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Biomedical subjects

Y C Cheng

Publications and source records attributed to Y C Cheng.

At least 217 records · Page 12Linked to original sources
PubMed1987

Synthesis and biological effects of 2'-fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil.

2'-Fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (FEAU) was synthesized, and its biological activities were compared with those of 2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FMAU). Earlier studies indicated that both compounds showed potent anti-herpes simplex virus activity, with a 50% effective dose (ED50) of less than 0.25 microM. In the present study the cell growth inhibitory activity of FEAU (ED50, 200 to 2,060 microM) was found to be about 100-fold less than that of FMAU. With an ED50 ranging from 630 to 3,700 microM, FEAU only weakly inhibited thymidine incorporation into DNA, as compared with FMAU with an ED50 of 9 to 28 microM. Following exposure to [2-14C]FEAU (100 microM), 0.48 pmol/10(6) cells per h was incorporated into the DNA of herpes simplex virus type 1-infected Vero cells, whereas no detectable incorporation was found in uninfected Vero cells or L1210 cells. The Ki of FEAU for thymidine kinase purified from human leukemic cells was greater than 150 microM. For herpes simplex virus type 1- and 2-encoded thymidine kinases, the Kis were 0.6 and 0.74 microM, respectively. Both FEAU and FMAU were relatively nontoxic for mice, with a 50% lethal dose of greater than 800 mg/kg per day (four intraperitoneal doses). However, the lethal dose of FEAU for dogs was 100 mg/kg per day (10 intravenous doses), a dose which is 40- to 80-fold greater than the toxic dose of FMAU. These results suggest that FEAU is a worthy candidate for further development as an antiherpetic agent.

Antiviral Agents↗
PubMed1987

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.

Antibodies, Monoclonal↗
PubMed1987

Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerases in vitro: visualization of chain elongation on a defined template.

1-beta-D-arabinofuranosyl-5-aza-cytosine (ara-5-aza-Cyd) is an analog of 1-beta-D-arabinofuranosylcytosine (ara-C), which resembles ara-C in anabolic metabolism, incorporation into DNA, and inhibition of DNA replication. Human T-lymphoblastic cells (Molt-4) incorporate three- to fivefold more ara-5-aza-Cyd than ara-C into DNA during 5-8 hr exposure. Although ara-5-aza-Cyd and its triphosphate metabolite are unstable in aqueous solution, the aza-analog was much more stable in solution when incorporated into native DNA isolated from Molt-4 cells. By using gapped duplex DNA as a substrate for purified human DNA polymerases alpha and beta, inhibition of [3H]-dCTP incorporation by ara-5-aza-CTP and ara-CTP was competitive, with Ki values for alpha of 11 and 1.5 microM, respectively. Ki values for polymerase beta were 39 and 7.6 microM, respectively. A DNA elongation assay was adapted from DNA sequencing technology, using singly primed bacteriophage M13mp19 or M13mp9 (+)-DNA. Elongation of 5'-[32P]-labeled primer by polymerase alpha is slowed considerably by incorporation of one ara-CMP and to a lesser extent after incorporation of one ara-5-aza-CMP. Neither analog significantly affected elongation by polymerase beta after a single incorporation. However, neither polymerase alone could appreciably extend the growing chain if two consecutive ara-5-aza-CMP or ara-CMP analogs were incorporated. Thus, if similar mechanisms are operant in intact cells, the greater incorporation of ara-5-aza-Cyd than ara-C into DNA may be due to a more facile elongation of the nascent DNA strand by polymerase alpha after incorporation of a single analog. The effect in vitro of incorporation of either analog on DNA chain elongation is widely variable, depending on the identity of the polymerase involved and the sequence of the DNA template being copied.

Arabinofuranosylcytosine Triphosphate↗
PubMed1987

Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs.

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.

Arabinonucleotides↗
PubMed1986

Inhibition of adenosine and thymidylate kinases by bisubstrate analogs.

Potential bisubstrate analogs, in which the 5'-hydroxyl group of adenosine was joined to the phosphoryl group acceptor by polyphosphoryl bridges of varying length (ApnX, where n is the number of phosphoryl groups and X is the nucleoside moiety of the acceptor), were tested as inhibitors of human liver adenosine kinase and of thymidylate kinase from peripheral blast cells of patients with acute myelocytic leukemia. Adenosine kinase was most strongly inhibited by P1,P4-(diadenosine 5')-tetraphosphate (Kd = 30 nM) and P1,P5-(diadenosine 5')-pentaphosphate (Kd = 73 nM). Thymidylate kinase was most strongly inhibited by P1-(adenosine 5')-P5-(thymidine 5')-pentaphosphate (Kd = 120 nM) and by P1(adenosine 5')-P6-(thymidine 5')-hexaphosphate (Kd = 43 nM). In these enzymes, as in adenylate and thymidylate kinases, strongest inhibition was achieved in compounds containing one or two more phosphoryl groups than the substrates combined. These results support the view that nucleoside and nucleotide kinases mediate direct transfer of phosphoryl groups from ATP to acceptors, rather than acting by a double displacement mechanism.

Adenine Nucleotides↗
PubMed1986

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate.

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.

Adenine Nucleotides↗
PubMed1986

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates.

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.

Antiviral Agents↗
PubMed1986

Synthesis of dihydrofolate reductase and metabolism of related RNA in a methotrexate resistant human cell line infected with herpes simplex virus type 2.

Using a human cell line with amplified gene copy for dihydrofolate reductase (DHFR) and permissive for herpes simplex virus type 2 (HSV-2), the effect of HSV-2 on DHFR synthesis and on the steady-state level of total cellular and nuclear DHFR RNA was examined. There was a reduction in DHFR synthesis accompanied by a proportional decrease in the levels of DHFR messenger RNA (mRNA) 1 hr after infection. Both effects could be induced by HSV pretreated with ultraviolet light and this early virus induced rapid turnover of DHFR mRNA was not dependent on de novo protein synthesis. Analysis of nuclear RNA (nRNA) from uninfected cells by Northern blot hybridization identified a large DHFR nRNA of about 23 kb which probably represents the primary transcript and four processed intermediates of DHFR mRNA ranging in size from 12 to 4.4 kb. The levels of these nRNAs were unchanged during the first hour; however, the 4.4-kb species accumulated in nuclei 2 hr after infection. This effect was induced in the absence of HSV gene expression.

Cell Line↗
PubMed1986

Antitumor agents. 78. Inhibition of human DNA topoisomerase II by podophyllotoxin and alpha-peltatin analogues.

It has been reported that the action of etoposide (VP-16) (14) as an antitumor agent is mediated through its interaction with DNA topoisomerase II which results in DNA breakage inside the cell. In order to understand the mechanism of action as well as structure-activity relationships of 14, several novel, synthetic and some naturally occurring analogues related to podophyllotoxin were examined for inhibition of the DNA topoisomerase II activity. Compound 2 exhibited enhanced activity and compound 5 slightly diminished activity relative to 14. A 4 beta-substituted ether at the C ring and O-demethylation at the E ring appear to enhance activity.

Antineoplastic Agents, Phytogenic↗
PubMed1986

Inhibitory action of 10-deazaaminopterins and their polyglutamates on human thymidylate synthase.

The action of 10-deazaaminopterin, its 10-alkyl derivatives, and their polyglutamates against thymidylate synthase (TMPS) from human acute myeloblastic leukemia was examined. Comparison of aminopterin with methotrexate showed that the methylation of the N10-position (methotrexate) increased the inhibitory effect of aminopterin on TMPS. In contrast, alkylation of the 10-position of 10-deazaaminopterin decreased inhibition of TMPS, and the 50% inhibitory concentration values were progressively higher, in the order 10,10-dimethyl-, 10-methyl-, and 10-ethyl-derivatives. The addition of gamma-glutamyl moieties to both 10-deazaaminopterin, and one of its alkylated analogs, 10-ethyl-10-deazaaminopterin, enhanced inhibition. The maximum inhibition was achieved with the addition of three glutamyl moieties to 10-deazaaminopterin and two glutamyl moieties to 10-ethyl-10-deazaaminopterin, respectively. Thus, 10-deazaaminopterin-tetraglutamate was 138-fold and 10-ethyl-10-deazaaminopterin-triglutamate was greater than 51-fold more active than their respective parental compound. The compounds 10-deazaaminopterin and its polyglutamates, 10-methyl- and 10,10-dimethyl-analogs, inhibited TMPS in a noncompetitive fashion with respect to 5,10-methylene-tetrahydropteroylglutamate. Ki values for the monoglutamates were 220 microM, 310 microM, and 225 microM, respectively. In contrast, 10-ethyl-10-deazaaminopterin and its polyglutamates inhibited TMPS in a competitive fashion with a Ki value of 410 microM for the monoglutamate. With 5,10-methylene-tetrahydropteroylpentaglutamate as a substrate, 10-deazaaminopterin and its polyglutamates behaved as mixed type inhibitors, and 10-ethyl-10-deazaaminopterin, monoglutamate and diglutamate, behaved as noncompetitive inhibitors, whereas its pentaglutamate behaved as a mixed-type inhibitor. These results suggest that the addition of gamma-glutamyl moieties to the substrate also caused the change in the mode of inhibitory action of these compounds. These findings also show that both replacement of the N10-position of the 4-aminopteroyl structure with a methylene group and its alkylation caused interesting and unexpected changes in the structure-activity relationships and the mode of action for these 4-aminopteroyl antifolates as inhibitors of TMPS, which may be therapeutically relevant.

Alkylation↗
PubMed1986

Oncogenes in human primary hepatic cancer.

Transfection assay of NIH 3T3 cells was performed with DNAs isolated from ten human PHC (primary hepatic cancer) specimens and a hepatoma 7402 cell line. Positive results were obtained in 7402 and in six out of ten PHC DNAs. Human N-ras gene was identified in transfectants from 7402 DNA and transformed cells from three PHC DNA samples, which had passed more than two cycles of transfection. The expression of N-ras was also remarkably enhanced in six out of nine poly(A)+RNA samples isolated from PHC tissues. P21 synthesis was elevated in 7402 cells as well as in transfectants derived from 7402 cells and PHC DNA. In analysis of PHC DNA, rearrangement and amplification of N-ras gene was observed in two PHC samples. The discrepancy of results of the transfection assay and mRNA expression was discussed. Furthermore, c-myc was also highly expressed in most PHC tissues. It implied that the cooperating activity of N-ras and c-myc might be responsible for the malignant phenotypic alteration in some or most cases in human primary liver cancer.

Autoradiography↗
PubMed1986

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.

Antibodies, Viral↗