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At least 217 records · Page 12Linked to original sources
PubMed1985

High-dose cytosine arabinoside therapy with and without anthracycline antibiotics for remission reinduction of acute nonlymphoblastic leukemia.

Seventy-eight patients with acute nonlymphoblastic leukemia in relapse were treated with high-dose cytosine arabinoside (3 g/m2 intravenously (IV) every 12 hours for 12 doses) alone, or with three days of anthracycline antibiotics (doxorubicin 20 mg/m2 or daunorubicin 30 mg/m2 IV daily) after completing the course of cytosine arabinoside. Consolidation and maintenance therapy was not given. When anthracyclines were added there was no increase in frequency or severity of nonhematologic toxicity including conjunctivitis, photophobia, dermatitis, cerebellar dysfunction, and gastrointestinal disturbance. All 78 patients achieved aplasia of the bone marrow. Five patients in each group died before bone marrow recovery. The use of anthracyclines did not prolong bone marrow recovery, with both groups demonstrating adequate granulocyte and platelet counts about four weeks after beginning treatment. Forty-one (53%) of the total 78 patients achieved a complete remission. In patients not clinically resistant to conventional-dose cytosine arabinoside, both regimens were equally effective inducing a complete remission (high-dose cytosine arabinoside alone, 12/19 [63%]; with anthracycline, 11/17 [65%], P = .270); in patients clinically resistant, the regimen including anthracycline was superior (15/27 [56%] v 3/15 [20%], P = .022). The duration of unmaintained response was similar (median, five months), but the longest remissions occurred when anthracyclines were used. Thus, high-dose cytosine arabinoside is effective in producing remissions in relapsed patients with acute nonlymphoblastic leukemia, and the addition of an anthracycline enhances this effect.

Acute Disease↗
PubMed1987

Cytosine arabinoside plus cisplatin and other drugs as chemotherapy for gliomas.

Twenty-five evaluable patients with gliomas were treated with a combination of cytosine arabinoside plus cisplatin administered intravenously (IV). Ten of the 25 patients (40%) responded, including three of 13 patients (23%) with prior cranial radiation, and seven of 12 patients (58%) without prior cranial radiation. In a second study, intramuscular (IM) and oral caffeine were added to IV cytosine arabinoside plus cisplatin. Caffeine-induced seizures prevented escalation of the dose of caffeine to a level that would have been anticipated to potentiate the cytotoxicity of the cytosine arabinoside plus cisplatin. Twelve of 25 treated patients (48%) responded. Most of the patients had not received prior cranial radiation. In a third study, IV cytosine arabinoside was administered in combination with intracarotid and IV cisplatin, plus BCNU and tenoposide (VM-26). This study is continuing to accrue patients. Myelosuppression has been pronounced, but has generally been rapidly reversible. It is too early to comment on efficacy of this combination. Our results to date permit us to conclude that the combination of cytosine arabinoside plus cisplatin is capable of inducing regression of gliomas in some patients. We feel that further studies are warranted.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1983

High-dose cytosine arabinoside therapy for refractory leukemia.

Fifty-seven patients with refractory acute leukemia were treated with high-dose cytosine arabinoside to establish the maximum tolerated dose and duration and to determine the antileukemic activity. The maximum tolerated regimen was found to be 3 g/sq m every 12 hr for 6 days. At this dose, nonhematologic toxicity was limited to conjunctivitis in approximately half of the patients, and liver toxicity (transient elevations in transaminase, alkaline phosphatase, or bilirubin) was frequently observed, but neither was dose-limiting. Extending the duration of treatment to 8 days resulted in excessive diarrhea and skin toxicity (painful erythema with bullae), while increasing the dose to 4.5 g/sq m q. 12 hr for 6 days resulted in severe cerebellar toxicity. Myelosuppression was severe, but was not related to the intensity of treatment; granulocyte recovery occurred a median of 28 days (range 22-40 days) after initiating therapy, and platelet recovery occurred after a median of 25 days (range 16-41 days). Antileukemic activity was evaluable in the 46 patients who survived at least 3 wk. Complete remissions were obtained in 1 of 6 patients with chronic myelogenous leukemia (CML) in accelerated phase and 1 of 3 acute lymphoblastic leukemia (ALL) patients. A more detailed analysis of response was possible for the 37 evaluable patients with acute nonlymphoblastic leukemia: 70% of these patients responded, with 51% complete remissions. The median unmaintained response was 4 mo (range 2-26+ mo). The complete response rate was higher in patients who received at least 12 doses of high-dose cytosine arabinoside compared to shorter regimens [17/28 (61%) versus 2/9 (22%), p less than 0.05]. Resistance to cytosine arabinoside in conventional doses was documented in 11 patients, 5 of whom responded (2 complete remissions) to high-dose regimens. We conclude that high-dose cytosine arabinoside in the maximally tolerated regimen of 3 g/sq m every 12 hr for 6 days has substantial antileukemic activity in patients refractory to standard therapy. Durable unmaintained remissions can be achieved, even in patients who fail to respond to cytosine arabinoside in conventional doses.

Acute Disease↗
PubMed1987

Infection with frog virus 3 allows transcription of DNA methylated at cytosine but not adenine residues.

The genome of the iridovirus, frog virus 3, is highly methylated at cytosine residues by a virus-encoded DNA methyltransferase. We have shown previously that an FV3-induced trans-acting protein alters either host RNA polymerase II or methylated template to allow transcription from promoters inactivated by methylation. We now present evidence that the ability of FV3-infected cells to transcribe methylated DNA is specific for DNA methylated at cytosine residues. Eukaryotic promoters were inactivated by methylation of either adenine or cytosine residues, and tested for transcriptional activity. Only promoters inactivated by cytosine methylation were transcribed in FV3-infected cells. We also show that the dinucleotide sequence in which the methylcytosine is found appears to have no effect on the ability of FV3 to trans-activate the methylated promoters.

5-Methylcytosine↗
PubMed2004

The value-added genome: building and maintaining genomic cytosine methylation landscapes.

Epigenetic marks, such as cytosine methylation and post-translational histone modifications, are important for interpreting and managing eukaryotic genomes. Recent genetic studies in plants have uncovered details on the different interwoven mechanisms that are responsible for specification of genomic cytosine methylation patterns. These mechanisms include targeting cytosine methylation using heterochromatic histone modifications and RNA guides. Genomic cytosine methylation patterns also reflect locus-specific demethylation initiated by specialized DNA glycosylases. While genetics continues to more fully define these mechanisms, genomic studies in Arabidopsis have yielded an unprecedented high-resolution view of how epigenetic marks are layered over a genome.

Arabidopsis↗
PubMed2002

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues.

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.

Adenine↗
PubMed2004

The mechanism of target base attack in DNA cytosine carbon 5 methylation.

We measured the tritium exchange reaction on cytosine C(5) in the presence of AdoMet analogues to investigate the catalytic mechanism of the bacterial DNA cytosine methyltransferase M.HhaI. Poly(dG-dC) and poly(dI-dC) substrates were used to investigate the function of the active site loop (residues 80-99), stability of the extrahelical base, base flipping mechanism, and processivity on DNA substrates. On the basis of several experimental approaches, we show that methyl transfer is the rate-limiting pre-steady-state step. Further, we show that the active site loop opening contributes to the rate-limiting step during multiple cycles of catalysis. Target base activation and nucleophilic attack by cysteine 81 are fast and readily reversible. Thus, the reaction intermediates involving the activated target base and the extrahelical base are in equilibrium and accumulate prior to the slow methyl transfer step. The stability of the activated target base depends on the active site loop closure, which is dependent on the hydrogen bond between isoleucine 86 and the guanine 5' to the target cytosine. These interactions prevent the premature release of the extrahelical base and uncontrolled solvent access; the latter modulates the exchange reaction and, by implication, the mutagenic deamination reaction. The processive catalysis by M.HhaI is also regulated by the interaction between isoleucine 86 and the DNA substrate. Nucleophilic attack by cysteine 81 is partially rate limiting when the target base is not fully stabilized in the extrahelical position, as observed during the reaction with the Gln(237)Trp mutant or in the cytosine C(5) exchange reaction in the absence of the cofactor.

Carbon↗
PubMed2005

A molecular dynamics exploration of the catalytic mechanism of yeast cytosine deaminase.

Yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, is investigated by a series of molecular dynamics simulations in its free form and complexed with its reactant (cytosine), product (uracil), several reaction intermediates, and an intermediate analogue. Quantum chemical calculations, used to construct a model for the catalytic Zn ion with its ligands (two cysteines, a histidine, and one water) show, by comparison with crystal structure data, that the cysteines are deprotonated and the histidine is monoprotonated. The simulations suggest that Glu64 plays a critical role in the catalysis by yCD. The rotation of the Glu64 side-chain carboxyl group that can be protonated or deprotonated permits it to act as a proton shuttle between the Zn-bound water and cytosine and subsequent reaction intermediates. Free energy methods are used to obtain the barriers for these rotations, and they are sufficiently small to permit rotation on a nanosecond time scale. In the course of the reaction, cytosine reorients to a geometry to favor nucleophilic attack by a Zn-bound hydroxide. A stable position for a reaction product, ammonia, was located in the active site, and the free energy of exchange with a water molecule was evaluated. The simulations also reveal small motions of the C-terminus and the loop that contains Phe114 that may be important for reactant binding and product release.

Binding Sites↗
PubMed1984

Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine.

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.

Azacitidine↗
PubMed2001

Cytosine methylation enhances mitoxantrone-DNA adduct formation at CpG dinucleotides.

Recently, we have shown that mitoxantrone can be activated by formaldehyde in vitro to form DNA adducts that are specific for CpG and CpA sites in DNA. The CpG specificity of adduct formation prompted investigations into the effect of cytosine methylation (CpG) on adduct formation, since the majority of CpG dinucleotides in the mammalian genome are methylated and hypermethylation in subsets of genes is associated with various neoplasms. Upon methylation of a 512-base pair DNA fragment (containing the lac UV5 promoter) using HpaII methylase, three CCGG sites downstream of the promoter were methylated at C5 of the internal cytosine residue. In vitro transcription studies of mitoxantrone-reacted DNA revealed a 3-fold enhancement in transcriptional blockage (and hence adduct formation) exclusively at these methylated sites. In vitro cross-linking assays also revealed that methylation enhanced mitoxantrone adduct formation by 2-3-fold, and methylation of cytosine at a single potential drug binding site on a duplex oligonucleotide also enhanced adduct levels by 3-fold. Collectively, these results indicate preferential adduct formation at methylated CpG sites. However, adducts at these methylated sites exhibited the same stability as nonmethylated sites, suggesting that cytosine methylation increases drug accessibility to DNA rather than being involved in kinetic stabilization of the adduct.

Base Pairing↗
PubMed1995

Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase.

When human DNA(cytosine-5)methyltransferase was used to methylate a series of snapback oligodeoxy-nucleotides of differing stem lengths, each containing a centrally located CG dinucleotide recognition site, the enzyme required a minimum of 22 base pairs in the stem for maximum activity. Extrahelical cytosines in slipped duplexes that were 30 base pairs in length acted as effective methyl acceptors and were more rapidly methylated than cytosines that were Watson-Crick paired. Duplexes containing hairpins of CCG repeats in cruciform structures in which the enzyme recognition sequence was disrupted by a C.C mispair were also more rapidly methylated than control Watson-Crick-paired duplexes. Since enzymes have higher affinities for their transition states than for their substrates, the results with extrahelical and mispaired cytosines suggest that these structures can be viewed as analogs of the transition state intermediates produced during catalysis by methyltransferases.

Base Composition↗
PubMed1990

A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.

Amino Acid Sequence↗
PubMed2007

A molecular dynamics study of the ligand release path in yeast cytosine deaminase.

Yeast cytosine deaminase, a zinc metalloenzyme, catalyzes the deamination of cytosine to uracil. Experimental and computational evidence indicates that the rate-limiting step is product release, instead of the chemical reaction step. In this work, we use molecular dynamics to suggest ligand exit paths. Simulation at 300 K shows that the active site is well protected by the C-terminal helix (residues 150-158) and F-114 loop (residues 111-117) and that on the molecular dynamics timescale water does not flow in or out of the active site. In contrast, simulation at 320 K shows a significant increase in flexibility of the C-terminal helix and F-114 loop. The motions of these two regions at 320 K open the active site and permit water molecules to diffuse into and out of the active site through two paths with one much more favored than the other. Cytosine is pushed out of the active site by a restraint method in two directions specified by these two paths. In path 1 the required motion of the protein is local-involving only the C-terminal helix and F-114 loop-and two residues, F-114 and I-156, are identified that have to be moved away to let cytosine out; whereas in path 2, the protein has to rearrange itself much more extensively, and the changes are also much larger compared to the path 1 simulation.

Binding Sites↗
PubMed2001

DNA (cytosine) methyltransferase overexpression is associated with acquired drug resistance of murine neuroblastoma cells.

We have previously reported that C-1300 murine neuroblastoma (rMNB) cells made resistant to the nucleoside analogue, (Z)-5'-fluoro-4', 5'-didehydro-5'deoxyadenosine (MDL), an irreversible inhibitor of S-adenosylhomocysteine (AdoHcy) hydrolase have an increased expression of the S-adenosylmethionine (AdoMet) synthetase gene. Results of the immunoblot analysis of DNA (cytosine) methyltransferase with anti-human DNA (cytosine) methyltransferase specific polyclonal antibody demonstrated a significant increase ( approximately 2-fold, p<0.01) in expression of DNA (cytosine) methyltransferase protein in rMNB/MDL cells compared to wild-type C1300 MNB (wMNB) cells. To rule out the possibility that multidrug resistance (MDR) genes are involved in development of acquired drug resistance in murine neuroblastoma (rMNB/MDL) cells made resistant to MDL, the expression of Mdr1a, Mdr1b, Mdr2 (multidrug resistance/P-glycoprotein), and Mrp-1 (multidrug resistance associated protein) was examined in rMNB-MDL cells. The analysis of Mdr and Mrp-1 expression was performed by RT-PCR using PCR specific primers to respective genes. No significant difference was observed in the expression of MDR1a, Mdr1b and Mrp-1 genes between wMNB and rMNB-MDL cells, however, a slight decrease was noticed in Mdr1 expression in some samples. Expression of the Mdr2 (human MDR3) gene, which is not associated with the acquired drug resistance phenotype, was significantly decreased in rMNB-MDL cells. These findings were also confirmed by the immunoblot analyses using specific monoclonal antibodies to Mdr1/3 proteins. Expression of N-Myc gene--a prognostic factor in neuroblastoma tumors was also not altered in rMNB-MDL cells. Results of the present study suggest that acquired drug resistance in rMNB-MDL cells to MDL is associated to the overexpression of DNA (cytosine) methyltransferase, and could be due to genetic or epigenetic changes in particular to DNA hypermethylation in response to an increased AdoMet synthetase gene expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Sensitivity of the restriction endonucleases HaeIII, BsrI, EaeI and CfrI to cytosine N4-methylation.

HaeIII, BsrI and NgoII are isochizomers that recognize the sequence GGCC while EaeI and CfrI recognize the overlapping sequence YGGCCR. It has previously been shown that all these enzymes are inhibited by cytosine C5-methylation within the recognition sequence. The methylation sensitivities of these enzymes to cytosine N4-methylation have not been previously reported. In this paper we present data demonstrating that all these enzymes, except NgoII, are inhibited by cytosine N4-methylation of the second 5' cytosine residue within the recognition sequence.

5-Methylcytosine↗
PubMed2006

Cytosine methylation and DNA repair.

Cytosine methylation is a common form of post-replicative DNA modification seen in both bacteria and eukaryotes. Modified cytosines have long been known to act as hotspots for mutations due to the high rate of spontaneous deamination of this base to thymine, resulting in a G/T mismatch. This will be fixed as a C-->T transition after replication if not repaired by the base excision repair (BER) pathway or specific repair enzymes dedicated to this purpose. This hypermutability has led to depletion of the target dinucleotide CpG outside of special CpG islands in mammals, which are normally unmethylated. We review the importance of C-->T transitions at non-island CpGs in human disease: When these occur in the germline, they are a common cause of inherited diseases such as epidermolysis bullosa and mucopolysaccharidosis, while in the soma they are frequently found in the genes for tumor suppressors such as p53 and the retinoblastoma protein, causing cancer. We also examine the specific repair enzymes involved, namely the endonuclease Vsr in Escherichia coli and two members of the uracil DNA glycosylase (UDG) superfamily in mammals, TDG and MBD4. Repair brings its own problems, since it will require remethylation of the replacement cytosine, presumably coupling repair to methylation by either the maintenance methylase Dnmt1 or a de novo enzyme such as Dnmt3a. Uncoupling of methylation from repair may be one way to remove methylation from DNA. We also look at the possible role of specific cytosine deaminases such as Aid and Apobec in accelerating deamination of methylcytosine and consequent DNA demethylation.

5-Methylcytosine↗
PubMed2003

Cytosine deaminase versus thymidine kinase: a comparison of the antitumor activity.

The efficacy of single and combination suicide gene therapy was evaluated using a Herpes simplex virus thymidine kinase/ganciclovir system and Escherichia coli cytosine deaminase/5-fluorocytosine system on the rat prostate tumor cell line R3327 AT-1. The wild-type R3327 AT-1 cell line was transfected with a bifunctional fusion gene CDglyTK, which had the advantage that the resulting R3327 AT-1/CDglyTK cell line has the same amount of cytosine deaminase and thymidine kinase molecules. The percentage of viable R3327 AT-1/CDglyTK cells after 96 h incubation with 0.1 micro g/ml ganciclovir or 10 micro g/ml 5-fluorocytosine were 85% and 52% of controls, respectively. The cell viability when both suicide genes systems were activated was 43%. For in vivo analysis, Copenhagen rats were injected subcutaneously with R3327 AT-1 or R3327 AT-1/CDglyTK cells and treated with 30 mg/kg ganciclovir, 500 mg/kg 5-fluorocytosine, or both prodrugs together. A survival of 83% with the thymidine kinase/ganciclovir and 57% with the CD/5-FC could be observed. Only co-administration of thymidine kinase- and cytosine deaminase-specific prodrugs resulted in a 100% recurrence-free survival of the Copenhagen rats with a Dunning R3327 AT-1/CDglyTK prostate tumor and showed an additive cytotoxic effect. Calculation of the degree of activation and the potential of activation can be used to predict the success of a suicide gene therapy. In our case, the cytosine deaminase/5-fluorocytosine system had a low degree of activation (value 40), which is also found in the low response to 5- fluorocytosine in vivo (57% tumor free).

Animals↗
PubMed1987

Hydrogen bonding between cytosine and peptides of threonine or serine: is it relevant to the origin of the genetic code?

13C, 15N, and 1H nuclear magnetic resonance measurements indicate that chloroform-soluble threonine-containing tripeptide derivatives, such as t-Boc-Thr-Gly-Gly-OBz, form three strong hydrogen bonds to the cytosine moiety of 2',3'-O-isopropylidene-5'-O-t-butyldimethylsilylcytidine. The C = O and NH of the central peptide residue plus the OH of the threonine side chain appear to form bonds to the N(4')H2, N(3), and C(2) = O, respectively, of the pyrimidine. An association constant calculated from the cytidine 15N(4') nuclear magnetic resonance response to added peptide is four times larger than the corresponding cytosine-guanine constant. It is suggested that cytosine-peptide bonding was part of the primitive genetic coding mechanism early in evolution and accounts for the origin of the cytosine-centered codons for the hydroxy amino acids, serine and threonine, in the present code.

Codon↗