Multiple conformations of the cytidine repressor DNA-binding domain coalesce to one upon recognition of a specific DNA surface.

The cytidine repressor (CytR) is a member of the LacR family of bacterial repressors with distinct functional features. The Escherichia coli CytR regulon comprises nine operons whose palindromic operators vary in both sequence and, most significantly, spacing between the recognition half-sites. This suggests a strong likelihood that protein folding would be coupled to DNA binding as a mechanism to accommodate the variety of different operator architectures to which CytR is targeted. Such coupling is a common feature of sequence-specific DNA-binding proteins, including the LacR family repressors; however, there are no significant structural rearrangements upon DNA binding within the three-helix DNA-binding domains (DBDs) studied to date. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the CytR DBD free in solution and to determine the high-resolution structure of a CytR DBD monomer bound specifically to one DNA half-site of the uridine phosphorylase (udp) operator. We find that the free DBD populates multiple distinct conformations distinguished by up to four sets of NMR peaks per residue. This structural heterogeneity is previously unknown in the LacR family. These stable structures coalesce into a single, more stable udp-bound form that features a three-helix bundle containing a canonical helix-turn-helix motif. However, this structure differs from all other LacR family members whose structures are known with regard to the packing of the helices and consequently their relative orientations. Aspects of CytR activity are unique among repressors; we identify here structural properties that are also distinct and that might underlie the different functional properties.

Ribavirin antagonizes the in vitro anti-hepatitis C virus activity of 2'-C-methylcytidine, the active component of valopicitabine.

Ribavirin antagonizes the in vitro anti-hepatitis C virus (HCV) activity of the pyrimidine nucleoside analogue 2'-C-methylcytidine, the active component of the experimental anti-HCV drug valopicitabine. In contrast, the combination of ribavirin with either the purine nucleoside analogue 2'-C-methyladenosine or the HCV protease inhibitor VX-950 resulted in an additive antiviral activity. These findings may have implications when planning clinical studies with valopicitabine.

Positive and negative transcriptional states of a variegating immunoglobulin heavy chain (IgH) locus are maintained by a cis-acting epigenetic mechanism.

Analyses of transgene expression have defined essential components of a locus control region (LCR) in the J(H)-C(mu) intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Ig(mu) H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Ig(mu) H chain genes were distinguished by a silent mutation and fused cells in which the mu gene was active with cells in which mu was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.

The effect of cyclopentenyl cytosine on human SK-N-BE(2)-C neuroblastoma cells.

Human neuroblastoma SK-N-BE(2)-C cell-line cells were cultured in the presence of various concentrations of cyclopentenyl cytosine (CPEC). In the absence of cytidine, the IC50 value of CPEC for SK-N-BE(2)-C cells was 100 nM after 72 hr drug exposure. The IC20 value was 1 microM after 24 hr of exposure to CPEC in the presence of 10 microM cytidine, whereas in the absence of cytidine, CPEC at 1 microM resulted in an IC40 value after 24 hr. Therefore, cytidine partially prevented the cytostatic effect of CPEC. Cells cultured with 1 microM CPEC for 72 hr were enriched by approximately 410% with mono- and oligonucleosomes in comparison with cells cultured without CPEC. This enrichment was partially prevented with 10 microM deoxycytidine and completely prevented with 10 microM cytidine.

Reversal by cytidine of cyclopentenyl cytosine-induced toxicity in mice without compromise of antitumor activity.

Among nine compounds surveyed, cytidine was found to be the most effective in reversing the antiproliferative effects of cyclopentenyl cytosine (CPEC) on human T-lymphoblasts (MOLT-4) in culture. Cytidine, at concentrations of 1-25 microM, enabled cells to maintain normal logarithmic growth when added up to 12 hr after exposure to a 200 nM concentration of the oncolytic nucleoside, CPEC. The most abundant CPEC metabolite, CPEC-5'-triphosphate, is a potent [K1 approximately 6 microM] inhibitor of CTP synthetase (EC 6.3.4.2). Accumulation of this inhibitor resulted in a depletion of CTP levels to 17% of their original cellular concentration. Exogenous cytidine reversed CPEC-induced cellular cytotoxicity by suppressing the formation of CPEC-5'-triphosphate by 70%, and by partially replenishing intracellular CTP to at least 60-70% of its original concentration. In vivo, cytidine (500 mg/kg) administered intraperitoneally 4 hr after each daily dose of CPEC (LD10-LD100) for 9 days reduced the toxicity and abolished the lethality of CPEC to non-tumored mice. Of greater practical importance is the finding that, under these experimental conditions, cytidine did not curtail the antineoplastic properties of CPEC in L1210 tumor-bearing mice. Moreover, the concentration range over which CPEC exhibited antineoplastic activity was extended with cytidine administration.

Cyclopentenyl cytosine and neuroblastoma SK-N-BE(2)-C cell line cells.

We studied the effect of cyclopentenyl cytosine (CPEC) on human neuroblastoma SK-N-BE(2)-C cell line cells. CPEC had an IC50 value of 100 nM for non-synchronised SK-N-BE(2)-C cells. These cells were arrested in G0/G1-phase or early S-phase of the cell cycle upon treatment with CPEC. After treatment of synchronised S-phase cells with 1 microM CPEC, the number of cells present after 3 days was less than 10% of that observed for the untreated cells. S-phase synchronised cells treated with CPEC and deoxycytidine showed an increased viability in comparison with cells treated with CPEC alone. Approximately 15% of the cells treated with CPEC and deoxycytidine traversed through one cell cycle. The amount of CTP declined to undetectable levels within 3 h after addition of 1 microM CPEC. The presence of cytidine prevented, to a large extent, the cytostatic effect of CPEC.

Metabolism and RNA incorporation of cyclopentenyl cytosine in human colorectal cancer cells.

We studied the cytotoxicity and metabolism of the investigational cytidine analogue cyclopentenyl cytosine (CPE-C) in three human colorectal cancer cell lines: HCT 116, SNU-C4, and NCI-H630. CPE-C potently inhibited cell growth and decreased clonogenic capacity at concentrations achieved in murine and primate pharmacologic studies. CPE-C produced a concentration-dependent depletion of CTP, accompanied by changes in the dCTP pools. CPE-C exposure was associated with an accumulation of cells in the S phase at 48 hr. [3H]CPE-C was metabolized predominantly to the triphosphate (CPE-CTP) form. Saturation of phosphorylation to the monophosphate form occurred above 5-10 microM. Plateau CPE-CTP pools were of a magnitude similar to that of the physiologic ribonucleotide triphosphate pools. The intracellular half-life of CPE-CTP was 24 hr. After a 24-hr exposure to 0.5 microM CPE-C, CPE-CTP was detected for up to 96 hr post-drug removal, accompanied by persistent depletion of the CTP pools. Cesium sulfate density centrifugation of purified nucleic acids indicated that [3H]CPE-C incorporated into RNA, but was not detected in DNA. Agarose-gel electrophoresis of RNA from [3H]CPE-C-treated cells indicated that it localized predominantly in low molecular weight (4-8 S) RNA species. When CPE-C was administered concurrently with [3H]adenosine (Ado), the proportion of [3H]Ado migrating with low molecular weight RNA species increased. Concurrent exposure to 10 microM cytidine (Cyd), sufficient to replete CTP pools, provided essentially complete protection against lethality resulting from a 24-hr exposure to less than or equal to 0.5 microM CPE-C. While 10 microM Cyd substantially decreased CPE-CTP formation and CPE-C-RNA incorporation during the initial 3 hr of exposure compared to CPE-C alone, after 24 hr the levels were not significantly different. Cyd rescue did not affect the accumulation of [3H]CPE-C or [3H]Ado into low molecular weight RNA species after a 24-hr exposure to CPE-C. Our results indicate that depletion of CTP and dCTP pools is an important component of CPE-C cytotoxicity. While CPE-C incorporation into RNA may not be the critical cytotoxic event during a 24-hr exposure to CPE-C, it may play a role during prolonged exposure to CPE-C. CPE-C is a highly potent new agent and merits clinical evaluation in the treatment of colorectal cancer.

Sequence-dependent interaction of 5-fluorouracil and arabinosyl-5-azacytosine or 1-beta-D-arabinofuranosylcytosine.

We studied the cytotoxicity of arabinosyl-5-azacytosine (Ara-AC), a dCyd antagonist which inhibits DNA synthesis, in combination with 5-fluorouracil (FUra) in two human colon cancer cell lines, HCT 116 and SNU-C4. Clonogenic assays done following sequential or concurrent 24-hr exposures to Ara-AC and FUra showed that the sequence Ara-AC followed by FUra resulted in more than additive lethality in the HCT 116 cell lines and additive lethality in the SNU-C4 cells. In contrast, the reverse sequence, FUra followed by Ara-AC, was antagonistic in both cell lines. A similar interaction between FUra and 1-beta-D-arabinofuranosylcytosine (Ara-C) was evident in HCT 116 cells; at concentrations which individually diminished viability by 34 and 62%, respectively, the sequence Ara-C followed by FUra decreased viability by 97%. Pulse-labeling with [3H]dUrd showed profound inhibition of DNA synthesis by the sequence Ara-AC followed by FUra, with over 90% inhibition lasting for up to 48 hr following Ara-AC exposure. When FUra preceded Ara-AC, however, earlier recovery from inhibition of DNA synthesis occurred. FUra pretreatment did not appreciably alter the quantity or distribution of [3H]Ara-AC or [3H]Ara-C nucleotides after a 4- to 6-hr exposure. Pre-exposure to FUra decreased Ara-AC incorporation into DNA by 37 and 73% at 6 hr in HCT 116 and SNU-C4, respectively. FUra pretreatment also inhibited Ara-C incorporation into DNA by over 50% at 6 and 24 hr. The antagonism of Ara-AC and Ara-C cytotoxicity by FUra pretreatment can thus be explained by diminished incorporation of the dCyd analogs into DNA resulting from inhibition of DNA synthesis by FUra-induced dTTP and dCTP depletion. In contrast, when Ara-AC or Ara-C preceded FUra, their incorporation into DNA was not disturbed, and prolonged inhibition of DNA synthesis was observed.

Cellular pharmacology of cyclopentenyl cytosine in Molt-4 lymphoblasts.

The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'-triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation was between 50 and 100 nM. Cytidine, uridine, and nitrobenzylthioinosine almost fully prevented the cytotoxicity of CPEC when introduced shortly before or together with the drug, but only cytidine was effective as an antidote when added 12 h after 200 nM CPEC. Studies of the cellular entry of CPEC revealed that nitrobenzylthioinosine fully blocked this process over a 60-s interval and for as long as 2 h, suggesting that the initial interiorization was mediated by facilitated diffusion. In Molt-4 cells incubated with tritiated CPEC, 9 metabolites could be distinguished: prominent among these was cyclopentenyl uridine (CPEU), the deamination product of CPEC; other major metabolites included the 5'-mono-, di-, and triphosphates of CPEC, and of CPEU, along with two phosphodiesters provisionally identified as CPEC-diphosphate choline and CPEC-diphosphate ethanolamine. When the accumulation of CPEC-5'-triphosphate was measured as a function of concentration of the drug in the medium, the process was found not to be saturable by levels of CPEC up to 1000 nM. In cells incubated with 200 nM drug, CPEC-5'-triphosphate accumulated rapidly and linearly for approximately 4 h, the time for doubling of the concentration being 2 h. After a 16-h incubation with 100 nM CPEC, the concentration of CPEC-5'-triphosphate was 50-fold that of the parent drug in the medium and could be readily monitored spectrophotometrically in high-pressure liquid chromatography effluents without recourse to radiolabeled nucleoside. In 2-h incubations, the concentration of free CPEC required to reduce CTP by 50% was 150 nM; this corresponded to a CPEC-5'-triphosphate level of 750 nM. After washout of extracellular CPEC, CPEC-5'-triphosphate decayed with a half-life that ranged from 9 to 14 h. Twenty-four h after washout of 200 nM CPEC (the concentration of drug reducing proliferation by 80%), cells had not resumed proliferation, and CTP pools were still depressed by 90%. Cytidine, uridine, and nitrobenzylthioinosine all strongly repressed the anabolic phosphorylation of CPEC when added to Molt-4 cells along with the drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Broad-spectrum antiviral activity of carbodine, the carbocyclic analogue of cytidine.

Carbocyclic cytidine (C-Cyd) is a broad-spectrum antiviral agent active against DNA viruses [pox (vaccinia)], (+)RNA viruses [toga (Sindbis, Semliki forest), corona], (-)RNA viruses [orthomyxo (influenza), paramyxo (parainfluenza, measles), rhabdo (vesicular stomatitis)] and (+/-)RNA viruses (reo). The target enzyme of C-Cyd is supposed to be CTP synthetase that converts UTP to CTP. In keeping with this assumption are the observations that (i) C-Cyd effects a dose-dependent inhibition of RNA synthesis in both virus-infected and uninfected cells, and (ii) exogenous addition of either Urd or Cyd reverses both the antiviral and cytocidal activity of C-Cyd, whereas addition of dThd or dCyd fails to do so. The selectivity of C-Cyd against Sindbis, vesicular stomatitis and reo virus is markedly increased when C-Cyd is combined with Cyd (10 micrograms/mL). This combination may therefore be worth pursuing as a chemotherapeutic modality for the treatment of virus infections.

Antileukemic effects of pseudoisocytidine, a new synthetic pyrimidine C-nucleoside.

Pseudoisocytidine, a new synthetic pyrimidine C-nucleoside, which might be considered a more stable analog of 5-azacytidine, is active in vitro and in vivo, i.p. and p.o., against various 1-beta-D-arabinofuranosylcytosine-resistant lines of mouse leukemia. This antileukemic activity is blocked by cytidine but not by deoxycytidine or thymidine.