine 5′-phos-phosulfate (PAPS) in the reaction and was inhibited by dehydroepiandrosterone, a physiological substrate for BAST I. Glutathione, a cellular nucleophile with important protective properties, decreased DNA adduct formation in the HMBA sulfation reaction in the absence of glutathione S-transferase activity. These results indicate the usefulness of BAST I to investigate the sulfation and activation of HMBA and probably other hydroxy-methylated polyaromatic hydrocarbons to electrophilic and mutagenic metabolites under defined reaction conditions.     

Short-period hypoxia increases mouse embryonic stem cell proliferation through cooperation of arachidonic acid and PI3K/Akt signalling pathways

Hypoxia plays important roles in some early stages of mammalian embryonic development and in various physiological functions. This study examined the effect of arachidonic acid on short-period hypoxia-induced regulation of G(1) phase cell-cycle progression and inter-relationships among possible signalling molecules in mouse embryonic stem cells. Hypoxia increased the level of hypoxia-inducible factor-1alpha (HIF-1alpha) expression and H2O2 generation in a time-dependent manner. In addition, hypoxia increased the levels of cell-cycle regulatory proteins (cyclin D(1), cyclin E, cyclin-dependent kinase 2 (CDK2) and CDK4). Maximum increases in the level of these proteins and retinoblastoma phosphorylation were observed after 12-24 h of exposure to hypoxic conditions, and then decreased. Alternatively, the level of the CDK inhibitors, p21(Cip1) and p27(Kip1) were decreased. These results were consistent with the results of [3H]-thymidine incorporation and cell counting. Hypoxia also increased the level of [3H]-arachidonic acid release and inhibition of cPLA(2) reduced hypoxia-induced increase in levels of the cell-cycle regulatory proteins and [3H]-thymidine incorporation. The level of cyclooxygenase-2 (COX-2) was also increased by hypoxia and inhibition of COX-2 decreased the levels of cell-cycle regulatory proteins and [3H]-thymidine incorporation. Indeed, the percentage of cells in S phase, levels of cell cycle regulatory proteins, and [3H]-thymidine incorporation were further increased in hypoxic conditions with arachidonic acid treatment compared to normoxic conditions. Hypoxia-induced Akt and mitogen-activated protein kinase (MAPK) phosphorylation was inhibited by vitamin C (antioxidant, 10(-3) M). In addition, hypoxia-induced increase of cell-cycle regulatory protein expression and [(3)H]-thymidine incorporation were attenuated by LY294002 (PI3K inhibitor, 10(-6) M), Akt inhibitor (10(-6) M), rapamycin (mTOR inhibitor, 10(-9) M), PD98059 (p44/42 inhibitor, 10(-5) M), and SB203580 (p38 MAPK inhibitor, 10(-6) M). Furthermore, hypoxia-induced increase of [(3)H]-arachidonic acid release was blocked by PD98059 or SB203580, but not by LY294002 or Akt inhibitor. In conclusion, arachidonic acid up-regulates short time-period hypoxia-induced G(1) phase cyclins D(1) and E, and CDK 2 and 4, in mouse embryonic stem cells through the cooperation of PI3K/Akt/mTOR, MAPK and cPLA(2)-mediated signal pathways.     

PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1.

BACKGROUND: Platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis within minutes. These events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully account for later responses, such as cell-cycle progression. RESULTS: We assessed when PI 3-kinase products accumulate in PDGF-stimulated cells. In addition to the previously identified early accumulation of products, we detected a second, prolonged wave of accumulation 3-7 hours after stimulation. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis, we first developed an assay in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a PDGF-receptor mutant. The lipids rescued DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors failed to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1 phase, prevented PDGF-dependent cell-cycle progression. CONCLUSIONS: PDGF induces distinct waves of PI 3-kinase activity. The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use PI 3-kinase to mediate distinct cellular responses seems to be by regulating when its products accumulate.