Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors.

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.

Benzo[a]pyrene diol epoxide forms covalent adducts with deoxycytidylic acid by alkylation at both exocyclic amino N(4) and ring imino N-3 positions.

The carcinogen 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) alkylates DNA at dGuo, dAdo, and dCyd. dCyd adducts, formed in small amounts, elute near the more abundant dGuo adducts. We isolated the dCyd adducts formed with dCMP. Each BPDE enantiomer forms three major adducts with dCMP, two cis and one trans. The trans adduct and one of the cis adducts form by alkylation at exocyclic N(4), while the second cis adduct is a dUrd adduct formed by alkylation at ring N-3 followed by deamination. Epoxide ring-opening geometries were assigned on the basis of halide and temperature effects on adduct yield, the sign of the major CD band, and benzo ring proton NMR coupling constants. One of each set of cis adducts is fluorescent (FL), and the other is nonfluorescent (NF). The trans and FL cis adducts have fluorescence quantum yields 40-50% of that of the BPDE hydrolysis product. The long wavelength UV maxima of the FL and NF cis adducts are red-shifted 1 and 3 nm relative to the trans adduct. (1)H NMR deuterium exchange experiments indicate that in the trans and FL cis adducts N(4)-H is coupled to C10-H. Adduct formation experiments with methyl-protected Cyd derivatives show that NF cis adducts result from alkylation at N-3. MS results, pK(a) measurements, and dUrd alkylation experiments indicate that the N-3 dCyd adducts spontaneously deaminate to dUrd adducts. NMR coupling constants show that in the NF cis adduct the C7 and C8 substituents are quasi equatorial and the C9 substituent is quasi axial, unlike in other cis BPDE adducts. (1)H NOESY spectra of the (-)-BPDE NF cis adduct reveal that it exists in two conformers. Molecular modeling shows that the conformers result from two low-energy conformations of very similar energies with the pyrimidine in opposite orientations, separated by significant barriers to rotation of the uracil moiety.

Probing the mechanistic consequences of 5-fluorine substitution on cytidine nucleotide analogue incorporation by HIV-1 reverse transcriptase.

Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2'-fluorine substitution on the pre-steady state incorporation of 2'-deoxycytidine-5'-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-beta-L-2',3'-dideoxy-3'-thia-5-fluorocytidine (L-FTC, Emtriva), and (+)-beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC, Reverset), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2' position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.

5-Substituted N(4)-hydroxy-2'-deoxycytidines and their 5'-monophosphates: synthesis, conformation, interaction with tumor thymidylate synthase, and in vitro antitumor activity.

Convenient procedures are described for the synthesis of 5-substituted N(4)-hydroxy-2'-deoxycytidines 5a,b,d-h via transformation of the respective 5-substituted 3', 5'-di-O-acetyl-2'-deoxyuridines 1a-c,e-h. These procedures involved site-specific triazolation or N-methylimidazolation at position C(4), followed by hydroxylamination and deblocking with MeOH-NH(3). Nucleosides 5a,b,d-h were selectively converted to the corresponding 5'-monophosphates 6a,b,d-h with the aid of the wheat shoot phosphotransferase system. Conformation of each nucleoside in D(2)O solution, deduced from (1)H NMR spectra and confirmed by molecular mechanics calculations, showed the pentose ring to exist predominantly in the conformation S (C-2'-endo) and the N(4)-OH group as the cis rotamer. Cell growth inhibition was studied with two L5178Y murine leukemia cell lines, parental and 5-fluoro-2'-deoxyuridine (FdUrd)-resistant, the latter 70-fold less sensitive toward FdUrd than the former. With FdUrd-resistant L5178Y cells, 5-fluoro-N(4)-hydroxy-2'-deoxycytidine (5e) caused almost 3-fold stronger growth inhibition than FdUrd; 5e was only some 3-fold weaker growth inhibitor of the resistant cells than of the parental cells. Thymidylate synthase inhibition was studied with two forms of the enzyme differing in sensitivities toward 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), isolated from parental and FdUrd-resistant L1210 cell lines. All N(4)-hydroxy-dCMP (6a,b,d-h) and dUMP analogues studied were competitive vs dUMP inhibitors of the enzyme. Analogues 6b,d-h and 5-hydroxymethyl-dUMP, similar to N(4)-hydroxy-dCMP (6a) and FdUMP, were also N(5), N(10)-methylenetetrahydrofolate-dependent, hence mechanism-based, slow-binding inhibitors. 5-Chloro-dUMP, 5-bromo-dUMP, and 5-iodo-dUMP, similar to dTMP, did not cause a time-dependent inactivation of the enzyme. Instead, they behaved as classic inhibitors of tritium release from [5-(3)H]dUMP. 5-Bromo-dUMP and 5-iodo-dUMP showed substrate activity independent of N(5), N(10)-methylenetetrahydrofolate in the thymidylate synthase-catalyzed dehalogenation reaction. The =N-OH substituent of the pyrimidine C(4) prevented the enzyme-catalyzed release from the C(5) of Br(-) and I(-) (the same shown previously for H(+)). While FdUMP and 6a showed a higher affinity and greater inactivation power with the parental cell than FdUrd-resistant cell enzyme, an opposite relationship could be seen with 5-hydroxymethyl-dUMP.

Colorimetric method for detecting amplified nucleic acids.

Methods for detecting and measuring the success of nucleic acid sequence amplifications can be developed by detecting the by-products of amplification procedures. One method includes the detection of inorganic phosphate (Pi) during or on completion of the PCR. The method requires modification of assay conditions to prevent thermal- and template-independent enzymatic activity from nonspecifically hydrolyzing dNTPs. Detection of Pi by the traditional Fiske-SubbaRow method provides a sensitivity similar to ethidium bromide staining of amplified products. The method offers a simple and rapid assay for amplified nucleic acids and can be useful in assays where confirmation of the amplified DNA product is not essential.

In vitro anti-human immunodeficiency virus and anti-hepatitis B virus activities and pharmacokinetic properties of heterodinucleoside phosphates containing AZT or ddC.

In vitro activities, against human immunodeficiency virus (HIV)- and hepatitis B virus (HBV)-infected cells, of four amphiphilic heterodinucleoside phosphates containing 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxycytidine (ddC) as antiviral monomers were evaluated. The four compounds were N4-hexadecyl-2'-deoxyribocytidylyl-(3'-->5')-3'-azido-2',3'-deoxyt hymidine (N4-hxddC-AZT), N4-palmitoyl-2'-deoxyribocytidylyl-(3'-->5')-3'-azido-2',3'-deoxyt hymidine (N4-pamdC-AZT), N4-hexadecyl-2'-deoxycytidylyl-(3'-->5')-2',3'-dideoxycytidine (N4-hxddC-ddC) and 2'-deoxythymidylyl-(3'-->5')-N4-palmitoyl-2',3'-dideoxycytidine (dT-N4-pamddC). All four dimers were active against HIV, dT-N4-pamddC being the most active and least toxic. dT-N4-pamddC also exhibited strong antiviral effects against a panel of eight AZT-resistant HIV strains. The ddC-containing heterodimers incorporated in liposomes additionally inhibited HBV replication by 50-80% in HepG2 2.2.15 cells. AZT and the AZT-containing dimers were ineffective. Differences in pharmacokinetic properties between the antiviral monomers and the heterodimers were evaluated using liposomal formulations of 3H-labelled AZT heterodimers as model compounds. The cellular distribution of AZT in H9 cells was predominantly cytoplasmic, whereas the amphiphilic dimers were distributed more evenly throughout the cytoplasm, nuclear membranes and microsomes. Blood levels of the heterodimers decreased at a rate two- to threefold slower than AZT and the areas-under-the-curves were five- to sevenfold higher for N4-pamdC-AZT and N4-hxddC-AZT, respectively. Compared to AZT, the peak levels of the dimers were three to four times higher in blood and five to six times higher in the liver. Analysis of blood samples showed that 34% of N4-pamdC-AZT was metabolized to AZT, whereas only 9% of N4-hxddC-AZT released AZT. Considering the antiviral potency and the favourable pharmacokinetic properties of the heterodimers, these compounds merit further exploration as antiviral drugs.

D4T-5'-[p-bromophenyl methoxyalaninyl phosphate] as a potent and non-toxic anti-human immunodeficiency virus agent.

Three aryl phosphate derivatives of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) were tested for their anti-human immunodeficiency virus (HIV) activity in peripheral blood mononuclear cells (PBMC) and thymidine kinase (TK)-deficient CEM T cells. Compared to the parent compound d4T, the lead compound d4T-5'-[p-bromophenyl methoxyalaninylphosphate] with a para-bromo substituent in the aryl moiety was 12.6-fold more potent in inhibiting p24 production (IC50 values: 44 nM versus 556 nM) and 41.3-fold more potent in inhibiting the reverse transcriptase (RT) activity (IC50 values: 57 nM versus 2355 nM) in HIV-infected TK-deficient CEM cells. None of the compounds exhibited any detectable cytotoxicity to PBMC or CEM cells at concentrations as high as 10,000 nM. To our knowledge, this is the first demonstration that the potency as well as selectivity index of the d4T aryl phosphate derivatives in TK-deficient cells can be substantially enhanced by introducing a single para-bromo substituent in the phenyl moiety.

Monitoring gene therapy with herpes simplex virus thymidine kinase in hepatoma cells: uptake of specific substrates.

UNLABELLED: This study investigates the application of PET with specific substrates for the assessment of enzyme activity after transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene. METHODS: After transfection of a rat hepatoma cell line with a retroviral vector containing the HSV-tk gene, different clones were established by G418 selection. Uptake measurements were performed up to 48 hr in a TK-expressing cell line and in a control cell line using thymidine (TdR; measured under therapy conditions), fluorodeoxycytidine (FdCyt) and ganciclovir (GCV). Additionally, bystander experiments and inhibition/competition studies were done. RESULTS: In TK-expressing cells GCV treatment caused an increased (up to 250%) TdR uptake in the acid-soluble fraction and a decrease to 5.5% in the acid-insoluble fraction. The FdCyt uptake was higher in the TK-expressing cells than in controls with a maximum after 4 hr (12-fold and 3-fold higher in the acid-insoluble and acid-soluble fraction). GCV accumulated up to 180-fold more in the acid-insoluble and 26-fold more in the acid-soluble fraction. GCV uptake occurred mainly by the nucleoside transport systems. Bystander experiments revealed a relation between growth inhibition or GCV uptake and the amount of TK-expressing cells. GCV uptake and growth inhibition were correlated with r = 0.96. CONCLUSION: Assessment of GCV accumulation may serve as an indicator of the enzyme activity and of therapy outcome. TdR may be useful to measure therapy effects on DNA synthesis, whereas the potential of FdCyt has to be investigated in further studies.

New lipophilic alkyl/acyl dinucleoside phosphates as derivatives of 3'-azido-3'-deoxythymidine: inhibition of HIV-1 replication in vitro and antiviral activity against Rauscher leukemia virus infected mice with delayed treatment regimens.

The antiretroviral activity of two new lipophilic derivatives of azidothymidine (AZT), N4-hexadecyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-hexadecyldC-AZT) and N4-palmitoyl-2'-deoxyribocytidylyl-(3',5')-3'-azido-2',3'-deoxythy midine (N4-palmitoyldC-AZT) was evaluated in comparison to AZT. In vitro the drugs were tested in human immunodeficiency virus 1 (HIV-1) infected CD4+ HeLa and H9 cells. The in vivo antiviral effect of these derivatives was analysed in Rauscher leukemia virus (RLV) infected mice. The derivatives were incorporated into small liposomes. In vitro both derivatives inhibited virus proliferation in both HIV-1 infected cell lines in a similar dose-responsive manner as AZT. In a plaque reduction assay, using HeLa cells, the IC50 values were 0.035 microM for AZT, 0.5 microM for N4-hexadecyldC-AZT and 4.5 microM for N4-palmitoyldC-AZT, whereas p24 antigen analysis on H9 cells gave IC50 values of 0.005 microM, 0.05 microM and 0.05 microM, respectively. RLV infected mice were treated with intermittent schedules i.p. or i.v. on days 1, 6, 11, and days 16 or 0, 3, 7, and 11 after infection. Regimens with further delayed drug application were on days 3, 7, and 11 and 7 and 11 only. While i.p. treatment with total doses of 380-1140 mg/kg free AZT resulted in 10-30% inhibition of RLV induced splenomegaly, the derivatives gave inhibitions of 37-94%. Late onset of treatment with the derivatives was significantly more effective as compared to free AZT. Intravenous treatment with N4-hexadecyldC-AZT was effective, but with AZT was inactive. The discrepancy in antiviral activity of the AZT derivatives found between the in vitro and in vivo test systems emphasizes the importance of investigating the activity of drug derivatives in vivo.

Properties of an affinity-column-purified human deoxycytidylate deaminase.

Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.

Mechanism of inhibition of mammalian tumor and other thymidylate synthases by N4-hydroxy-dCMP, N4-hydroxy-5-fluoro-dCMP, and related analogues.

N4-Hydroxy-dCMP (N4-OH-dCMP), N4-methoxy-dCMP (N4-OMe-dCMP), and their 5-fluoro congeners (syntheses of which are described) were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N4-OH-dCMP was not a substrate (no dihydrofolate produced; no tritium released with 5-3H-labeled molecule), and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based, with arrest of a step posterior to addition of cofactor and blocking abstraction of the C(5) hydrogen. Ki values for N4-OH-dCMP and its 5-fluoro analogue were in the range 10(-7) - 10(-8) M, 2-3 orders of magnitude higher for the corresponding N4-OMe analogues. The 5-methyl analogue of N4-OH-dCMP was 10(4)-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N4-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N4-OH-dCMP, suggesting interaction of both N4-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N4-OH-dCMP increased its affinity from 2- to 20-fold for the enzyme from different sources.(ABSTRACT TRUNCATED AT 250 WORDS)

2'-Fluoro-2'-deoxypolynucleotides as templates and inhibitors for RNA- and DNA-dependent DNA polymerases.

Poly(2'-fluoro-2'-deoxyadenylic acid) (poly(dAfl)) and poly(2'-deoxycytidylic acid) (poly(dCfl)) were tested as templates in DNA synthesis reactions catalyzed by Xenopus laevis oocytes DNA polymerase alpha, mouse cell DNA polymerase gamma and avian myeloblastis virus (AMV) reverse transcriptase. Poly(dAfl).(dT)12 can fully substitute for poly(rA).(dT)12 as template with DNA polymerase gamma, to 50% with reverse transcriptase, but was poorly recognized by DNA polymerase alpha. DNA synthesis by reverse transcriptase with poly(dCfl).(dG)12 as template was 50% of that with poly(rC).(dG).(dG)12. The use of 2'-fluoropolymers as templates was more efficient at 37 degrees C than at 25 degrees C. No appreciable differences on the fidelity of DNA synthesis by reverse transcriptase were observed when dCMP misincorporation was measured with poly(dAfl).(dT)12 or poly(rA).(dT)12 as template primers. Poly(C) and poly-2'-O-methylcytidylic acid had no significant effect on the reaction catalyzed by DNA polymerase gamma and reverse transcriptase, independent of the synthetic polynucleotide complex utilized as template. On the other hand, poly(dCfl) was an inhibitor when poly(rA).(dT)12 or poly(dA).(dT)12 were used as templates, but not when poly(dAfl).(dT)12 was employed. Analogous results have been obtained with activated DNA and AMV 70 S RNA as templates in the reverse transcriptase reaction. The inhibition by poly(dCfl) was noncompetitive with regard to TTP, poly(dA) and poly(rA). Xenopus laevis oocytes DNA polymerase alpha was not inhibited by poly(dCfl).

Mutation induction by excess deoxyribonucleotides in Saccharomyces cerevisiae.

Excess dTMP is toxic and mutagenic with exponentially growing dTMP efficient uptaking yeast strains 831 rho+ and 833 rho. The respiratory deficient strain 833 exhibits a tenfold sensitivity to the genotoxicity of excess dTMP. Mutant yield in the forward mutation system CAN1----can1 after dTMP excess is comparable to that found after irradiation with UV254nm. Excess dTMP is a poor mutagen in stationary phase cells of both strains. Mutagenicity of excess dTMP is not found in an ochre mutant allele (ade2-1). Exposure of exponentially growing cells to other deoxyribonucleotides (dCMP, dAMP, and dGMP) reveal these nucleotides to have mutagenic potential as well.

Interaction of N4-hydroxy-2'-deoxycytidylic acid with thymidylate synthetase.

The interaction of dTMP synthetase with N4-hydroxy-2'-deoxycytidylate (N4-HOdCMP) has been investigated. With use of standard assay conditions, N4-HOd-CMP is a competitive inhibitor with an apparent Ki of 8.0 microM. Incubation of N4-HOdCMP with dTMP synthetase in the presence of 5,10-methylenetetrahydrofolate (CH2-H4folate) resulted in a rapid time-dependent inactivation of the enzyme which was not first order and the formation of complexes which could be isolated on nitrocellulose filter membranes. With use of radioactive ligands, the isolable native complex was shown to possess 2 mol of N4-HOdCMP and 2 mol of CH2-H4folate/mol of dimeric enzyme; the apparent dissociation constant of N4-HOdCMP was 1.0 microM. Ultraviolet difference spectroscopy of the ternary complex showed a loss of the pyrimidine chromophore which did not reappear upon denaturation with NaDodSO4. The rate of dissociation of N4-HOdCMP from the ternary complex was biphasic in which one-half of the initially bound ligand dissociated with t 1/2 congruent to 2.3 min and the remainder with t 1/2 congruent to 13 min. When the N4-HOdCMP-CH2-H4folate-enzyme complex was denatured, one-half of the CH2-H4folate dissociated whereas all of the N4-HOdCMP remained bound to the enzyme. Taken together, our results indicate that N4-HOdCMP forms a covalent bond with dTMP synthetase and reveal an unusual asymmetry in the two subunits of the N4-HOdCMP-CH2-H4folate-enzyme complex. It appears that one subunit is covalently bound to N4-HOdCMP, which, in turn, is covalently linked to CH2-H4folate whereas the other subunit is covalently bound to N4-HOdCMP but CH2-H4folate is bound by noncovalent interactions.

The influence of deoxycytidine monophosphate (dCMP) on the cytotoxicity of hydroxyurea in the embryonic spinal cord of the mouse.

On day 10 of pregnancy NMRI mice received an intraperitoneal injection of either 500 mg/kg hydroxyurea (HU) or 500 mg/kg HU plus 500, 700, 800, or 900 mg/kg deoxycytidine monophosphate (dCMP). Three hours after application of the substances, the animals received 5 microCi/g 3H-thymidine i.p. One hour later the animals were sacrificed and the embryos were removed. Autoradiographs of the embryos served to determine the percentage of necroses, labelled necroses, mitoses, labelled mitoses, and labelled neuroepithelial cells of the spinal cord. Optimal inhibition of HU effects was achieved by application of 700 mg/kg dCMP. The cytotoxic effects of HU on neuroepithelial cells could be partially but not completely inhibited by simultaneous application of dCMP. The duration of HU-induced DNA synthesis inhibition was shortened after simultaneous dCMP application. A strong increase of the labelling index and shortening of the G2-phase duration of neuroepithelial cells after simultaneous application of HU and dCMP was striking as compared to the results obtained from untreated or HU-treated animals.

Factors affecting deoxycytidylate deaminase activity in some transplantable rat hepatomas.

Dunning hepatoma has a low activity of deoxycytidylate deaminase, comparable to that of normal adult rat liver. This activity seems inconsistent with the rapid proliferation rate of the tumor. Factors which might affect the activity of deoxycytidylate deaminase in the Dunning hepatoma have been examined in it and compared to the Novikoff hepatoma which has high activity of this enzyme. The low activity in Dunning hepatoma does not appear to be the result of any inhibition or, possibly, proteolytic enzyme as judged by mixing experiments, nor does it appear to be due to in vivo differences in nucleotide concentrations especially deoxycytidine 5'-monophosphate, deoxycytidine 5'-triphosphate, or deoxyguanosine 5'-monophosphate which might either help stabilize the enzyme, allosterically increase its activity, or inhibit it. The Dunning hepatoma does not convert cytosine deoxyriboside to uridine deoxyriboside at a significant rate, and the formation of uridine deoxyriboside from deoxyuridine monophosphate is 1% or less during a 30-min incubation of high-speed supernatant fraction from the tumor in either the presence or absence of fluoride. It is concluded that the Dunning hepatoma probably has intrinsically low deoxycytidylate deaminase activity.

Inhibition of de novo pyrimidine nucleotide and DNA synthesis and growth of cultured Novikoff rat hepatoma cells and other cell lines by pyrazofurin (NSC 143095).

Pyrazofurin (PYF), a C-riboside, inhibited the replication of cultured Novikoff rat hepatoma cells, HeLa cells, and mouse L-cells at concentrations as low as 0.1 to 10 muM, but Novikoff cells were more sensitive than the cells of the other two cell lines. Inhibition of cell replication was completely prevented by the presence of 0.1 to 1 mM uridine in the medium, and partly by the presence of other pyrimidine, but not purine nucleosides. A 2- to 4-hr treatment of the cells with 10 muM PYF resulted in a 2-fold increase in the rate of incorporation of uridine into the acid-soluble pool and nucleic acids, while the rate of incorporation of adenosine into RNA was reduced about 85%. The incorporation of adenosine and deoxyuridine into DNA were reduced about 85 and 50%, respectively. The results are consistent with the view that PYF inhibits the de novo synthesis of pyrimidine nucleosides. The inhibition of cell replication seems to be due mainly to an inhibition of DNA rather than RNA synthesis, resulting from a rapid depletion of the pyrimidine deoxynucleotide pool, since addition of thymidine and deoxycytidine reversed the inhibition of DNA synthesis and cell replication by PYF. PYF must enter the cells to exert its toxicity since the toxicity of PYF was reduced 70 to 80% by the presence of 8 muM Persantin, a potent inhibitor of the facilitated and simple diffusion of various substrates, in the medium. If PYF is incorporated via normal nucleoside salvage pathways, its affinity for the nucleoside transport system(s) and kinases, must be low since, even at a concentration of 1 mM, it had only a slight effect on the initial rates of incorporation of various nucleosides into the nucleotide pool.