Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CYTOSINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,063 records · Page 59Linked to original sources
PubMed2002

High-affinity peptide nucleic acid oligomers containing tricyclic cytosine analogues.

[structure: see text] Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and 9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were incorporated into PNA oligomers using Boc-chemistry for solid-phase synthesis. PNAs containing single G-clamp modifications exhibit significantly enhanced affinity toward RNA and DNA targets relative to unmodified PNA while maintaining mismatch discrimination. These PNA G-clamp modifications exhibit the highest increase in affinity toward nucleic acid targets reported so far for PNA modifications.

Cytosine↗
PubMed1999

Implications of cytosine methylation on (+)-anti-Benzo[a]pyrene 7, 8-dihydrodiol 9,10-epoxide N(2)-dG adduct formation in 5'-d(CGT), 5'-d(CGA), and 5'-d(CGC) sequence contexts of single- and double-stranded oligonucleotides.

Covalent binding of (+)-anti-benzo[a]pyrene 7,8-dihydrodiol 9, 10-epoxide (anti-BPDE) to the N(2)-amino group of deoxyguanine in the oligonucleotides 5'-d(CCTATCGXTATCC) and 5'-d(CCTATm(5)CGXTATCC) (X being T, A, or C) has been studied. The extent of formation of the (+)-trans-anti-BPDE-N(2)-dG adduct in single-stranded 13-mer oligonucleotides with 5'-d(m(5)CGT) and 5'-d(m(5)CGA) sequence contexts was significantly higher (1.5- and 2.4-fold, respectively) relative to that of the nonmethylated sequences. With the 5'-d(CGC) sequence context, m(5)dC had no significant effect on adduct formation. When the reaction was allowed to proceed in the presence of oligonucleotide duplexes (composed of a 13-mer parent strand and a 9-mer complement), a significant increase in the extent of adduct formation was observed with 5'-d(m(5)CGT)/d(CGA) and 5'-d(m(5)CGA)/d(CGT), but not with 5'-d(CGC)/d(GCG), relative to those of the nonmethylated duplexes. Independent of sequence context, no clear effect of m(5)dC on diol epoxide binding to the opposite dG in the complementary strand was observed. The level of diol epoxide binding to the dG target in the 13-mer oligonucleotides is in general higher in single-stranded sequences than in the duplexes. With 5'-d(CGA) and 5'-d(m(5)CGA), for instance, adduct yields were 3- and 4-fold higher, respectively. The thermodynamic stability of the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex (composed of a 13-mer parent strand and a full complement) was substantially higher than in the 5'-d(CGT)/d(GCA) sequence context. The stimulating effect of cytosine methylation on the formation of DNA adducts of anti-BPDE has previously been demonstrated in other experimental systems. The increase in yield could possibly be rationalized in terms of prestacking of the pyrenyl ring system with the nucleobases prior to the nucleophilic addition reaction of the exocyclic amino group. The results from induced circular dichroism studies with the (+)-trans-anti-BPDE-N(2)-dG adduct in the 5'-d(m(5)CGT)-containing duplex are consistent with substantial heterogeneity of adduct conformations, including both external minor groove-localized and intercalated structures.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

5-Fluorocytosine-mediated apoptosis and DNA damage in glioma cells engineered to express cytosine deaminase and their enhancement with interferon.

To explore the antitumor mechanism of bacterial cytosine deaminase plus 5-fluorocytosine (CD/5-FCyt) in combination with interferons (IFNs), glioma cells were transduced with recombinant retroviruses expressing CD. The transduced glioma cells become sensitive to the nontoxic prodrug 5-FCyt. Apoptosis, DNA damage, bystander effect, and inhibition of thymidylate synthase (TS) and DNA synthesis are associated with CD/5-FCyt-mediated glioma cell killing. Furthermore, IFNs enhance this effect by increasing DNA damage and further inhibiting TS activity. The bystander effect is mediated by the release of cytotoxic metabolites of 5-FCyt into the extracellular milieu triggering apoptosis and DNA damage. Our data indicate that the use of CD/5-FCyt in combination with IFNs may provide a more effective approach for the treatment of brain tumors.

3T3 Cells↗
PubMed2004

Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation.

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.

Animals↗
PubMed1999

Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila.

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

Animals↗
PubMedDate not supplied

Structure of an adenine-cytosine base pair in DNA and its implications for mismatch repair.

Mutational pathways rely on introducing changes in the DNA double helix. This may be achieved by the incorporation of a noncomplementary base on replication or during genetic recombination, leading to substitution mutation. In vivo studies have shown that most combinations of base-pair mismatches can be accommodated in the DNA double helix, albeit with varying efficiencies. Fidelity of replication requires the recognition and excision of mismatched bases by proofreading enzymes and post-replicative mismatch repair systems. Rates of excision vary with the type of mismatch and there is some evidence that these are influenced by the nature of the neighbouring sequences. However, there is little experimental information about the molecular structure of mismatches and their effect on the DNA double helix. We have recently determined the crystal structures of several DNA fragments with guanine X thymine and adenine X guanine mismatches in a full turn of a B-DNA helix and now report the nature of the base pairing between adenine and cytosine in an isomorphous fragment. The base pair found in the present study is novel and we believe has not previously been demonstrated. Our results suggest that the enzymatic recognition of mismatches is likely to occur at the level of the base pairs and that the efficiency of repair can be correlated with structural features.

Adenine↗
PubMed2000

The extended and eccentric E-DNA structure induced by cytosine methylation or bromination.

Cytosine methylation or bromination of the DNA sequence d(GGCGCC)2 is shown here to induce a novel extended and eccentric double helix, which we call E-DNA. Like B-DNA, E-DNA has a long helical rise and bases perpendicular to the helix axis. However, the 3'-endo sugar conformation gives the characteristic deep major groove and shallow minor groove of A-DNA. Also, if allowed to crystallize for a period of time longer than that yielding E-DNA, the methylated sequence forms standard A-DNA, suggesting that E-DNA is a kinetically trapped intermediate in the transition to A-DNA. Thus, the structures presented here chart a crystallographic pathway from B-DNA to A-DNA through the E-DNA intermediate in a single sequence. The E-DNA surface is highly accessible to solvent, with waters in the major groove sitting on exposed faces of the stacked nucleotides. We suggest that the geometry of the waters and the stacked base pairs would promote the spontaneous deamination of 5-methylcytosine in the transition mutation of dm5C-dG to dT-dA base pairs.

Base Sequence↗
PubMed1995

Extension of the four-stranded intercalated cytosine motif by adenine.adenine base pairing in the crystal structure of d(CCCAAT).

The crystal structure of d(CCCAAT), refined at 2.0 A resolution, shows a four stranded molecule in which two parallel duplexes intercalate with opposite polarity, using cytosine.protonated cytosine base pairs. The intercalation motif in this structure is extended by adenine.adenine base pairs. Two topologically distinct broad grooves are found in the lath-like central part of the molecule with the phosphate groups on one side bent over towards each other, stabilized by bridging water molecules. At the 3' ends, two arrangements of intermolecular A.A.T base triplets are found, involving both asymmetric and symmetric A.A base pairs joined to thymine residues by Watson-Crick and reverse Hoogsteen base pairing, respectively.

Adenine↗
PubMed2003

A33scFv-cytosine deaminase: a recombinant protein construct for antibody-directed enzyme-prodrug therapy.

A recombinant fusion protein of colon carcinoma binding A33 single chain antibody with cytosine deaminase displayed specific antigen binding and enzyme activity in surface plasmon resonance and is catalytic activity assay. In vitro, it selectively increased the toxicity of 5-FC to A33 antigen-positive cells by 300-fold, demonstrating the potency of this ADEPT strategy.

Antigens, Neoplasm↗
PubMedDate not supplied

Analysis of the human carcinoembryonic antigen promoter core region in colorectal carcinoma-selective cytosine deaminase gene therapy.

We isolated a 204-base pair carcinoembryonic antigen (CEA) promoter core region from a CEA-producing human colorectal carcinoma (CRC) and constructed retrovirus vectors carrying the expression cassette consisting of the CEA promoter core region and the cytosine deaminase (CD) gene. pCD2 retrovirus carrying the CD gene directed by the retrovirus long terminal repeat promoter served as a control vector. An in vitro study showed that the CEA promoter conferred CEA-producing cell-selective CD expression, specifically when the CD expression cassette was inserted into the 3' long terminal repeat of the retrovirus vector. CD-modified CRC xenografts in nude mice were sensitive to 5-fluorocytosine and caused a profound bystander effect on the unmodified CRC. When nude mice harboring intraperitoneally disseminated CRCs were injected intraperitoneally with the CD expression cassette-carrying retrovirus-producing cells, CD transduction into the disseminated CRCs and bone marrow (BM) was observed. CD expression was, however, restricted to CRCs, and it was observed in both CRCs and BM of mice injected with pCD2 retrovirus-producing cells, resulting in better therapeutic outcomes without BM suppression. These results indicate that effective and safe in vivo gene therapy for CRC may be feasible by transferring the CD gene controlled by the CEA promoter core region.

Animals↗
PubMed2000

Cytosine deaminase suicide gene therapy for peritoneal carcinomatosis.

Gene therapy is a novel therapeutic approach that might soon improve the prognosis of some cancers. We investigated the feasibility of cytosine deaminase (CD) suicide gene therapy in a model of peritoneal carcinomatosis. DHD/K12 colorectal adenocarcinoma cells transfected in vitro with the CD gene were highly sensitive to 5-fluorocytosine (5-FC), and a bystander effect could also be observed. Treating CD+ cells with 5-FC resulted in apoptosis as detected by terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick-end labeling. In vitro, several human cell lines derived from ovarian or colorectal carcinomas, as well as the rat glioblastoma 9 L cell line, responded to CD/5-FC and showed a very strong bystander effect. 5-FC treatment of peritoneal carcinomatosis generated in syngeneic BDIX rats by CD-expressing DHD/K12 cells led to a complete and prolonged response and to prolonged survival. Our study thus demonstrated the efficacy of CD suicide gene therapy for the treatment of peritoneal carcinomatosis.

Animals↗
PubMed2000

Enhanced antitumoral effect of adenovirus-mediated cytosine deaminase gene therapy by induction of antigen-presenting cells through stem cell factor/granulocyte-macrophage colony-stimulating factor gene transfer.

Suicide gene therapy has been studied intensively for the treatment of cancer. A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC). To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy. We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma. AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration. The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently. A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01). Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs. Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy.

Adenoviridae↗
PubMed2000

Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase.

Colon carcinoma accounts for 20% of deaths due to malignancies in the Western world. Once metastases occur, therapeutic options are limited, with an approximate 5-year survival of only 5%. To investigate the potential of new gene therapeutic approaches, a hepatic micrometastasis model of colon carcinoma in BALB/c mice was established. Inoculation of syngeneic MCA26 colon carcinoma cells into the spleens of 18- to 20-week-old mice resulted in the formation of multiple hepatic metastases. Selective transduction of developing hepatic metastases was demonstrated using a beta-galactosidase-expressing recombinant adenovirus. Cytosine deaminase (CD) can metabolize 5-fluorocytosine into the chemotherapeutic reagent 5-fluorouracil (5FU). The antitumoral potential of this suicide gene therapy approach was explored by systemic application of a recombinant replication-deficient adenovirus encoding for the bacterial CD gene under the control of the cytomegalovirus promoter (Ad.CMV-CD). Injection into the tail vein of tumor-bearing mice resulted in delayed tumor growth with significant reduction in hepatic metastases. The potential of this experimental approach for possible future clinical applications was evaluated by investigating adenoviral transduction efficiency, 5FU sensitivity, and 5-fluorocytosine-dependent Ad.CMV-CD toxicity in a variety of human colon cancer cell lines. Although the murine cell lines MCA26 and CC36 were highly sensitive to 5FU, the human colon cancer cell lines showed a 1-100 times higher resistance to 5FU. Specific Ad.CMV-CD toxicity correlates with 5FU toxicity. Transduction efficiency in human colon carcinoma cell lines was shown to be 10-1700 times higher compared with murine cell lines, thus compensating for 5FU resistance. In conclusion, suicide gene therapy using CD may be promising as an adjuvant treatment regimen for hepatic micrometastases of human colon carcinoma.

Adenoviridae↗
PubMed2000

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine.

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.

Adenoviridae↗
PubMed2000

Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach.

We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Adenoviridae↗
PubMed2000

Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene.

The use of the cytosine deaminase (CD)/5-fluorocytosine suicide system as a cancer gene therapy approach enables selective killing of CD-modified cells as well as the ablation of non-modified tumor cells due to a bystander effect that has been suggested to involve the immune system in vivo. Using a stable CD transfectant of the tumorigenic rat adenocarcinoma cell line AS (AS/CD), an antitumoral response against the CD expressing cell line as well as the parental cell line could be induced by stepwise vaccinations in syngeneic animals. AS/CD tumor regression occurred independently of 5-fluorocytosine treatment and was sufficient to protect 37% of the animals against subsequent challenge with tumorigenic doses of the parental AS cell line. Immune rats contained lymphocytes able to specifically lyse CD modified as well as unmodified AS tumor cells in vitro, most likely contributing to the in vivo antitumoral reaction. Thus, the CD suicide system seems to be suitable not only for a local tumor gene therapy but also for the application as therapy of metastatic tumors and minimal residual disease.

Adenocarcinoma↗
PubMed2003

Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

Adenoviridae↗