Biomedical subjects

Y C Cheng

Publications and source records attributed to Y C Cheng.

At least 235 records · Page 13Linked to original sources

PubMed1985

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.

Animals↗

PubMed1985

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.

Antiviral Agents↗

PubMed1985

Interaction of DNA polymerase and nucleotide analog triphosphates.

The properties of virus and host DNA polymerases are important factors in determining the selectivity of deoxynucleotide analogs used in antiviral chemotherapy. The high affinity of herpes DNA polymerase for nucleotide analogs may be particularly important in CMV and EBV-infected cells, since these viruses do not induce the synthesis of a virus-specified thymidine kinase. In general, the effect of nucleotide analog incorporation into DNA may be summarized as follows: analogs with modifications at the base moiety do not affect the rate of DNA chain elongation whereas those modified at the sugar moiety will inhibit the rate of chain elongation. ACGTP and DHPGTP competitively inhibit incorporation of dGTP into DNA; however, steric freedom of the acyclic phosphate may allow these nucleotides to bind virus enzyme in a conformation similar to that assumed by dGTP only at the transitional stage of the enzyme reaction. This may explain the high affinity of virus enzyme for these inhibitors. The interaction of aphidicolin with virus enzyme differs from that with host enzyme. These differences suggest new strategies for antiviral chemotherapy using aphidicolin derivatives.

Antiviral Agents↗

PubMed1985

Analysis of dihydrofolate reductase gene amplification in a methotrexate-resistant human tumor cell line.

Detailed cytogenetic and molecular biologic studies have been performed on the human KB tumor cell line and four methotrexate-resistant subclones. Results are presented, demonstrating that the gene encoding the target enzyme dihydrofolate reductase is increasingly amplified in progressively methotrexate-resistant subclones, and that dihydrofolate reductase sequences are localized to a homogeneously staining region on chromosome 10q.

Cell Line↗

PubMed1985

Synthesis and biological activities of 2-pyrimidinone nucleosides. 2. 5-Halo-2-pyrimidinone 2'-deoxyribonucleosides.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-bromo-2-pyrimidinone (BrPdR) and 1-(2-deoxy-beta-D-ribofuranosyl)-5-iodo-2-pyrimidinone (IPdR) have been synthesized by condensation of the appropriate silylated bases 2a and 2b, respectively, with 3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-alpha-D-ribofuranosyl chloride (8) in 1,2-dichloroethane, in the presence of SnCl4, followed by separation of the anomeric blocked nucleosides via column chromatography and subsequent deprotection with methanolic ammonia. Both BrPdR and IPdR exhibited significant antiherpes activities against various strains of HSV-1 and HSV-2, the latter compound (IPdR) showing the higher activity as well as the stronger binding to the virus-specific thymidine kinase.

Animals↗

PubMed1985

Demonstration of a stimulatory protein for virus-specified DNA polymerase in phorbol ester-treated Epstein-Barr virus-carrying cells.

A heat-labile Epstein-Barr virus-specific DNA polymerase stimulatory protein having a molecular mass of 45 kDa was purified from phorbol 12-myristate 13-acetate-treated P3HR-1 cells by column chromatography. The virus DNA polymerase stimulatory protein was precipitated by sera from patients with nasopharyngeal carcinoma but not by sera from healthy donors. The interaction of the stimulatory protein with DNA polymerase was stoichiometric. Furthermore, this protein stimulated Epstein-Barr virus DNA polymerase but not herpes simplex virus type 1 or type 2 or human DNA polymerase alpha. The stimulatory protein did not alter the Km value of dTTP or DNA but did increase the Vmax of DNA polymerase. Salt concentrations between 100 mM and 150 mM KCl were optimal for this protein-induced stimulation of Epstein-Barr virus DNA polymerase activity. The presence of the stimulatory protein in the reaction mixture enhanced the sensitivity of virus DNA polymerase to phosphonoformate.

Carcinoma↗

PubMed1985

Interaction of Epstein-Barr virus DNA polymerase and 5'-triphosphates of several antiviral nucleoside analogs.

The 5'-triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methylcytosine, 9-[(2-hydroxyethoxy)methyl]guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine had lower Ki values for Epstein-Barr virus DNA polymerase than has been reported elsewhere for host DNA polymerase. Inhibition of DNA elongation by these analogs ranged from moderate to strong, suggesting that preferential incorporation of these analogs into DNA by virus DNA polymerase may contribute to antiviral selectivity.

Acyclovir↗

PubMed1985

Mutually exclusive inhibition of herpesvirus DNA polymerase by aphidicolin, phosphonoformate, and acyclic nucleoside triphosphates.

Dual inhibitor studies were performed to examine the interaction of aphidicolin, phosphonoformate, 9-(2-hydroxyethoxymethyl)guanine triphosphate, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate with herpes simplex virus DNA polymerase. Kinetic data indicated that inhibition by one agent prevents simultaneous inhibition by a second agent, producing a mutually exclusive inhibition pattern. This suggested that binding sites on the DNA polymerase molecule for these compounds are kinetically overlapping. These findings should be taken into consideration for the design of future antiviral compounds and combination chemotherapy protocols.

Acyclovir↗

PubMed1985

Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.

Acyclovir↗

PubMed1985

Human cytomegalovirus-induced DNA polymerase and its interaction with the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil, -5-iodocytosine, and -5-methylcytosine.

Human cytomegalovirus-induced DNA polymerase and cellular DNA polymerase alpha were purified by successive chromatography on DEAE-cellulose, phosphocellulose, heparin agarose, and single-stranded DNA agarose columns. The purified virus-induced DNA polymerase was resolved to consist of two polypeptides corresponding to molecular weights of 140,000 and 58,000, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Virus-induced DNA polymerase and cellular alpha polymerase were examined for their sensitivities to the triphosphates of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAUTP), -5-iodocytosine (FIACTP), and -5-methylcytosine (FMACTP). The inhibitive effects of these triphosphates on the DNA polymerases were competitive with regard to the natural substrates; thus FMAUTP competes with dTTP, and FIACTP and FMACTP compete with dCTP. The inhibition constants (Ki) for FMAUTP, FIACTP, and FMACTP of virus-induced DNA polymerase are 0.06, 0.30, and 0.47 microM, respectively. Cellular DNA polymerase alpha is much less sensitive to these inhibitors, and its Ki values for FMAUTP, FIACTP, and FMACTP are 0.45, 3.10, and 2.90 microM, respectively. In addition, human cytomegalovirus-induced DNA polymerase, but not cellular DNA polymerase alpha, can utilize these analog triphosphates as alternate substrates for their corresponding natural deoxyribonucleoside triphosphates in in vitro DNA synthesis.

Antiviral Agents↗

PubMed1985

Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia.

Pretreatment of L5178Y murine leukemia cells with uracil arabinoside (ara-U) enhances the cytotoxicity of cytosine arabinoside (ara-C). This effect is mediated by the cytostatic effect of ara-U, which causes a delay of cell progression through S-phase. Consequently, the specific activity of enzymes that peak during S-phase increases, and deoxycytidine kinase increases 3.6-fold over untreated controls. This allows enhanced anabolism of ara-C to nucleotides, as well as increased incorporation into DNA with ultimate synergistic cytotoxicity. It is postulated that the systemic metabolism of high-dose ara-C to sustained high levels of ara-U in patients with acute leukemia may enhance the activity of subsequent doses of ara-C, and thus contribute to a means for pharmacologic self-potentiation, contributing to the unique therapeutic activity of high-dose ara-C.

Animals↗

PubMed1985

Metabolism of 1-beta-D-arabinofuranosyl-5-azacytosine and incorporation into DNA of human T-lymphoblastic cells (Molt-4).

1-beta-D-Arabinofuranosyl-5-azacytosine (ara-5-aza-Cyd) had potent cytotoxicity against human T-type lymphoblastic cells in culture. When Molt-4 cells were exposed to ara-5-aza-Cyd for 24 h, clonogenic survival was reduced by 50 and 98% at initial concentrations of 10(-7) and 10(-6) M, respectively, compared to 3 X 10(-8) and 10(-6) M, respectively, for the same effect with 1-beta-D-arabinofuranosylcytosine (ara-C). The analogue is chemically unstable, with a t1/2 of 12 h at 37 degrees C in phosphate-buffered saline. ara-5-aza-Cyd is not significantly deaminated by human Cyd-deoxycytidine (dCyd) deaminase, in contrast to ara-C. It is phosphorylated by human cytoplasmic dCyd kinase, with a Km of 55 microM and a relative Vmax of 310% compared to dCyd. The primary metabolite (70%) in Molt-4 cells was identified as ara-5-aza-Cyd triphosphate. Thymidine but not uridine or amino acid incorporation was inhibited by ara-5-aza-Cyd. ara-5-aza-Cyd was incorporated in a dose-dependent manner into DNA, but not RNA, primarily in internucleotide linkage as the original compound. Incorporation into the cellular methanol-insoluble fraction was 3- to 5-fold higher at 8 h than was ara-C incorporation. ara-5-aza-Cyd may have a unique activity against tumor cells resistant to ara-C, particularly where high Cyd-dCyd deaminase activity is a factor. The mode of action, like that of ara-C, is probably mediated through its incorporation into DNA and inhibition of DNA synthesis.

Antineoplastic Agents↗

PubMed1985

Characterization of dihydrofolate reductase-related DNA and RNA from human KB cell subclones containing different amounts of enzyme.

The present study was aimed to characterize dihydrofolate reductase (DHFR)-related DNA and RNA in a series of human KB cell sublines and to further understand the mechanisms of DHFR regulation. We used two probes, one which could recognize the 5' flanking sequence (p5') of the human DHFR gene and one derived from mouse complementary DNA (pDHFR 26) which contains the coding sequence for DHFR, to identify the DHFR-related DNA and RNA. Our results revealed no major differences in DNA gene structure as the copy number increases. The recognizable fragments of DHFR gene were similar including the 5' flanking sequence upstream from the first exon. We observed that all DHFR mRNA species identified were present in the subclones. The content of cytoplasmic DHFR mRNA does not always correlate with the relative gene copy number in these cell lines. Furthermore, we were able to detect high-molecular-weight RNA related to DHFR which might be the precursors of the DHFR mRNAs. Finally, using different RNA extraction procedures, we observed that different patterns of cytoplasmic DHFR mRNA can be obtained, which is probably due to differential breakdown of RNA species during extraction procedures.

Base Sequence↗

PubMed1985