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Biomedical subjects

Y C Cheng

Publications and source records attributed to Y C Cheng.

At least 253 records · Page 14Linked to original sources
PubMed1985

Activity of the new antifolate N10-propargyl-5,8-dideazafolate and its polyglutamates against human dihydrofolate reductase, human thymidylate synthetase, and KB cells containing different levels of dihydrofolate reductase.

The action of N10-propargyl-5,8-dideazafolate (PDDF) and its gamma-polyglutamyl analogues against human thymidylate synthetase and dihydrofolate reductase was examined. PDDF inhibited thymidylate synthetase in a noncompetitive fashion with respect to 5,10-methylenetetrahydrofolate and dihydrofolate reductase in a competitive fashion with respect to dihydrofolate. Ki values were estimated to be 20 and 250 nM, respectively. The addition of glutamyl moieties through gamma-linkage enhanced the inhibitory activity of PDDF against thymidylate synthetase without significant effect on dihydrofolate reductase. PDDF inhibited human KB cell growth, and its potency was found to be influenced less than that of methotrexate by the amount of cellular dihydrofolate reductase.

Animals↗
PubMed1984

Novel interaction of aphidicolin with herpes simplex virus DNA polymerase and polymerase-associated exonuclease.

DNA polymerases induced by herpes simplex virus (HSV)-1 (KOS) and by three phosphonoformic acid-resistant strains were purified and the interaction of these enzymes with aphidicolin was examined. Incorporation of dATP, dCTP, and dTTP into activated DNA by parental enzyme was inhibited competitively by aphidicolin whereas dGTP incorporation was inhibited noncompetitively. Phosphonoformic acid-resistant enzymes were altered in KM and KI values for substrate and inhibitor, and two were inhibited by aphidicolin via the same modes as parental enzyme. However, aphidicolin competitively inhibited incorporation of dGTP by the third phosphonoformic acid-resistant enzyme under identical assay conditions. Two phosphonoformic acid-resistant enzymes were more sensitive than parental enzyme to inhibition by aphidicolin, indicating a close association between binding determinants for aphidicolin and for phosphonoformic acid on the virus DNA polymerase molecule. Aphidicolin inhibited hydrolysis of polynucleotide by HSV-1 DNA polymerase-associated nuclease. Inhibition was uncompetitive with DNA and the KI value (0.09 microM) was within the range of those calculated during nucleotide incorporation (0.071-0.74 microM). Therefore, aphidicolin may produce antiviral effects both by inhibition of deoxynucleotide incorporation and by deleterious effects resulting from inhibition of polymerase-associated nuclease.

Animals↗
PubMed1984

Differential binding affinities of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine for herpes simplex virus-induced and human cellular deoxythymidine kinases.

The affinity of a large number of sugar-modified derivatives of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was determined towards deoxythymidine (dThd) kinases (TK) of various origin, i.e. human cytosol and mitochondrial TK, as well as herpes simplex virus (HSV) type 1 and type 2 TK. Substitution at the 3'- and 5'-position had differential effects on the interaction of BVDU with TK from different sources. The binding affinity of the nucleoside analogs for these different TKs was also influenced by the nature of the 5-substituent (2-bromovinyl vs 2- chlorovinyl ). The 5'-azido and 5'-amino derivatives of BVDU showed affinity for HSV-1 TK only and may, therefore, be useful to differentiate HSV-1 TK from all other TKs . There was no stringent correlation between the antiviral effects of the compounds and their binding constants for viral TK, suggesting that phosphorylation by viral TK is an essential but not sufficient factor in determining the antiviral activity of these analogs.

Bromodeoxyuridine↗
PubMed1984

Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.

Animals↗
PubMed1984

The intracellular content of dihydrofolate reductase: possibilities for control and implications for chemotherapy.

Intracellular levels of DHFR can be modulated by mechanisms other than gene amplification. We found that MTX itself has an effect and the important features of this mechanism are as follows: (a) Sub-saturating doses of MTX induce intracellular DHFR activity by increasing DHFR synthesis; (b) The time-dependent effect seems quite specific for DHFR and is reversible (7); (c) Elevated DHFR synthesis is accompanied by disproportionate increases in DHFR mRNA; (d) The time scale for maximum induction is appreciably longer than the cell generation time. We suggest that part of the control involved is translational and we postulate that DHFR may regulate its own biosynthesis through feedback mechanisms. It is conceivable that the induction phenomenon could affect the clinical efficacy of MTX-therapy in some instances.

Cell Line↗
PubMed1984

A potent multisubstrate analogue inhibitor of human thymidylate synthetase.

The synthesis of an 8-deazafolate analogue of the intermediate in the methylation of 2'-deoxyuridylate is described. Alkylation of diethyl 5,6,7,8-tetrahydro-8-deazafolate with 3'-O-acetyl-5-(bromomethyl)-2'-deoxyuridine 5'-[bis-(trichlorethyl) phosphate], followed by removal of the trichloroethyl groups with a Zn/Cu couple and mild saponification, gave the target inhibitor N-[4-[[[2-amino-3,4,5,6,7, 8-hexahydro-4-oxo-5-[(2'-deoxyuridin-5-yl)methyl]-pyrido[3,2-d] pyrimidin-6-yl]methyl]amino]benzoyl]-L-glutamic acid 5'-monophosphate. The free nucleoside and the 5'-(methyl phosphate) diester were similarly prepared. Each of these reactions yielded a pair of diastereoisomers about C-6 of the reduced deazafolate in approximately a 1:1 ratio. These diastereoisomeric mixtures were evaluated as inhibitors of thymidylate synthetase derived from human tumor (HeLa) cells. The 5'-monophosphate was a potent inhibitor, competitive with respect to both 2'-deoxyuridylate (Ki = 0.06 microM) and tetrahydrofolate (Ki = 0.25 microM). In contrast, the nucleoside and the nucleotide methyl ester were poorer inhibitors by more than 3 orders of magnitude, attesting to the importance of the anionic function at the nucleoside 5'-position in the affinity of an inhibitor for the enzyme active site.

HeLa Cells↗
PubMed1984

Effects of certain nucleoside analogues on human cytomegalovirus replication in vitro.

Four nucleoside analogues, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), -5-iodouracil (FIAU), -5-methylcytosine (FMAC) and -5-iodocytosine (FIAC), were studied for their effect on human cytomegalovirus (HCMV) replication in vitro. FMAU, FIAU, FMAC and FIAC showed antiviral activities for four strains of HCMV (Major, Clegg, D550 and Towne) in a plaque reduction assay, with a dose required for 50% inhibition (ED50) in the range of 0.1 to 0.65 microM. At a concentration of 1 microM-FMAU or -FIAC, the synthesis of five virus-specific late polypeptides of molecular weights 150 000, 120 000, 67000, 54000 and 27000 was entirely blocked. Quantification of Towne viral DNA synthesis, using complementary RNA-DNA hybridization with a Towne-specific cRNA probe, demonstrated a complete inhibition of HCMV DNA replication at 1 microM of FMAU or FIAC. After the removal of the inhibitors, however, viral DNA synthesis resumed, and infectious virus reappeared, indicating that the inhibition of HCMV replication by these nucleoside analogues was of a virostatic reversible type.

Antiviral Agents↗
PubMed1984

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.

Acyclovir↗
PubMed1984

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.

Animals↗
PubMed1983

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.

Acyclovir↗
PubMed1983

Epstein-Barr virus: inhibition of replication by three new drugs.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with three human malignancies. Acyclovir [9-(2-hydroxyethoxymethyl)guanine], the first clinically useful drug effective against replication of EBV, is without effect against latent or persistent EBV infection. Three nucleoside analogs, E-5-(2-bromovinyl)-2'-deoxyuridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine, and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil are potent inhibitors of EBV replication in vitro. Moreover, in contrast to the reversibility of viral inhibition by Acyclovir, these three drugs have prolonged effects in suppressing viral replication even after the drugs are removed from persistently infected cell cultures.

Acyclovir↗
PubMed1983

Studies of amino-acid sequence in dihydrofolate reductase from a human methotrexate-resistant cell line KB/6b. Structural and kinetic comparison with mouse L1210 enzyme.

The partial amino acid sequence of dihydrofolate reductase (DHFR, EC 1.5.1.3) from human KB/6b cells has been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradations and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequences. A comparison of the proposed human DHFR sequence with the previously known sequences of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed.

Amino Acid Sequence↗
PubMed1983

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.

Drug Synergism↗
PubMed1983

Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus. Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase.

The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.

Acyclovir↗
PubMed1983

Thymidylate synthase, dihydrofolate reductase, folyl binder, 10-formyl-H4PteGlu synthetase, 5,10-methenyl-H4PteGlu cyclohydrolase and 5,10-methylene-H4PteGlu dehydrogenase derived from cells of human origin.

In the past few years we have been engaged in studying several folate cofactor requiring enzymes derived from human cells; namely thymidylate synthase, dihydrofolate reductase, folyl binder, 10-formyl-H4PteGlu synthetase, 5,10-methenyl-H4PteGlu cyclohydrolase and 5,10-methylene-H4PteGlu dehydrogenase. These have been purified and several properties have been examined, in particular the interactions with folyl-polyglutamate forms as substrates and inhibitors. Folylpolyglutamates are better substrates for thymidylate synthase, 10-formyl-H4PteGlu synthetase, and 5,10-methylene-H4PteGlu dehydrogenase when NADP is lower than 30 microM, whereas dihydrofolate reductase and membrane associated folyl binder do not distinguish between folylmono- and polyglutamates. Analog studies with thymidylate synthase suggest that there are two types of folate binding sites and this led us to propose a model for the subunit association.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1983

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.

Cell Line↗