Incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine into deoxyribonucleic acids of varicella-zoster virus (TK+ and TK- strains)-infected cells

The incorporation of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) into DNA of varicella-zoster virus (VZV)-infected human embryo fibroblasts was studied, using thymidine kinase-positive (TK+) and thymidine kinase-negative (TK-) VZV strains. [125I]IVDU was taken up by cells infected with TK+ VZV-, but not by TK- VZV- or mock-infected cells. [125I]IVDU was incorporated into both VZV DNA and cellular DNA of TK+ VZV-infected cells. When the cells were exposed to 0.3 microM IVDU, a more marked shift was noted in the buoyant density of viral DNA than of host DNA. In contrast, the DNAs isolated from TK- VZV- or mock-infected cells did not exhibit a detectable incorporation of [125I]IVDU. [125I] IVDU-labeled VZV DNA was purified from the viral nucleocapsids of TK+ VZV-infected cells. Substitution of no more than 0.1-1% of the thymidine residues in the VZV DNA by IVDU seemed to suffice to inhibit the replication of VZV.     

Molecular basis of the antineoplastic activity of 3'-amino-3'-deoxythymidine

3'-Amino-3'-deoxythymidine decreased the incorporation of [2-14C]thymidine into DNA of L1210 cells in vitro, and produced an accumulation of [2-14C]thymidine di- and triphosphate. The extent of these effects varied with the amount of recovery time after removal of 3'-amino-3'-deoxythymidine prior to addition of labeled thymidine. The distribution of radioactivity in the acid-soluble fraction derived from [3H]3'-amino-3'-deoxythymidine was as follows: 50% as 3'-amino-3'-deoxythymidine, 20% as the monophosphate, 10% as the diphosphate, and 20% as the triphosphate derivatives. No incorporation of [3H]3'-amino-3'-deoxythymidine into L1210 DNA could be detected. 3'-Amino-3'-deoxythymidine-5'-triphosphate is a competitive inhibitor against dTTP with a Ki of 3.3 microM, whereas the Km for dTTP was 8 microM using activated calf thymus DNA as the template and DNA polymerase-alpha. These data indicate that a major site of inhibition by 3'-amino-3'-deoxythymidine is inhibition of the DNA polymerase reaction.     

Exclusion of exogenous 5-methyl-2'-deoxycytidine from DNA in human leukemic cells. A study with [2(-14)C]- and [methyl-14C]5-methyl-2'-deoxycytidine

Modification of DNA-cytosine by a 5-methyl group is thought to be an important mechanism which regulates the expression of eukaryotic genes. This modification takes place after semiconservative replication. There is very little evidence, if any, that 5MeCyt could be naturally incorporated into mammalian DNA in semiconservative replication. We have clarified the possibility of incorporating 5MedCyd pharmacologically into human leukemic cells in vitro. To this end, we developed a novel small-scale synthesis method for 14C-labeled 5MedCyd starting from commercially available [14C]dThd derivatives. Particular attention was focused upon possible incorporation of radioactive 5MedCyd derivatives into the acid-soluble cellular fraction as well as into nucleic acids and protein in human cells. The results showed that [2(-14)C]- and [methyl-14C]5MedCyd were incorporated into human leukemic cells to a similar extent. The radioactivity originating from these compounds was incorporated mainly into the acid-soluble pool and nucleic acids. The exact nature of the intracellular radioactive molecules in RNA is not known, but the radioactive label in DNA hydrolyzate co-chromatographed exclusively with thymine. Hence, 5MedCyd is deaminated to thymidine before incorporating into DNA. This deamination had taken place already (partially) in the culture medium. Human leukemic cells do effectively protect their DNA from incorporation of exogenous 5MedCyd.     

Susceptibility of protein kinase (ORF47)-deficient varicella-zoster virus strains to anti-herpesvirus nucleosides

To clarify whether varicella-zoster virus (VZV) protein kinase (PK; ORF47) takes part in phosphorylation of anti-herpesvirus nucleosides, thymidine kinase (TK) deficient, and PK/TK double deficient recombinant VZV strains were isolated and their susceptibility, and that of wild type and PK-deficient strains to various nucleoside analogs was evaluated. The PK-deficient VZV strains showed a sensitivity equal to that of the wild type strain against all compounds tested, including ganciclovir. This indicates that PK is not involved in phosphorylation of the tested nucleosides in VZV-infected cells.     

Famciclovir, from the bench to the patient--a comprehensive review of preclinical data

Famciclovir is converted rapidly and efficiently after oral administration to the selective antiviral compound, penciclovir. In cell culture, penciclovir is a potent inhibitor of herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and hepatitis B virus (HBV). Phosphorylation of penciclovir and aciclovir in uninfected cells is limited, and penciclovir, like aciclovir, has minimal effect on replicating cells in culture as expected for a selective antiviral agent. Mode of action studies with VZV and HSV have shown that the phosphorylation of penciclovir in infected cells is far more efficient than for aciclovir. This compensates for differences observed between penciclovir triphosphate and aciclovir triphosphate in the inhibition of HSV and VZV DNA polymerases. Because HBV is not known to encode a thymidine kinase, a different rationale for the selective inhibition of this virus by penciclovir is required. Recent data indicate that the DNA polymerase of HBV is far more sensitive to inhibition by penciclovir triphosphate than cellular DNA polymerases, suggesting that for this virus, selectivity operates at the DNA polymerase. Penciclovir triphosphate is more stable within infected cells than aciclovir triphosphate, and consequently penciclovir has more prolonged antiviral activity than aciclovir. Similarly, famciclovir is more effective than aciclovir or valaciclovir in suppressing HSV replication when given at a lower dosing frequency in certain animal models. These preclinical properties have helped to provide the foundation for the famciclovir clinical programme.     

Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells

The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Instability of (ara-C)DNA under alkaline conditions

We employed both [5-³H]ara-C and [2-¹⁴C]ara-C labeled L1210 DNA for analysis following exposure to alkali under various conditions. The results demonstrated that the tritium label on C₅ of ara-C molecules incorporated in DNA was exchanged with water under alkaline conditions and, therefore, radioactivity was subsequently detectable in the acid-soluble fraction. The [¹⁴C]ara-C labeled DNA, however, was not susceptible to loss of radioactivity by this mechanism, and the appearance of this isotope in the acid-soluble fraction required degradation of the DNA strand or pyrimidine ring. Our results indicated that the [¹⁴C]ara-C labeled DNA was degraded by alkali, suggesting structural instability of this abnormal nucleic acid. These findings provide useful technical information on the purification of (ara-C)DNA labeled with different isotopes.     

Murine cytomegalovirus DNA polymerase: purification, characterization and role in the antiviral activity of acyclovir.

Murine cytomegalovirus (MCMV) neither induces a viral thymidine kinase (TK) nor enhances the activity of a cellular TK. Nevertheless, MCMV is highly susceptible to 9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV). The cellular TK is neither responsible for phosphorylation of ACV nor its anti-MCMV activity. This is clear from the findings that little ACV triphosphate is formed in MCMV-infected mouse embryo fibroblasts (MEF) and that the replication of MCMV is inhibited equally well by ACV in TK+ and TK- cells. Even if trace amounts of ACV triphosphate would be formed by enzymes other than TK, and ACV triphosphate would be responsible for the anti-MCMV activity of ACV, then the MCMV DNA polymerase ought to be highly sensitive to ACV triphosphate. To examine this possibility, the MCMV DNA polymerase was partially purified and characterized. The apparent Ki value of the MCMV DNA polymerase for ACV triphosphate indicates that the sensitivity of the MCMV DNA polymerase to ACV triphosphate is equivalent to that of the HSV DNA polymerase. Therefore, the trace amounts of ACV triphosphate that are formed in MCMV-infected MEF seem to be insufficient to inhibit MCMV DNA polymerase and may not play a key role in the anti-MCMV activity of ACV.     

Effects of tumor necrosis factor alpha on replication of varicella-zoster virus.

Replication of varicella-zoster virus (VZV) and expression of VZV nuclear antigen are inhibited in human embryonic lung fibroblast (HEL) cells pretreated with recombinant tumor necrosis factor (TNF) alpha for 24 h. This antiviral activity is completely blocked by the addition of monoclonal antibodies against TNF. TNF acts synergistically with interferons alpha and gamma. When TNF is added to HEL cells after VZV adsorption, virus replication is still inhibited. When VZV-infected HEL cells are co-cultured with HEL cells which have been pretreated with TNF or grown in the presence of TNF, spread of VZV from VZV-infected HEL cells to uninfected cells is unaffected. No interferon is detected in the supernatants or cell lysates of HEL cells cultured with TNF and antibodies to alpha-, beta- and gamma-interferon have no effect on the antiviral action of TNF.     

Studies of chlorambucil-DNA adducts.

Chlorambucil (CLB) is a bifunctional nitrogen mustard whose therapeutic and major side-effects are thought to be caused by binding to DNA. HPLC analysis of hydrolyzed DNA from L1210 cells incubated with [14C]CLB generated two peaks of radioactivity, indicating the formation of two or more major adducts. Since DNA incubated with [14C]CLB in a cell-free system gave rise to the same profile, experiments were conducted with DNA from cells exposed to radiolabeled DNA precursors, which was then reacted with CLB. DNA containing [8-14C]guanine gave rise to one peak of radioactivity, while DNA containing [2,8-3H]adenine gave rise to two peaks. These peaks corresponded to the peaks seen in the experiment with intact L1210 cells treated with [14C]CLB. Experiments with DNA containing [5-3H]cytosine indicated that no cytosyl adducts were formed. No adducts were seen in hydrolysates prepared from labeled DNA incubated with drug solvent alone. These data indicate that the majority of adducts induced by CLB are guanyl adducts, but a substantial quantity of adenyl adducts has also been identified.     

Structure-activity relationship between (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil (BV-araU, V-araU) in inhibition of Epstein-Barr virus replication.

The structure-activity relationship between (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil (BV-araU and V-araU) in inhibition of Epstein-Barr virus (EBV) was evaluated. Both V-araU and BV-araU effectively inhibited EBV replication in virus-producer P3HR-1(LS) cells, as determined by DNA-DNA hybridization. The 50% effective doses (ED50) for viral DNA replication were 0.005 and 0.3 microM for V-araU and BV-araU, respectively. The in vitro therapeutic index was 4000 for V-araU and 1300 for BV-araU. Synthesis of EBV-induced polypeptides with molecular weights of 145,000 (145, 140, 130, and 110 kDa) was significantly inhibited by both drugs. Only V-araU inhibited the synthesis of 85-, 55-, and 32-kDa polypeptides by approx. 50%. Kinetic analysis of inhibition and reversibility of EBV DNA replication after removal of the drugs indicated that BV-araU has a more prolonged inhibitory effect than V-araU. These results indicate that the substitution of H by Br in the 5-vinyl group results in marked reduction in anti-EBV activity while prolonging the drug effect and diminishing cytotoxicity.     

Comparative inhibition of DNA polymerases from varicella zoster virus (TK+ and TK-) strains by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

The inhibitory effect of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) 5'-triphosphate on varicella zoster virus (VZV) DNA polymerase was studied using the parent strain (TK+-VZV) and the mutant strain (TK--VZV). The mutant strain was deficient in thymidine kinase (TK)-inducing activity and resistant to BVDU. In the absence of BVDU, TK--VZV and TK+-VZV induced an equivalent level of viral DNA polymerase activity in human embryo fibroblasts. In the presence of 5 microM BVDU, TK--VZV still induced viral DNA polymerase activity, whereas TK+-VZV failed to do so. BVDU 5'-triphosphate (BVDUTP) was considerably more inhibitory to the TK+- and TK--VZV DNA polymerases than to the cellular DNA polymerases. There were no significant differences in the affinity for dTTP as substrate and the sensitivity to BVDUTP as inhibitor between the TK+- and TK--VZV DNA polymerases. The Km value for dTTP and the Ki value for BVDUTP of the VZV DNA polymerases were 1.43 microM and 0.55 microM, respectively. The inhibitory effect of BVDUTP to VZV DNA polymerase was competitive with respect to the natural substrate.     

Current pharmacological approaches to the therapy of varicella zoster virus infections: a guide to treatment

Varicella zoster virus (VZV), a member of the herpesvirus family, is responsible for both primary (varicella, chickenpox) as well as reactivation (zoster, shingles) infections. In immunocompetent patients, the course of varicella is generally benign. For varicella zoster, post-herpetic neuralgia is the most common complication. In immunocompromised patients (particularly those with AIDS), transplant recipients and cancer patients, VZV infections can be life-threatening. For these patients and also for immunocompetent patients at risk such as pregnant women or premature infants, the current treatment of choice is based on either intravenous or oral aciclovir (acyclovir). The low oral bioavailability of aciclovir, as well as the emergence of drug-resistant virus strains, have stimulated efforts towards the development of new compounds for the treatment of individuals with VZV infections. Among these new compounds, penciclovir, its oral prodrug form famciclovir and the oral pro-drug form of aciclovir (valaciclovir), rank among the most promising. As with aciclovir itself, all of these drugs are dependent on the virus-encoded thymidine kinase (TK) for their intracellular activation (phosphorylation), and, upon conversion to their triphosphate form, they act as inhibitors/alternative substrate of the viral DNA polymerase. Therefore, cross-resistance to these drugs may be expected for those virus mutants that are TK-deficient and thus resistant to aciclovir. Other classes of nucleoside analogues dependent for their phosphorylation on the viral TK that have been pursued for the treatment of VZV infections include sorivudine, brivudine, fialuridine, fiacitabine and netivudine. Among oxetanocins, which are partially dependent on viral TK, lobucavir is now under clinical evaluation. Foscarnet, which does not require any previous metabolism to interact with the viral DNA polymerase, is used in the clinic when TK-deficient VZV mutants emerge during aciclovir treatment. TK-deficient mutants are also sensitive to the acyclic nucleoside phosphonates (i.e. [s]-1-[3-hydroxy-2-phosphonylmethoxypropyl]cytosine; HPMPC); these agents do not depend on the virus-encoded TK for their phosphorylation but depend on cellular enzymes for conversion to their diphosphoryl derivatives which then inhibit viral DNA synthesis. Vaccination for VZV has now come of age. It is recommended for healthy children, patients with leukaemia, and patients receiving immunosuppressive therapy or those with chronic diseases. The protection induced by the vaccine seems, to some extent, to include zoster and associated neuralgia. Passive immuniatin based on specific immunoglobulins does not effectively prevent VZV infection and is therefore restricted to high risk individuals (i.e. immunocompromised children and pregnant women).     

Cell-protecting effect against herpes simplex virus-1 and cellular metabolism of 9-(2-phosphonylmethoxyethyl)adenine in HeLa S3 cells.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a selective and potent inhibitor of retrovirus and herpesvirus replication in vitro and in vivo. In cell culture studies, pretreatment of HeLa S3 cells with PMEA before infection enhanced its antiviral potency by almost 10-fold, compared with treatment of the cells only after viral infection. To elucidate the basis for this observation, the uptake, metabolism, and retention of PMEA metabolites were examined in uninfected and herpes simplex virus type 1-infected cells, by using [2,8-3H]PMEA. Uptake of the drug into both acid-soluble and acid-insoluble fractions was slow and did not begin to plateau until close to 24 hr. High performance liquid chromatographic analysis of acid-soluble extracts revealed at least four metabolites in addition to PMEA itself, designated as X, Y, DP, and TP. Metabolites X and Y, which were distinct from PMEA and its mono- and diphosphoryl derivatives, represented almost 90% of the radioactivity associated with the cells after 24 hr of incubation. Dephosphorylation of acid-soluble metabolites resulted in accumulation of radioactivity in the peaks associated with PMEA and X. Most of the radioactivity in the acid-insoluble fraction was associated with DNA. Enzymatic digestion of [3H] PMEA-labeled DNA from either infected or uninfected cells yielded both metabolite X and PMEA itself. The role of newly discovered PMEA metabolites in its antiviral activity and cytotoxicity is not clear.     

Differential metabolism of (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) by equine herpesvirus type 1- and herpes simplex virus-infected cells

Thymidine kinase (TK) enzymes encoded by herpes simplex viruses types 1 and 2 (HSV-1, HSV-2), and equine herpesvirus type 1 (EHV-1) catalyze the phosphorylation of thymidine (dThd) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The replication of HSV-1 is sensitive to BVDU, but the replication of HSV-2 and EHV-1 is not. To investigate the differential sensitivity of the viruses to halogenated vinyldeoxyuridine drugs, the phosphorylation of 125I-labeled (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) was studied. Cytosol enzymes from cells infected by HSV-2 and EHV-1 phosphorylated [125I]IVDU to the monophosphate, IVDUMP, but did not convert IVDUMP to higher di- plus triphosphates (IVDUDP plus IVDUTP) forms. In contrast, enzymes from HSV-1-infected cells converted [125I]IVDU to radioactive IVDUMP and IVDUDP plus IVDUTP. Experiments with mixtures of EHV-1- and HSV-1-induced enzymes showed that the EHV-1 enzyme did not inhibit formation of the IVDUDP plus IVDUTP by the HSV-1 enzyme. With [125I]IVDU as substrate, the Km values for the EHV-1 and HSV-1 TKs were 1.82 and 0.34 microM, respectively, and the Ki (dThd) value for the EHV-1 TK was 0.35 microM. In vivo experiments showed that HSV-1-infected cells converted IVDU to the mono- and the di- plus triphosphate forms. In contrast, EHV-1-infected cells converted IVDU to the monophosphate to a lesser extent than did HSV-1-infected cells, and did not produce the di- plus triphosphates. Thus, inefficient phosphorylation of the monophosphates probably contributes to the insensitivity of EHV-1 replication to IVDU, as it does to the insensitivity of HSV-2 replication to this drug.     

Disposition of methacrylonitrile in rats and distribution in blood components

The interaction of 2[14C]methyl-2,3[14C]acrylonitrile (MeAN) with the components of blood and its disposition in male Sprague-Dawley rats has been investigated. Following an oral administration of 100 mg/kg (0.5 LD50, 8 microCi/kg), the rats excreted 43% of the [14C] in the urine, 15% in the feces and 2.5% in the expired air as 14CO2 in 5 days. Hydrogen cyanide (H14CN) was not detectable. The red blood cells retained significant amounts of radioactivity for more than five days after administration, whereas the [14C]-activity in plasma declined sharply. More than 50% of the radioactivity in erythrocytes was detected as covalently bound to cytoplasmic (hemoglobin) and membrane proteins. A small amount of radioactivity was also found in the heme fraction. About 13% of the total dose administered was recovered as thiocyanate in the plasma and the urine. These results suggest that the toxicity of MeAN may be attributable to the whole molecule and not entirely to the in vivo liberation of cyanide.     

Metabolic competition studies of 2'-fluoro-5-iodo-1-beta-d-arabinofuranosylcytosine in vero cells and herpes simplex type 1-infected vero cells

2'-Fluoro-5-iodo-1-beta-D-arabinofuranosylcytosine (FIAC) is a potent antiviral agent with minimal cytotoxicity. In Vero cells, incorporation of labeled dCyd and dThd into the acid-insoluble DNA fraction was, respectively, competitively and noncompetitively inhibited by FIAC. In herpes simplex type 1 (HSV-1) infected Vero cells, these inhibition patterns became noncompetitive. The inhibition constants of FIAC on dThd and dCyd incorporation into the acid-insoluble fraction during a 15-min period were greater than 30 microM which were much higher than the antiviral concentration of FIAC (ED90 = 0.003-0.013 microM) for continuous exposure. Incorporation of dUrd into acid-insoluble DNA was inhibited by 10 microM FIAC in HSV-1-infected Vero cells, but not in uninfected cells. The radioactivity of [2-14C]FIAC was incorporated into the acid-insoluble DNA fraction, and this incorporation in uninfected cells was strongly inhibited by 10 microM dCyd but not by dThd. By contrast, the incorporation in HSV-1-infected Vero cells was strongly inhibited by 10 microM dThd but not by dCyd. These data indicate that FIAC behaves metabolically like dThd, dUrd, or 5-iodo-dUrd in HSV-1-infected cells but like dCyd in noninfected cells. Thus, combined use of dCyd and FIAC may reduce cytotoxicity of FIAC or incorporation of FIAC into host cell DNA without affecting its antiviral activity. This finding is of significance since, for practical reasons, incorporation of FIAC into host cell DNA needs to be reduced as much as possible.     

Terpentecin, an inhibitor of DNA synthesis

Terpentecin at a concentration of 0.78 microgram/ml decreased the number of viable cells of Escherichia coli NIHJ to less than one thousandth the starting number in an hour when added to an exponentially growing culture in a nutrient broth. During this time, the turbidity of the cell suspension kept increasing as fast as the control. Microscopic inspection of the cells exposed to terpentecin under these conditions revealed that the cells were elongated. Terpentecin at a concentration of 6.25 micrograms/ml inhibited incorporation of [14C]thymidine into the acid-insoluble material of cells of E. coli NIHJ by 70% in 30 minutes in contrast to little or no inhibition of the incorporation of [14C]uridine or [14C]leucine. Under similar conditions, terpentecin did not inhibit either membrane transport (uptake) of [14C]thymidine into the cells or the metabolic conversion of the precursor into various cellular acid-soluble components. Terpentecin at a higher concentration (70 micrograms/ml) inhibited by 40% in 30 minutes the incorporation of [methyl-3H]thymidine triphosphate into the DNA fraction of toluene-treated cells of E. coli JE6296 (pol A-). Terpentecin showed stronger antibacterial activities against Bacillus subtilis M45T (rec-) and E. coli BE1121 (rec A-) than against their corresponding wild type strains. However, terpentecin showed no mutagenicity by the Ames test with Salmonella typhimurium strains TA100, TA98, TA92, TA1538, TA1537 and TA1535, and with E. coli WP2 (uvr A). Terpentecin at a lower concentration (0.07 micrograms/ml) inhibited growth in vitro of mouse leukemia L1210 cells by 50%. With the mammalian cells again the incorporation of [14C]thymidine into the acid-insoluble cell material was inhibited more strongly than incorporation of [14C]uridine and [14C]leucine. There was no sign of mutagenicity by the micronucleus test using mice.     

Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.