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PubMed2005

An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation.

We have previously described a multiprotein complex termed the BHC or BRAF-HDAC complex, which is required for the repression of neuronal-specific genes. We have shown that the BHC complex is recruited by a neuronal silencer, REST (RE1-silencing transcription factor), and mediates the repression of REST-responsive genes. BHC is a multiprotein complex consisting of two enzymatic activities: a histone deacetylase (HDAC1 or 2) and a recently described histone demethylase (BHC110, also known as LSD1 or AOF2). Here we show that BHC110-containing complexes show a nearly fivefold increase in demethylation of histone H3 lysine 4 (H3K4) compared to recombinant BHC110. Furthermore, recombinant BHC110 is unable to demethylate H3K4 on nucleosomes, but BHC110-containing complexes readily demethylate nucleosomes. In vitro reconstitution of the BHC complex using recombinant subunits reveals an essential role for the REST corepressor CoREST, not only in stimulating demethylation on core histones but also promoting demethylation of nucleosomal substrates. We find that nucleosomal demethylation is the result of CoREST enhancing the association between BHC110 and nucleosomes. Depletion of CoREST in in vivo cell culture results in de-repression of REST-responsive gene expression and increased methylation of H3K4. Together, these results highlight an essential role for CoREST in demethylation of H3K4 both in vitro and in vivo.

Acetylation↗
PubMed1986

The effect of antidepressants on ethylmorphine and imipramine N-demethylation in rat liver microsomes.

The effects of single and multiple doses of desipramine, amitriptyline or citalopram on the rat liver microsomal cytochrome P-450 level and on the rate of ethylmorphine and imipramine demethylation in-vitro have been investigated. Desipramine, amitriptyline or citalopram when given to rats as a single dose, did not affect the level of cytochrome P-450 in the liver microsomes, however, there was a tendency towards acceleration of imipramine, and particularly ethylmorphine, demethylation. Prolonged administration of desipramine and citalopram, but not amitriptyline, elevated the microsomal level of cytochrome P-450 and accelerated the rate of ethylmorphine demethylation. All the drugs investigated, when given chronically, inhibited the rate of imipramine demethylation. Since demethylation of ethylmorphine and imipramine in a CO atmosphere was inhibited by ca 90% for the former and only by 58% for the latter, it can be assumed that prolonged administration of the drugs investigated has two different effects on the oxygenase systems in rat liver microsomes: on the one hand they stimulate the cytochrome P450 oxygenase system involved in ethylmorphine demethylation and, on the other, they inhibit the other microsomal oxygenase system involved in demethylation of imipramine.

Animals↗
PubMedDate not supplied

Effects of phenothiazine neuroleptics on the rate of caffeine demethylation and hydroxylation in the rat liver.

The primary metabolic pathways of caffeine are 3-N-demethylation to paraxanthine (CYP1A2), 1-N-demethylation to theobromine and 7-N-demethylation to theophylline (CYP1A2 and other enzymes), and 8-hydroxylation to 1,3,7-trimethyluric acid (CYP3A). The aim of the present study was to investigate the influence of phenothiazine neuroleptics (chlorpromazine, levomepromazine, thioridazine, perazine) on cytochrome P-450 activity measured by caffeine oxidation in rat liver microsomes. The obtained results showed that all the investigated neuroleptics competitively inhibited caffeine oxidation in the rat liver, though their potency to inhibit particular metabolic pathways was not equal. Levomepromazine exerted the most potent inhibitory effect on caffeine oxidation pathways, the effect on 8-hydroxylation being the most pronounced. This indicates inhibition of CYP 1 A2 (inhibition of 3-N- and 1-N-demethylation; Ki = 36 and 32 microM, respectively), CYP3A2 (inhibition of 8-hydroxylations; Ki = 20 microM), and possibly other CYP isoenzymes (inhibition of 7-N-demethylation; Ki = 58 microM) by the neuroleptics. The potency of inhibition of caffeine oxidation by perazine was similar to levomepromazine. Thioridazine was a weaker inhibitor of caffeine 3-N- and 7-N-demethylation, while chlorpromazine was weaker in inhibiting caffeine 1-N- and 7-N-demethylation, compared to levomepromazine. In summary, the obtained results showed that all the investigated neuroleptics had a broad spectra of CYP inhibition in the rat liver. The isoenzymes CYP1A2 and CYP3A2 were distinctly inhibited by all the investigated neuroleptics, while other CYP isoenzymes (CYP2B and/or 2E1) by perazine and levomepromazine. The CYP3A2 inhibition was most pronounced. (Ki = 20-40 microM).

Animals↗
PubMed2003

Catalyzation of cocaine N-demethylation by cytochromes P4502B, P4503A, and P4502D in fish liver.

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.

Animals↗
PubMed1999

Antitumor agents. 194. Synthesis and biological evaluations of 4-beta-mono-, -di-, and -trisubstituted aniline-4'-O-demethyl-podophyllotoxin and related compounds with improved pharmacological profiles.

As a continuation of our structure-activity relationship studies, several new 4-beta-substituted 4'-O-demethyl-4-desoxypodophyllotoxins bearing mono-, di-, or trisubstituted anilines have been synthesized and evaluated as inhibitors of DNA topoisomerase II and tumor cell growth in tissue culture. Selected compounds were further evaluated as cytotoxic agents using a clonogenic survival assay. The target compounds include 4'-O-demethyl-4beta-[(4' '-(benzimidazol-2' '-yl)anilino]-4-desoxypodophyllotoxin (21), 4'-O-demethyl-4beta-(-)-(4' '-camphanamido-anilino)-4-desoxypodophyllotoxin (25), 4-beta-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxins (18-20, 26), 4-alpha-disubstituted-anilino-4'-demethyl-4-desoxypodophyllotoxin (27), 4-beta-trisubstituted-anilino-4'-demethyl-desoxypodophyllotoxin (22, 23), and 4'-O-demethyl-4beta-[4' '-(benzimidazol-2' '-yl)amino]-4-desoxypodophyllotoxin (24). Among the target series, 19, 21, and 24 displayed significant growth inhibitory action against a panel of tumor cell lines including human epidermoid carcinoma of the nasopharynx (KB) and its etoposide-resistant (KB7B) and vincristine-resistant (vin20c KB) subclones, lung carcinoma (A549), human ileocecal carcinoma (HCT-8), human kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), and human malignant melanoma (SK-MEL-2) cells. Compounds 19, 21, 24, and 25 were "cleavable-complex"-forming DNA topoisomerase II inhibitors with either improved or similar activity compared with the prototype drug etoposide (VP-16). Compound 21 was the most active analogue, being 10-fold more potent than etoposide in both cell killing and topoisomerase II inhibition in vitro assays. Using mouse models of antitumor activity, 21 was effective against (P388/0) leukemia but not against the growth of a (MCF7) mammary tumor.

Animals↗
PubMed2005

Epigenetic treatment of hematopoietic malignancies: in vivo targets of demethylating agents.

Although the first studies using DNA demethylating agents at low doses in hematologic neoplasia and hemoglobinopathies were initiated more than 20 years ago, development of this type of nonintensive treatment has only been spurred in the last 6 to 8 years by the discovery of many genes that are specifically hypermethylated in cancer. These provide a powerful rationale for using azanucleosides (and other small molecules being developed for DNA demethylation) as a novel means of pharmacologic targeting of cancer cells that is distinct from low-dose chemotherapy. Encouraging response rates of about 50% in myelodysplasia with 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine or DAC) have resulted in a number of phase III studies being initiated in this disorder. The development of such drugs for the treatment of acute myeloid leukemia (AML) is ongoing. While the specificity of DNA demethylation has been delineated by studying distinct genes or sets of genes, and proof-of-principle studies of in vivo methylation report demethylation and reactivation of genes like p15/INK4b and gamma-globin, responses to demethylating agents may be more complex. Specifically, so-called cancer testis antigens (CTAs) are intriguing targets for demethylation, since they are silenced in many hematopoietic disorders and may be reactivated by epigenetic therapy. Thus, demethylating agents and histone deacetylase inhibitors may also induce a T-cell-mediated antileukemic or antitumor effect.

Antigens, Neoplasm↗
PubMed2000

Human term placental lipoxygenase-mediated N-demethylation of phenothiazines and insecticides in the presence of linoleic acid.

This study investigated the hypothesis that human term placental lipoxygenase (HTPLO) and soybean lipoxygenase (SLO) are capable of mediating N-demethylation of selected phenothiazines and insecticides in the presence of linoleic acid (LA). In addition to being LA dependent, the N-demethylation reaction mediated by HTPLO and SLO was limited by incubation time, pH of the medium, concentration of the enzyme and the substrate. Using Nash reagent to monitor formaldehyde production, the specific activity for LA-dependent N-demethylation of chlorpromazine, a model phenothiazine, was determined to be 1.7+/-0.3 nmoles/min/mg HTPLO. Besides chlorpromazine, N-demethylation of promazine, promethazine and trimeprazine was also observed. The insecticide, aminocarb, displayed a specific activity of 2.2+/-0.3 nmoles/min/mg HTPLO for N-demethylation. Other insecticides, namely chlordimeform, dicrotophos and zectran, were oxidized in a similar manner. As compared with HTPLO, the rates of N-demethylation of phenothiazines and insecticides mediated by SLO were higher. Classical inhibitors of lipoxygenase, as well as antioxidants and free radical scavengers, caused a dose-dependent reduction in the production of formaldehyde from chlorpromazine and aminocarb by HTPLO. These results clearly demonstrate the ability of polyunsaturated free fatty acids to support N-demethylation of xenobiotics via the lipoxygenase pathway.

Carbamates↗
PubMed2006

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks.

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3' to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

Animals↗
PubMed2001

Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene.

We have shown that the DNA demethylation complex isolated from chicken embryos has a G(.)T mismatch DNA glycosylase that also possesses 5-methylcytosine DNA glycosylase (5-MCDG) activity. Herein we show that human embryonic kidney cells stably transfected with 5-MCDG cDNA linked to a cytomegalovirus promoter overexpress 5-MCDG. A 15- to 20-fold overexpression of 5-MCDG results in the specific demethylation of a stably integrated ecdysone-retinoic acid responsive enhancer-promoter linked to a beta-galactosidase reporter gene. Demethylation occurs in the absence of the ligand ponasterone A (an analogue of ecdysone). The state of methylation of the transgene was investigated by Southern blot analysis and by the bisulfite genomic sequencing reaction. Demethylation occurs downstream of the hormone response elements. No genome-wide demethylation was observed. The expression of an inactive mutant of 5-MCDG or the empty vector does not elicit any demethylation of the promoter-enhancer of the reporter gene. An increase in 5-MCDG activity does not influence the activity of DNA methyltransferase(s) when tested in vitro with a hemimethylated substrate. There is no change in the transgene copy number during selection of the clones with antibiotics. Immunoprecipitation combined with Western blot analysis showed that an antibody directed against 5-MCDG precipitates a complex containing the retinoid X receptor alpha. The association between retinoid receptor and 5-MCDG is not ligand dependent. These results suggest that a complex of the hormone receptor with 5-MCDG may target demethylation of the transgene in this system.

Azacitidine↗
PubMed1979

Attractants and repellents control demethylation of methylated chemotaxis proteins in Escherichia coli.

A group of methylated proteins, the methyl-accepting chemotaxis proteins (MCP), has been shown to play a central role in bacterial chemotaxis. Both methylation and demethylation of MCP occur continuously in the absence of added stimuli; these two processes are in balance such that a basal level of methylation is maintained. Attractants cause the methylation level to increase to a new value, whereas repellents bring about a decrease in level. Therefore, attractants and repellents must somehow perturb the balance between methylation and demethylation of MCP. In this report the effect of attractants on demethylation of MCP was monitored in two ways: (i) by following the disappearance of [methyl-3H]MCP and (ii) by measuring formation of [3H]methanol, the product of MCP demethylation. Both methods showed that addition of attractants causes a transient inhibition of MCP demethylation. Repellent addition has previously been shown to stimulate MCP demethylation. It is therefore concluded that control of demethylation plays a crucial role in changing the level of methylation of MCP in response to attractants and repellents.

Bacterial Proteins↗
PubMed1986

Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis.

Genomic sequencing was used to study the extent of cytosine methylation of four CpG sites within the regulatory region of the estradiol-inducible avian vitellogenin II gene. Three of these sites, two of which lie within the estradiol-receptor binding site and one in a short stretch of alternating purines and pyrimidines, were initially fully methylated. Analysis of DNA isolated from liver nuclei revealed that hormone treatment of immature White Leghorn roosters resulted in a demethylation of these sites, which occurred initially in only one DNA strand. This demethylation correlated well with the induction of vitellogenin mRNA synthesis. The demethylation of the complementary DNA strand lagged approximately equal to 24 hr behind. The fourth CpG, located within an overlapping glucocorticoid-receptor binding site, was already hemimethylated at the onset of the experiment. The demethylation of this site also occurred with kinetics similar to the rate of vitellogenin mRNA synthesis. All four CpGs remained demethylated even after cessation of gene transcription. A comparison of the methylation state of these four sites in DNA from different tissues demonstrated a clear dependence of the demethylation on estradiol. Our results suggest that this hormone-dependent event occurs via an active pathway through excision repair and/or enzymatic demethylation.

5-Methylcytosine↗
PubMed2003

In vitro inhibition of human small intestinal and liver microsomal astemizole O-demethylation: different contribution of CYP2J2 in the small intestine and liver.

1. The effects of chemical agents on the metabolism of the antihistamine drug astemizole were investigated to evaluate drug-drug interactions. 2. Chemical inhibitors of astemizole O-demethylation were screened using the small intestinal and liver microsomes from rabbit as an animal model for the first-pass metabolism of humans. In the rabbit small intestine, astemizole O-demethylation was clearly inhibited by ebastine, arachidonic acid, alpha-naphthoflavone, ketoconazole, tranylcypromine, troglitazone and terfenadine. 3. In humans, these inhibitors also reduced microsomal astemizole O-demethylation in both the small intestine and liver. However, the inhibition rate of almost all these chemicals were clearly greater in the small intestine than in the liver. Thus, a different contribution of cytochrome p450 in each tissue is suggested. 4. All the chemicals inhibited astemizole O-demethylation in recombinant CYP2J2 microsomes. The results suggest that CYP2J2 is involved in astemizole O-demethylation in both the human small intestine and liver; however, the contribution in the liver is lower than in the small intestine. The effects of the CYP2J2 inhibitors during first-pass metabolism may be more important in the small intestine than in the liver. Since all the inhibition profiles of astemizole O-demethylation were different in the liver and small intestine, involvement of another p450 in astemizole O-demethylation in human liver may be speculated. 5. In the rabbit microsomal systems, the same metabolites found in humans were qualitatively detected and the inhibition profiles of the chemical agents in the microsomes resembled that of humans.

Animals↗
PubMed1998

Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts.

The mechanism for demethylation of DNA in rat myoblasts has recently been studied using a new in vitro system that monitors demethylation in whole cell extracts. Previous investigations using this system had indicated that demethylation is resistant to conditions that are normally assumed to denature or digest proteins. Remarkably, it was reported that the activity appeared to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA. This manuscript reports that, upon further purification of the extract, demethylation activity has properties that are different. When subjected to more rigorous procedures for digestion of proteins, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is added to the reaction mix or when extracts treated with RNase are subsequently treated with protease. Although a role for RNA cannot be completely discounted, it is unlikely that this demethyl-ation reaction involves RNA cofactors or ribozyme components. These results have important implications for the mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RNase sensitivity experiments.

Animals↗
PubMed1989

Codeine O-demethylation co-segregates with polymorphic debrisoquine hydroxylation.

1. A single oral dose of codeine (25 mg) was given to 132 healthy Swedish Caucasians who had previously been phenotyped with respect to debrisoquine hydroxylation. The 'metabolic ratios' (MR) in urine of codeine O-demethylation (codeine/(morphine (M) + morphine-3- and 6-glucuronides (M3G and M6G) + normorphine], N-demethylation (codeine/(norcodeine (NC) + norcodeine glucuronide + normorphine (NM]) and glucuronidation (codeine/codeine-6-glucuronide (C6G] were calculated following h.p.l.c. analysis of urine samples collected over 8 h. 2. There was a significant correlation between the log MR for debrisoquine hydroxylation and the log MR for codeine O-demethylation (rs = 0.77, P less than 0.001). The poor debrisoquine hydroxylators had MRs of codeine O-demethylation between 8.3 and 55.1, while the values for extensive hydroxylators were between 0.4 and 5.5. 3. The poor debrisoquine hydroxylators excreted significantly less M, M3G, M6G and NM, while the urinary recovery of C6G and NC was significantly higher in these subjects compared to the extensive hydroxylators. 4. The MRs for glucuronidation and N-demethylation did not exhibit a bimodal distribution, and were not related to the MR of debrisoquine hydroxylation. 5. No associations were found between sex, body-weight, smoking habits, age, urine volume or urine pH and the O-demethylation of codeine. 6. The O-demethylation of codeine to form M appears to be under the same polymorphic genetic control as the 4-hydroxylation of debrisoquine.

Adolescent↗
PubMed1994

O-demethylation by the homoacetogenic anaerobe Holophaga foetida studied by a new photometric methylation assay using electrochemically produced cob(I)alamin.

The previously studied complete methyl transfer sequence of tetrahydrofolate-dependent O-demethylation catalyzed by Holophaga foetida strain TMBS4 extracts was separated into two steps using cobalamins as non-physiological substrates: electrochemically produced cob(I) alamin served as methyl acceptor for phenyl methyl ether demethylation, yielding methylcob(III)alamin (reaction I), and methylcob(III)alamin served as donor for tetrahydrofolate methylation, yielding 5-methyl tetrahydrofolate (reaction II). Both reactions were measured with a new and direct photometric assay of cob(I)alamin methylation (or the reverse reaction) at 540 nm, the isobestic wavelength of the cob(II)alamin/cob(I)alamin redox couple (delta epsilon 540 = 4.40 nM-1.cm-1. The rates of reactions I and II were proportional to protein concentration, unlike the complete reaction sequence. Small components of cell extract did not affect activity of reactions I and II. Isovanillate demethylation by extracts of synringate-grown cells (reaction I) required reductive activation by cob(I)alamin and was inhibited and inactivated by cob(II)alamin, indicating that the reaction mechanism was a nucleophilic attack of an enzyme-bound corrinoid in the reduced Co(I) state on the methyl carbon of the ether, rather than a radical attack. Only phenyl methyl ethers were demethylated; demethylation rates were enhanced by ortho-hydroxyl or para-carboxyl groups, but reduced by additional meta substituents. The rate of isovanillate demethylation was 81 nmol.min-1.(mg protein)-1 [0.76 mM cob(I)alamin] and apparent kinetic constants for cob(I)alamin were: Km = 1.2 mM, Vmax = 220 nmol min-1.(mg protein)-1, and Vmax/Km = 180 nmol.min-1.(mg protein) 1.mM-1 3,5-Dihydroxyanisole demethylation by extracts of 3,5-dihydroxyanisole-grown cells (also reaction I) was much slower. Reaction II did not require activation; specific activity and the specificity constant for methylcob(III)alamin were much lower.

Anisoles↗
PubMed2006

Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell.

AIM: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. METHODS: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. RESULTS: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner. N-demethyl-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. CONCLUSION: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.

Anti-Inflammatory Agents↗
PubMed2004

Interactions between CYP2C9 and CYP2C19 in reconstituted binary systems influence their catalytic activity: possible rationale for the inability of CYP2C19 to catalyze methoxychlor demethylation in human liver microsomes.

Previous studies in our laboratory showed that among cDNA-expressed human cytochrome P450 (P450) supersomes, CYP2C19 was the most active in methoxychlor-O-demethylation. However, based on the lack of inhibition of methoxychlor-O-demethylation by monoclonal anti-CYP2C19 antibodies in human liver microsomes (HLM), CYP2C19 did not seem to catalyze that reaction in HLM. By contrast, CYP2C9, much less active than CYP2C19 in supersomes, was the most active in HLM. The current study examines whether the lack of methoxychlor-O-demethylation by CYP2C19 in HLM was due to CYP2C19 exhibiting inferior competition for the NADPH-cytochrome P450 reductase (CPR) versus CYP2C9 and explores the interactions between CYP2C9 and CYP2C19 in a singular and binary complex of a reconstituted system. When reconstituted with CPR, cytochrome b(5), and lipid, purified CYP2C19 and CYP2C9 catalyzed methoxychlor-O-demethylation. However, whereas equimolar CPR to CYP2C9 supported maximal rates of methoxychlor demethylation and diclofenac hydroxylation, the rate of methoxychlor demethylation by CYP2C19 was not fully saturated, even with a 9-fold molar excess of CPR over CYP2C19. This behavior of CYP2C19 was also observed with S-mephenytoin as the substrate. When a binary reconstitution system was prepared by mixing CYP2C9 and CYP2C19 enzymes, methoxychlor-O-demethylation and S-mephenytoin hydroxylation by CYP2C19 were dramatically inhibited. Inhibition depended on the amount of CPR and substrate used. By contrast, in the incubation containing CYP2C9, diclofenac hydroxylation was activated by the presence of CYP2C19. These results show that interactions among P450 enzymes can modulate their catalytic rates, which depend on the substrate undergoing metabolism.

Aryl Hydrocarbon Hydroxylases↗
PubMed2005

Characterization of DNA demethylation effects induced by 5-Aza-2'-deoxycytidine in patients with myelodysplastic syndrome.

Azanucleoside drugs such as 5-azacytidine (Vidaza) and 5-aza-2'-deoxycytidine (decitabine, Dacogen) function as DNA methyltransferase inhibitors in vitro and represent promising new drugs for the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia. In this study, we aimed to determine the effect of decitabine on the genomic methylation level in MDS patients. Comparison of different assays established micellar electrokinetic chromatography as a reliable method for the analysis of genomic methylation levels. When used for the determination of DNA methylation levels in bone marrow DNA from MDS patients during various time points of decitabine treatment, the results revealed a significant (up to 70%) demethylation in five of seven patients. Interestingly, genome-wide demethylation appeared after karyotype normalization, which suggests demethylation of nonclonal cells. Drug-induced demethylation dynamics were also confirmed by bisulfite sequencing of pericentromeric satellite elements. Our results are the first to show a genome-wide demethylating activity of decitabine in tumor material. In addition, our data uncovers novel targets of decitabine-mediated demethylation that are important for the refinement of treatment schedules with demethylating drugs.

Aged↗