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PubMed2002

Effect of divalent cations and cytosine protonation on thermodynamic properties of intermolecular DNA double and triple helices.

The contribution of divalent cations and cytosine protonation to conformation and stability of duplex and triplex formation were intensively investigated and characterized by ultraviolet (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and electrophoresis mobility shift assay (EMSA). CD spectra showed that the divalent cations investigated would not significantly distort nucleotide geometry, while UV and DSC melting experiments revealed that the cation binding abilities to duplexes and triplexes were clearly dependent on the types of cations under near physiological conditions. The calorimetric enthalpies were generally underestimated relative to the corresponding van't Hoff enthalpies for Hoogsteen and Watson-Crick transitions, but free energy changes derived from the DSC measurements were in good agreement with those derived from the UV measurements. The adjacent placing of the C(+) x G.C triplets in triplexes lowered the stabilities of not only Hoogsteen base-pairing but also Watson-Crick base-pairing. The protonation contribution of the given cytosine residues might depend on the local and global structure of the protonated cytosine complex. A rigid structural targeted-strand would favor the protonation of cytosine residues. The apparent pK(a) values for parallel duplex and triplex investigated were determined to be 6.4 and 7.6, respectively, which are considerably heightened by 2.1 and 3.3 pH unit as compared to the intrinsic pK(a) value of the free cytosine residues.

Calorimetry, Differential Scanning↗
PubMed2001

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles.

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.

Autoradiography↗
PubMed2003

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair.

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.

Algorithms↗
PubMedDate not supplied

Combined cytosine deaminase expression, 5-fluorocytosine exposure, and radiotherapy increases cytotoxicity to cholangiocarcinoma cells.

Cholangiocarcinoma is a malignancy that is resistant to current therapy. We applied the toxin gene therapy strategy of cytosine deaminase conversion of the nontoxic producing 5-fluorocytosine to 5-fluorouracil combined with radiotherapy to cholangiocarcinoma. The transduction efficiency of SK-ChA-1 cholangiocarcinoma cells was determined by fluorescence-activated cell-sorting analysis following infection with recombinant adenovirus AdCMVLacZ, which encodes thc gene for Beta-galactosidase. To evaluate cytosine deaminase-mediated conversion of 5-fluorocytosine to 5-fluorouracil and subsequent cytotoxicity, SK-ChA-1 cells were infected with the recombinant adenovirus AdCMVCD, which encodes cytosine deaminase, and exposed to 5-fluorocytosine for 6 to 8 days. Additive cytotoxicity of radiation therapy was evaluated by cobalt-60 exposure following AdCMVCD infection and 5-fluorocytosine treatment. SK-ChA-1 cells were transduced (98.4%) by AdCMVLacZ at 100 plaque-forming units per cell. Following infection with AdCMVCD and exposure to 5 to 100 microgram/ml of 5-fluorocytosine, 20% to 64% of SK-ChA-1 cells were killed. A combination of radiation and cytosine deaminase/5-fluorocytosine therapy resulted in enhanced cell killing (83.5% to 91.5%). Cholangiocarcinoma cells were transduced by recombinant adenoviral vectors and were killed by cytosine deaminase-mediated production of 5-fluorouracil. Enhanced cytotoxicity was seen with the addition of external beam radiation. These results provide a foundation for multimodality therapy for human cholangiocarcinoma that combines gene therapy technology with radiation therapy.

Adenoviridae↗
PubMed1993

Cytosine deamination in mismatched base pairs.

The rate of deamination of cytosine in mismatched base pairs has been determined. Incubation of M13mp2 nicked heteroduplex DNA molecules containing T.C or C.C mispairs in the lacZ alpha-complementation gene results in deamination of cytosine to uracil, producing T.U or C.U mispairs. Strands which have undergone deamination at the target site to produce uracil will yield dark blue plaque revertants, while all other strands yield faint blue or colorless plaque phenotypes upon transfection of an ung- alpha-complementation Escherichia coli host strain. Rate constants were calculated from the reversion frequencies for several different heteroduplexes incubated at either 60 or 37 degrees C. For the 60 degrees C incubations, the hydrolytic deamination rate constants for mispairs in three different local sequence environments ranged from 8 x 10(-10) to 40 x 10(-10) s-1. For incubations at 37 degrees C, the rate constants were between 0.4 x 10(-10) and 1.3 x 10(-10) sec-1. At both temperatures and for all mispairs, these rate constants are significantly greater than deamination rate constants in properly matched Watson-Crick G.C base pairs and are similar to those constants determined for cytosine deamination in single-stranded DNA. Since deamination most likely occurs via a single-stranded intermediate, the data suggest that, at 37 degrees C, the T.C and C.C mispairs exhibit from 20% to 100% single-stranded character. We conclude that cytosine residues involved in a mispair in DNA are 1-2 orders of magnitude more prone to deaminate to uracil than are cytosines in double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed2002

Regulation of one-electron oxidation rate of guanine by base pairing with cytosine derivatives.

Effects of base pairing on the one-electron oxidation rate of guanine derivatives, guanine, 8-bromoguanine, and 8-oxo-7,8-dihydroguanine have been studied. The one-electron oxidation rate of guanine derivatives was determined by triplet-quenching experiments, using N,N'-dibutylnaphthaldiimide (NDI) in the triplet excited state (3NDI*) and fullerene (C(60)) in the triplet excited state ((3)C(60*)) as oxidants. In all three guanine derivatives studied here, acceleration of the one-electron oxidation was observed upon hydrogen bonding with cytosine, which demonstrates lowering of the oxidation potential of guanine derivatives by base pairing with cytosine. When a methyl or bromo group was introduced to the C5 position of cytosine, acceleration or suppression of the one-electron oxidation relative to the guanine:cytosine base pair was observed, respectively. The results demonstrate that the one-electron oxidation rate of guanine in DNA can be regulated by introducing a substituent on base pairing cytosine.

Base Pairing↗
PubMed2006

Quadruply hydrogen bonded cytosine modules for supramolecular applications.

In view of the range of properties required from supramolecular materials, there is clearly a need for new strong quadruple hydrogen bonded modules, which can be used in polymer or copolymer synthesis via the self- or hetero-association of complimentary units. A cytosine-based module has been prepared for supramolecular applications using a straightforward synthetic approach. The cytosine module was designed such that it does not undergo tautomeric changes observed with ureidopyrimidinones. The cytosine module was capable of forming quadruple hydrogen bonded assemblies both in solution and in the solid state, and the structure of the dimeric self-assembled unit was confirmed by single-crystal X-ray and solution NMR techniques. The dimerization constant was estimated to be greater than 9 x 106 M-1 in deuterated benzene. The capacity of the cytosine-based module to strongly hetero-associate with the ureidopyrimidinone module was demonstrated, and a supramolecular polymer of a bifunctional unit incorporating the cytosine module and PEG-based linker was described.

Cytosine↗
PubMed2005

Marked influences on the adenine-cytosine base pairs by electron attachment and ionization.

The reverse wobble and the reverse Hoogsteen adenine-cytosine mispairs regarding their radical cations and anions are studied with the hybrid three-parameter B3LYP density functional method and 6-31+G(d), 6-311+G(2df,2p) basis sets. Hydrogen bonding mispairs are remarkably influenced by electron attachment and ionization. Only one stronger hydrogen bond N6-H (in adenine)...N3 (in cytosine) exists in the radical pair, while the strengths of two N-H...N hydrogen bonds in the neutral pair are comparable. Geometrical coplanarity is found for the neutral and cationic pairs, in contrast to the anionic pairs in which the cytosine moiety exhibits significant deformation due to electron attachment. Dissociation energies for the neutral and radical pairs are slightly higher than those of the adenine-thymine pairs but much smaller than those of the guanine-cytosine pairs. Valence-bound anions of these two adenine-cytosine pairs are thermodynamically stable by 0.1-0.2 eV with respect to the neutral pairs. On the basis of the comparison between the experimental data of the solvated clusters and the calculated values, these two pairs can be quantitatively equivalent to the clusters in which each base is solvated by five water molecules.

Adenine↗
PubMed2006

Na+, Mg2+, and Zn2+ binding to all tautomers of adenine, cytosine, and thymine and the eight most stable keto/enol tautomers of guanine: a correlated ab initio quantum chemical study.

Interactions of adenine, cytosine, guanine, and thymine with Na(+), Mg(2+), and Zn(2+) cations were studied using an approximate resolution of identity correlated second-order MP2 (RI-MP2) method with the TZVPP ([5s3p2d1f/3s2p1d]) basis set. All existing tautomers of adenine, cytosine, and thymine and the eight most stable keto/enol tautomers of guanine were considered. Cations bind mostly in a bidentate manner, and stabilization energies of these complexes are larger than those in the case when cations bind in a unidentate manner. The cation...Y (Y equal to N or O) distances for divalent metals are shorter than those for Na(+) and for Zn(2+) are mostly shorter than the Mg(2+)...Y distance. The intermolecular distances between the cation and the base for complexes containing adenine and cytosine are systematically shorter than those for complexes containing guanine and thymine. Only for cytosine the canonical keto/amino tautomer structure with ions represents the global minimum. For guanine, the metalated canonical form is again the most stable, but its stabilization energy is within less than 5% of the stabilization energies of the two other rare tautomers, which indicates that the canonical form and these two rare tautomers could coexist. The canonical structures of adenine and thymine in the presence of ions are considerably less stable (by more than 10%) than the complexes of the rare tautomers. It can be concluded that the interaction of Na(+), Mg(2+), and Zn(2+) cations with cytosine in the gas phase will not induce the change of the canonical form to the rare tautomeric form. In the case of isolated guanine, the equilibrium of the canonical form with rare tautomers can be found. For isolated adenine and thymine the presence of rare tautomers is highly probable.

Adenine↗
PubMed1983

Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes.

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.

Base Sequence↗
PubMed1995

Deamination of cytosine-containing pyrimidine photodimers in UV-irradiated DNA. Significance for UV light mutagenesis.

The realization that cytosine in cyclobutyl pyrimidine dimers rapidly deaminates to uracil raised the possibility that this chemical transformation, rather than an enzymatic polymerase error, is the major mutagenic step in UV mutagenesis. We have established a sensitive bioassay system that enabled us to determine the rate of deamination of cytosine in cyclobutyl pyrimidine dimers in plasmid DNA. This was done by in vitro UV irradiation and deamination of a plasmid carrying the cro gene, followed by photoreactivation, and assaying uracils in DNA by their ability to cause Cro- mutations in an indicator strain that was deficient in uracil DNA N-glycosylase. DNA sequence analysis revealed that 27 out of 29 Cro- mutants carried GC --> AT transitions, as expected from deamination of cytosine. Deamination of cytosines in the cro gene in UV-irradiated plasmid pOC2 proceeded at 37 degrees C with first-order kinetics, at a rate of (3.9 +/- 0.6) x 10(-5) s-1, corresponding to a half-life of 5 h. Physiological salt conditions increased the half-life to 12 h, whereas decreasing the pH increased deamination. The temperature dependence of the rate constant yielded an activation energy of 13.6 +/- 3.3 kcal/mol. These kinetics data suggest that deamination of cytosine-containing dimers is too slow to play an important role in UV mutagenesis in Escherichia coli. However, it is likely to play an important role in mammalian cells, where the mutagenic process is slower.

Base Composition↗
PubMed2005

Alginate-based in situ gelling suspensions and emulsions comprising N4-alkyloxycarbonyl derivatives of cytosine: zero-order release and effect of physicochemical properties.

As an alternative to incorporation of various excipients, N(4)-alkyloxycarbonyl-cytosine derivatives possessing various physicochemical properties and cytosine regeneration rates have been examined to modify release rate and kinetics from in situ gelling alginate formulations, e.g., liquid formulations that gel in acidic gastric juice and release the entrapped derivative or parent cytosine. Linear relationships were obtained between the release rate constants and the square root of the solubility for suspension formulations. Calculated diffusion coefficients were observed to be similar for suspension and solution formulations; however, for in situ gelling emulsion formulations, diffusivity correlated linearly to log P. Zero-order release of parent cytosine was observed from in situ gelling suspensions of the poorly soluble acid-labile N(4)-adamantyloxycarbonyl-cytosine prodrug.

Acetamides↗
PubMed1995

Effect of selective cytosine methylation and hydration on the conformations of DNA triple helices containing a TTTT loop structure by FT-IR spectroscopy.

5-Methylcytosines have been introduced into triplex-forming-oligonucleotides and shown to extend the pH range over which a triplex forms with a homopurine-homopyrimidine tract of duplex DNA. As a host strand, an oligodeoxypyrimidine with a base sequence of 5'-d(TC)3T4(CT)3 ([CC]) was designed to form a hairpin triplex with a 5'-d-A(GA)2G ([AG6]) purine strand at acidic pH (Tsay, et al., (1995) J. Biomol. Str. Dyn., 13, 1235-1245). We here present results obtained by FT-IR spectroscopy concerning the conformation of the hairpin triplex as a function of the selective substitution of cytosines by 5-methylcytosines in the host strand. Namely, cytosines are substituted by 5-methylcytosines in either the 3'-pyrimidine portion ([CM]) or the 5'-pyrimidine portion ([MC]) or in both ([MM]) of the host strand. The acidic-induced transitions of the equimolar mixtures of the purine target with either of the four pyrimidine oligomers gives rise to different apparent pK values, i.e., [MM].[AG6] (6.2) > [MC].[AG6] (6.0) > [CM].[AG6] (5.7) > [CC].[AG6] (5.2) > single-stranded oligopyrimidines (4.6 +/- 0.2), indicating that cytosine methylation expands the pH range compatible with the hairpin triplex formation regardless of whether the substitution is in the 5'-pyrimidine (Hoogsteen) portion or in the 3'-pyrimidine (Watson-Crick) portion. Thermal denaturation profiles indicated that all the triplexes denatured in a monophasic manner in the pH range of 4.0 to 7.0, and that cytosine methylations in any position of the 16-base pyrimidine oligomer increase the stability of the hairpin triplex DNA. IR spectra recorded in D2O and H2O solutions revealed that cytosine methylation does not significantly influence the conformation of triplex DNA in solution, i.e., all the four triplexes accept a similar sugar conformation, and predominately take on a S-type sugar pucker with a relative proportion of two S-type sugars for one N-type. Furthermore, we also investigated the effect of relative humidity (RH) on the conformation of triplex MC.AG6 in hydrated films, and found that the conformational change induced by the decrease of RH, from predominant S-type to primary N-type sugar pucker, might first occur in the purine strand at 86% RH.

Absorption↗
PubMed2000

Evidence that cytosine residues within 5'-CCTGG-3' pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5'-CG-3' dinucleotides.

In contrast to the complex sequence specificities of the prokaryotic DNA methylating systems, the mammalian machinery identified thus far methylates cytosine residues within the context of a 5'-CG-3' dinucleotide. To explore the possibility that cytosine residues that do not precede guanine may be independently methylated in mammalian DNA, we have examined a region of the human myogenic gene, Myf-3, which is not targeted by the methylating system that methylates 5'-CG-3' dinucleotides. Our investigations have revealed cytosine methylation within the 5'-CCTGG-3' pentanucleotides specified by the 0.8-kb Myf-3 probe. We have also found that in DNA from neoplastic cells, in which 5'-CG-3' dinucleotides within Myf-3 become abnormally hypermethylated, cytosine residues within 5'-CCTGG-3' pentanucleotides are not methylated. Moreover, methylation of 5'-CCTGG-3' pentanucleotides was not detected within the closely related Myf-4 gene, which is normally 5'-CG-3' hypermethylated. These findings indicate the existence of a system that methylates 5'-CCTGG-3' pentanucleotides independently of the system that methylates cytosine residues within 5'-CG-3' dinucleotides. It is possible that the 5'-CCTGG-3' methylating system influences the fate of foreign integrated DNA.

Animals↗
PubMed1995

In vivo adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene to human colon carcinoma-derived tumors induces chemosensitivity to 5-fluorocytosine.

To evaluate the concept that in vivo transfer of the Escherichia coli cytosine deaminase gene will confer sensitivity of a solid tumor to the prodrug 5-fluorocytosine (5FC), we constructed an adenovirus vector (AdCMV.CD) carrying the cytosine deaminase gene driven by the cytomegalovirus (CMV) promoter, infected HT29 colon carcinoma cells in vitro and in vivo, and evaluated cell growth over time. AdCMV.CD produced a functional cytosine deaminase protein in HT29 cells in vitro as evidenced by the ability of lysates from the infected cells to convert [3H]5FC to its active metabolite 5-fluorouracil (5FU). The AdCMV.CD vector effectively suppressed HT29 cell growth in vitro in the presence of 5FC in a dose-dependent manner. Infection with AdCMV.CD, when as few as 10% of cells expressed the cytosine deaminase gene, was associated with a bystander effect when combined with 5FC in cell mixing studies. Further, this bystander effect was not dependent on cell-to-cell contact as demonstrated by suppression of [3H]thymidine incorporation in HT29 cells when supernatant from AdCMV.CD-infected cells treated with 5FC was transferred cells. Consistent with these in vitro observations, when AdCMV.CD was directly injected into established subcutaneous HT29 tumors in nude mice receiving 5FC, there was a four-fold reduction in tumor size at day 15 compared to controls, and a five-fold reduction at day 28. These observations suggest that adenovirus-mediated gene transfer of the E. coli cytosine deaminase gene and concomitant administration of 5FC may have potential as a strategy for local control of the growth of tumor cells susceptible to 5FU.

Adenoviridae↗
PubMed1991

Evidence for the covalent binding of aflatoxin B1-dichloride to cytosine in DNA.

In vitro studies of the effect of aflatoxin B1-dichloride (AFB1-Cl2) on the template function for RNA synthesis of several single- and double-stranded synthetic DNAs containing cytosine and/or hypoxanthine bases are reported. The results indicate: (i) AFB1-Cl2 strongly inhibits the template function of the single-stranded homopolymer polydC and has no effect on polydI, (ii) the inhibition is stronger when cytosine is in the double-stranded alternating copolymer poly[d(I-C)], and (iii) polydI directed RNA synthesis can be inhibited if it is in the double-stranded homopolymer polydI.polydC, although the template function of the polydC strand is still inhibited to a greater extent. The evidence that the selective inhibition of the DNA template function is a direct reflection of the binding specificities of AFB1-Cl2 is provided by the binding studies of [3H]AFB1-Cl2 to these DNAs. The binding of AFB1-Cl2 to polydC is substantiated by the dose-response template inhibition and by the dose-response template binding studies. Additionally, these results show that AFB1 per se has neither inhibitory nor binding activity. Auto radiography of [alpha-32P]GTP labeled RNAs suggests that the mechanism of inhibition of polydC template function by AFB1-Cl2 is mainly due to the inhibition of the elongation of RNA synthesis. Spectrum measurement of the products of enzyme digestion of the AFB1-Cl2 modified polydC reveals that the deoxycytidine fraction gives a typical cytosine absorption peak at 275 nm followed by a broad peak between 300 and 400 nm with a maximum at 390 nm. High performance liquid chromatography confirms the existence of a cytosine-AFB1 adduct which absorbs strongly in the regions between 250 and 400 nm with peaks identifiable at 260, 350 and 390 nm. These results strongly suggest that AFB1 in the activated form of AFB1-Cl2 is able to covalently bind to cytosine in DNA.

Aflatoxin B1↗
PubMed1995

Differential sensitivity to loss of cytosine methyl groups within the hepatic p53 gene of folate/methyl deficient rats.

Dietary folate/methyl deficiency provides a unique model of endogenous hepatocarcinogenesis in which to study progressive alterations in DNA methylation patterns during tumor progression in vivo. Weanling male F344 rats were given a semi-purified diet deficient in the methyl donors choline, methionine and folic acid for a period of 9 weeks. Using a genomic sequencing procedure based on the PCR amplification of bisulfite-modified DNA, the methylation status of individual CpG sites within exons 6 and 7 of the p53 gene in liver samples from control and deficient rats was determined. Treatment of denatured nuclear DNA with sodium bisulfite quantitatively converts all cytosine residues to uracil which are then amplified as thymine in the PCR reaction. In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine. Automated sequencing of bisulfite-modified DNA will then elucidate the methylation status of each cytosine residue within a defined gene sequence. In addition to evaluation of the methylation status of the p53 gene, the relative activity of the DNA methyltransferase was also quantified in nuclear extracts from control and folate/methyl deficient rats. The results indicate that specific 5-methyl cytosines within the hepatic p53 gene from methyl deficient rats are resistant to demethylation despite the diet-induced decrease in S-adenosylmethionine and the increase in cell proliferation associated with this dietary intervention. Progressive demethylation was observed at other methylated cytosine residues in folate/methyl deficient rats after 9 weeks despite a paradoxical increase in DNA methyltransferase activity. The application of this sequence-specific technology will allow the definition of the methylation status of every CpG site within a coding sequence or promoter region and should provide new insights into mechanisms and consequences of methylation dysregulation during progressive multistage carcinogenesis.

Animals↗
PubMed1999

Cytosine deaminase/5-fluorocytosine-based vaccination against liver tumors: evidence of distant bystander effect.

BACKGROUND: The cytosine deaminase gene of Escherichia coli converts the nontoxic compound 5-fluorocytosine into 5-fluorouracil (5-FU), thereby acting as a suicide gene when introduced into cancer cells, killing the cells when they are exposed to 5-fluorocytosine. We analyzed the efficacy of using cytosine deaminase-bearing cancer cells as an autologous tumor vaccine in a rat model that mimics liver metastasis from colon carcinoma. METHODS: We introduced a plasmid vector containing the E. coli cytosine deaminase gene into a BDIX rat colon carcinoma cell line. Intrahepatic injection of the modified cells in syngeneic animals generates a single experimental liver "suicide tumor." We then analyzed the effect of 5-fluorocytosine treatment in terms of regression of cytosine deaminase-expressing cells in vivo as well as protection against wild-type cancer cells. RESULTS: Treatment with 5-fluorocytosine induced regression of cytosine deaminase-expressing (CD+) tumors, with seven of 11 treated animals being tumor free at the end of 30 days and a statistically significant difference in tumor volumes between treated and control animals (two-sided P<.0001). Intrahepatic injection of CD+ cells followed by 5-fluorocytosine treatment rendered the treated animals resistant to challenge with wild-type tumor cells, with no (zero of seven) treated animals developing wild-type tumors in contrast to all (four of four) control animals. Moreover, in animals with established wild-type liver tumors, injection of CD+ tumor cells followed by 5-fluorocytosine treatment produced a statistically significant increase in survival time (two-sided P<.0001). In vivo immunodepletion and immunohistologic analysis of experimental tumors indicate that natural killer cells are the major immune component involved in this antitumor effect. CONCLUSIONS AND IMPLICATIONS: Taken together, these results suggest the potential use of suicide gene-modified tumor cells as therapeutic vaccines against liver metastasis from colon carcinoma.

Animals↗