Anti-Epstein-Barr virus (EBV) activity of beta-L-5-iododioxolane uracil is dependent on EBV thymidine kinase.

beta-L-5-Iododioxolane uracil was shown to have potent anti-Epstein-Barr virus (EBV) activity (50% effective concentration = 0.03 microM) with low cytotoxicity (50% cytotoxic concentration = 1,000 microM). It exerts its antiviral activity by suppressing replicative EBV DNA and viral protein synthesis. This compound is phosphorylated in cells where the EBV is replicating but not in cells where the EBV is latent. EBV-specific thymidine kinase could phosphorylate beta-L-5-iododioxolane uracil to the monophosphate metabolite. The K(m) of beta-L-5-iododioxolane uracil with EBV thymidine kinase was estimated to be 5.5 microM, which is similar to that obtained with thymidine but about fivefold higher than that obtained with 2' fluoro-5-methyl-beta-L-arabinofuranosyl uracil, the first L-nucleoside analogue discovered to have anti-EBV activity. The relative V(max) is seven times higher than that of thymidine. The anti-EBV activity of beta-L-5-iododioxolane uracil and its intracellular phosphorylation could be inhibited by 5'-ethynylthymidine, a potent EBV thymidine kinase inhibitor. The present study suggests that beta-L-5-iododioxolane uracil exerts its action after phosphorylation; therefore, EBV thymidine kinase is critical for the antiviral action of this drug.

Metabolism and mode of inhibition of varicella-zoster virus by L-beta-5-bromovinyl-(2-hydroxymethyl)-(1,3-dioxolanyl)uracil is dependent on viral thymidine kinase.

A nonnaturally occurring L-configuration nucleoside analog, L-beta-5-bromovinyl-(2-hydroxymethyl)-1,3-(dioxolanyl)uracil (L-BVOddU) selectively inhibited varicella-zoster virus growth in human embryonic lung (HEL) 299 cell culture with an EC(50) of 0.055 microM, whereas no inhibition of CEM and HEL 299 cell growth or mitochondrial DNA synthesis was observed at concentrations up to 200 microM. L-BVOddU was phosphorylated by viral thymidine kinase but not by human cytosolic thymidine kinase, and the antiviral activity of this compound is dependent on the viral thymidine kinase. Unlike other D-configuration bromovinyl deoxyuridine analogs, such as E-5-(2-bromovinyl)-2'-deoxyuridine and 1-beta-arabinofuranosyl-E-5-(2-bromovinyl)uracil, this compound was metabolized only to its monophosphate metabolite. The di- or triphosphate metabolites were not detected. This suggested that the inhibitory mechanism may be unique and different from other anti-herpesvirus nucleoside analogs.

Triapine (3-aminopyridine-2-carboxaldehyde- thiosemicarbazone): A potent inhibitor of ribonucleotide reductase activity with broad spectrum antitumor activity.

Previous studies from our laboratories have shown that (a) Triapine() is a potent inhibitor of ribonucleotide reductase activity and (b) hydroxyurea-resistant L1210 leukemia cells are fully sensitive to Triapine. In an analogous manner, Triapine was similarly active against the wild-type and a hydroxyurea-resistant subline of the human KB nasopharyngeal carcinoma. Triapine was active in vivo against the L1210 leukemia over a broad range of dosages and was curative for some mice. This agent also caused pronounced inhibition of the growth of the murine M109 lung carcinoma and human A2780 ovarian carcinoma xenografts in mice. Optimum anticancer activity required twice daily dosing due to the duration of inhibition of DNA synthesis which lasted about 10 hr in L1210 cells treated with Triapine in vivo. DNA synthesis in normal mouse tissues (i.e. duodenum and bone marrow) uniformly recovered faster than that in L1210 leukemia cells, demonstrating a pharmacological basis for the therapeutic index of this agent. Triapine was more potent than hydroxyurea in inhibiting DNA synthesis in L1210 cells in vivo, and the effects of Triapine were more pronounced. In addition, the duration of the inhibition of DNA synthesis in leukemia cells from mice treated with Triapine was considerably longer than in those from animals treated with hydroxyurea. Combination of Triapine with various classes of agents that damage DNA (e.g. etoposide, cisplatin, doxorubicin, and 1-acetyl-1,2-bis(methylsulfonyl)-2-(2-chloroethyl)hydrazine) resulted in synergistic inhibition of the L1210 leukemia, producing long-term survivors of tumor-bearing mice treated with several dosage levels of the combinations, whereas no enhancement of survival was found when Triapine was combined with gemcitabine or cytosine arabinoside. The findings demonstrate the superiority of Triapine over hydroxyurea as an anticancer agent and further suggest that prevention by Triapine of repair of DNA lesions created by agents that damage DNA may result in efficacious drug combinations for the treatment of cancer.

Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine.

Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.

Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile.

Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.

Anti-herpes simplex virus activity of 5-substituted 2-pyrimidinone nucleosides.

Several 5-substituted 2-pyrimidinone 2'-deoxyribonucleoside (PdR) analogs were examined for their anti-herpes simplex virus (HSV) activity in cell culture. The order of potency of their antiviral activities against HSV type 1 (HSV-1) and HSV-2 was iodo PdR approximately ethynyl PdR approximately propynyl PdR. The antiviral action of iodo PdR is dependent on the ability of HSV to induce virus-specified thymidine kinase in infected cells. Several HSV-1 variants with altered thymidine kinase changed their sensitivity to iodo PdR, whereas HSV-1 variants with altered DNA polymerase were as sensitive as the parental virus to iodo PdR. Continuous presence of iodo PdR for more than one virus replication cycle was required for optimal antiviral activity. Iodo PdR (100 microM) had no activity against Epstein-Barr virus DNA replication in P3HR-1 cells. With an oral, an intraperitoneal, or a subcutaneous route of injection, iodo PdR administered twice a day for 2.5 days could prevent the death of mice infected with HSV-2. This in vivo activity is unlikely to be related to the potential conversion of iodo PdR to iododeoxyuridine, since iodo PdR is not a substrate of xanthine oxidase.

Study of ribonucleotide reductase in cells infected with six clinical isolates of herpes simplex virus type 2 (HSV-2) with mutations in its larger subunit.

Herpes simplex virus type 2 (HSV-2) induces a novel ribonucleotide reductase (RR) composed of two subunits (140 and 38 kDa) in infected cells. Other investigators have developed a monoclonal antibody, A6, against the 140-kDa subunit of RR and have found, in about 1% of the cases, an inability to detect this protein in cells infected with clinical isolates of HSV-2. We therefore investigated whether in such cases the clinical isolates were capable of inducing viral RR activity and whether the lack of detection of the 140-kDa protein by the monoclonal antibody was due to an alteration in the antigenic site of this protein. Six such isolates were examined and were found to induce RR activity, similar to HSV-2 (strain 333) RR, which did not require ATP for CDP reduction. Western blot analyses using A6 failed to detect the protein. However, R1, a polyclonal antibody raised against viral RR was capable of detecting this subunit. In addition, R1 was also capable of neutralizing RR activity induced by all the isolates and HSV-2 (strain 333). In conclusion, the lack of detection of the large subunit of RR was not due to the lack of induction but was due to an alteration in the antigenic site recognized by A6; this alteration did not appear to affect the properties of the induced RR activity.

Association of Epstein-Barr virus early antigen diffuse component and virus-specified DNA polymerase activity.

The role of Epstein-Barr virus (EBV) early antigen diffuse component (EA-D) and its relationship with EBV DNA polymerase in EBV genome-carrying cells are unclear, EBV-specified DNA polymerase was purified in a sequential manner from Raji cells treated with phorbol-12,13-dibutyrate and n-butyrate by phosphocellulose, DEAE-cellulose, double-stranded DNA-cellulose, and blue Sepharose column chromatography. Four polypeptides with molecular masses of 110,000, 100,000, 55,000, and 49,000 daltons were found to be associated with EBV-specified DNA polymerase activity. A monoclonal antibody which could neutralize the EBV DNA polymerase activity was prepared and found to recognize 55,000- and 49,000-dalton polypeptides. An EA-D monoclonal antibody, R3 (G. R. Pearson, V. Vorman, B. Chase, T. Sculley, M. Hummel, and E. Kieff, J. Virol. 47:183-201, 1983), was also able to recognize these same two polypeptides associated with EBV DNA polymerase activity. It was concluded that EBV EA-D polypeptides, as identified by R3 monoclonal antibody, are critical components of EBV DNA polymerase.

Induction of virus enzymes by phorbol esters and n-butyrate in Epstein-Barr virus genome-carrying Raji cells.

Phorbol esters and n-butyrate (SB) together could induce Epstein-Barr virus (EBV) DNA polymerase and DNase activities in Raji cells (virus nonproducer). Neither 12-O-tetradecanoylphorbol-13-acetate (TPA) nor SB alone could induce these EBV enzyme activities, transcription of the EcoRI C-region or other EBV proteins in Raji cells. The enzyme induction caused by exposure of Raji cells to TPA-SB was the result of the synthesis of virus-specified RNA, and the increase of linear EBV DNA content in Raji cells caused by TPA alone was not sufficient for induction of EBV-enzyme activities. Temporal characteristics of the TPA-SB induction process, but not the phorbol 12,13-dibutyrate-SB induction process, in Raji cells were observed; a critical phase (10-24 h) postphorbol ester treatment in phorbol 12,13-dibutyrate-SB-treated Raji cells which was responsible for the synthesis of virus RNA and enzymes was found. Phospholipase C, which increases intracellular diacylglycerols (and subsequently activates protein kinase C) was able to partially substitute for TPA in the TPA-SB induction for EBV polymerase and DNase activities. Sphingosine, a protein kinase C inhibitor, partially prevented the induction of virus enzyme activities in Raji cells treated with phorbol 12,13-dibutyrate and SB. No apparent changes in the methylation state of EBV DNA (EcoRI C region) were observed when Raji cells were treated with SB and TPA, alone or in combination. These results suggest that induction of EBV polymerase and DNase activities by TPA-SB may involve protein kinase C activation and another factor triggered by SB which together increase transcription of EBV DNA.

Demonstration of viral thymidine kinase inhibitor and its effect on deoxynucleotide metabolism in cells infected with herpes simplex virus.

The thymidine analog 5'-ethynylthymidine was a potent inhibitor of herpes simplex virus type 1 (strain KOS)-induced thymidine kinase with a Ki value of 0.09 microM. 5'-Ethynylthymidine was less inhibitory against herpes simplex virus type 2 (strain 333)-induced thymidine kinase with a Ki of 0.38 microM and showed no inhibition against human cytosolic thymidine kinase under the conditions tested. The compound was effective against the altered thymidine kinase induced by acyclovir- and bromovinyldeoxyuridine-resistant virus variants. At 100 microM 5'-ethynylthymidine, the cellular pool size of dTTP in herpes simplex virus type 1-infected cells was 5% that of infected cells receiving no drug treatment, while there was no significant effect on the pool sizes of dATP, dGTP, and dCTP. There was a positive correlation between dTTP pools and the intracellular thymidine kinase activity of herpes simplex virus type 1-infected cells. When tested alone, 5'-ethynylthymidine exhibited no antiviral activity, but it antagonized the antiviral efficacy of five compounds which require viral thymidine kinase for their action.

Demonstration of Epstein-Barr virus-specific DNA polymerase in chemically induced Raji cells and its antibody in serum from patients with nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) has been found to be associated with nasopharyngeal carcinoma (NPC), and antibodies with high frequency and titer to EBV proteins have been found in sera from NPC patients. Raji cells, an EBV genome-carrying nonproducer cell line, treated with 12-O-tetradecanoylphorbol-13-acetate and n-butyrate induced a unique EBV DNA polymerase which has properties similar to the EBV DNA polymerase induced by 12-O-tetradecanoylphorbol-13-acetate in P3HR-1 cells, an EBV producer cell line. The possible presence of antibodies to this EBV DNA polymerase in NPC patient serum was examined. The mean number of EBV DNA polymerase units neutralized was 380 +/- 168 units/ml serum (mean +/- SD) in 48 sera from patients with NPC, whereas that in the sera from 52 healthy donors was 62 +/- 56 units/ml (p less than 0.01). The EBV DNA polymerase antibody was found to be associated with the immunoglobulin G but not the immunoglobulin A fraction, and its titer was not correlated with the titers against EBV DNase or virus capsid antigen-immunoglobulin A. Whether the EBV DNA polymerase antibody is against the EBV DNA polymerase core protein or its stimulating protein is still being investigated. This study demonstrated the high frequency and high titer of antibody against EBV DNA polymerase in serum from NPC patients and suggested the potential of utilizing this antibody titer to complement other methods for the early diagnosis or prognosis of NPC.

The sources of thymidine nucleotides for virus DNA synthesis in herpes simplex virus type 2-infected cells.

The thymidine nucleotide sources present during herpes simplex virus type 2 (HSV-2) infection were examined. It was concluded that the source of dTTP in HSV-2-infected cells is not only derived from the ribonucleotide reductase-catalyzed de novo pathway, but also from host DNA. When the de novo pathway was inhibited by the addition of hydroxyurea, an inhibitor of ribonucleotide reductase, the dTTP levels were maintained by a compensatory increase in dTTP derived from host DNA. The utilization of host DNA-derived dTTP for viral DNA synthesis was demonstrated. In spite of an increased contribution of dTTP from host DNA in the presence of hydroxyurea, the level of utilization of host DNA-derived dTTP appeared to remain constant. More than one dTTP pool in virus-infected cells is implicated.

Can ribonucleotide reductase be considered as an effective target for developing antiherpes simplex virus type II (HSV-2) compounds?

Herpes simplex viruses are known to induce virus specified ribonucleotide reductase (RR) in infected cells. RR is considered as a possible target for the development of antiviral agents. In this study, the role of RR in virus replication has been investigated. The sensitivity of RR to hydroxyurea (HU) from virus infected and uninfected HeLa S3 cells was similar with IC50 values of 0.12 and 0.14 mM. In the presence of 2 mM HU, and 10 microM tetrahydrouridine (THU), a cytidine deaminase inhibitor, the incorporation of [14C]cytidine into viral DNA was found to be inhibited by 95%; [32P]-incorporation into viral DNA under the same conditions was inhibited by 75%. The pool size of dCTP and dGTP was 50 and 70%, respectively, with no significant effect on dATP and dTTP pools in virus infected cells treated with 2 mM HU, as compared with virus infected cells receiving no drug treatment. HU at 2 mM could not inhibit HSV-2 yield by more than one log. These results suggest that virus RR is not an effective target for developing anti HSV-2 compounds.

Effects of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new antiherpesvirus compound, on synthesis of macromolecules in herpes simplex virus-infected cells.

We examined the effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on viral DNA, RNA, protein, and enzyme synthesis in HeLa cells infected with herpes simplex virus type 1 and type 2. DHPG inhibited virus DNA synthesis in a dose-dependent fashion. This inhibition was not due to the lack of deoxynucleoside triphosphates which are required for DNA synthesis. This compound has no apparent effect on early and late viral RNA synthesis, viral protein synthesis, or viral thymidine kinase, DNA polymerase, and DNase induction in virus-infected cells.

Utilization of an immunostaining technique to demonstrate heterogeneity in the content of dihydrofolate reductase in peripheral blast cells from a patient with acute lymphocytic leukemia.

Cultured human KB cells which developed resistance to increasing concentrations of methotrexate showed increased levels of dihydrofolate reductase (DHFR) as demonstrated by an immunobridge staining technique. Peripheral blast cells from a leukemic patient were examined for DHFR content during several courses of methotrexate therapy. The number of cells which demonstrated high levels of DHFR increased at the end of treatment. The content in each individual cell was heterogeneous, based on varied intensity of staining in each cell. This immunostaining technique, which utilizes an antibody to DHFR, may have the potential to be a useful tool in the clinic for predicting a patient's possible response to methotrexate therapy.

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells.

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.

Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy.

A series of methotrexate (MTX)-resistant human sublines developed by step increases in selected MTX concentrations have been cloned and examined for dihydrofolate reductase (DHFR) content, relative DNA copy number, and sensitivity to MTX. These cloned sublines had increased DHFR levels which were dependent on the presence of MTX in the medium. The increased levels of DHFR in the absence of MTX were stable in all the clones examined for over a year. Antibody immunolocalization on Western blots showed good correlation of the intensity of the immunostained DHFR band with enzyme activities. Relative gene copy number in these sublines was low relative to the DHFR increases and was not dependent on the presence of MTX in the medium. The increase in gene copy number in these sublines did not correlate with either the levels of DHFR or the sensitivity to MTX.

Unique spectrum of activity of 9-[(1,3-dihydroxy-2-propoxy)methyl]-guanine against herpesviruses in vitro and its mode of action against herpes simplex virus type 1.

A guanosine analog, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG), was found to inhibit herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, cytomegalovirus, and Epstein-Barr virus replication by greater than 50% at concentrations that do not inhibit cell growth in culture. The potency of the drug against all of these viruses is greater than that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir). DHPG was active against HSV-1 growth during the early phase of virus replication and had no activity when added at a later time after infection. Its antiviral activity was irreversible. Thymidine partially neutralized its action. The anti-HSV-1 activity of DHPG was dependent on the induction and the properties of virus-induced thymidine kinase. Virus variants that induced altered virus thymidine kinase and became resistant to acyclovir were still as sensitive to DHPG as the parental virus. DHPG is active against five different HSV variants with induced altered DNA polymerase and resistance to acyclovir.

Roles of thymidylate synthetase activity in herpes simplex virus-infected HeLa cells.

Thymidylate synthetase was not essential for herpes simplex virus replication. However, it appeared to contribute to the formation of a portion of thymidine nucleotides for DNA synthesis in virus-infected cells. Therefore, it is possible that the enzyme plays an important role in determining the potency of several selective antiviral thymidine analogs. Since synergistic effects of 5-fluoro-2'-deoxyuridine with other analogs on virus reproduction were observed, it is suggested that the effects of these analogs depend to a certain degree on the abilities of the monophosphate derivatives to inhibit thymidylate synthetase.

Quantitative correlation of homogeneously stained regions on chromosome 10 with dihydrofolate reductase enzyme in human cells.

Gene amplification may be visualized within a chromosome as a homogeneously stained region (HSR) and HSRs have rarely been reported in human tumor cells with identification of the amplified gene. A parental line and seven clones derived from KB cells resistant to methotrexate (MTX) contain dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; EC 1.5.1.3), ranging from 0.007 unit/mg in the parent to 0.369 unit/mg in clone 7A with a 13,000-fold increase in resistance to MTX. The enzyme is identical to DHFR from other human sources, including that from leukemic patients. A HSR localized to the long arm of chromosome 10(q26) is present in clones selected at or above 2.5 microM MTX. Increase in number of 10q per cell, increase in number of HSR, and increasing amounts of DHFR correlate well. The chromosome change is stable with time as is enzyme production even in the absence of selection by MTX. No clone has shown double minutes. The gene copy number is low. The stability and low gene copy in the presence of large HSRs differ from the pattern described for murine tumors. A human gene for DHFR may be associated with the long arm of chromosome 10.