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A Fridland

Publications and source records attributed to A Fridland.

At least 55 records · Page 3Linked to original sources
PubMed1987

2',3'-Dideoxynucleoside phosphorylation by deoxycytidine kinase from normal human thymus extracts: activation of potential drugs for AIDS therapy.

As a first step toward improving dideoxynucleoside inhibition of human immunodeficiency virus replication in human lymphocytes, we examined the kinetics of 5'-phosphorylation of a series of 2',3'-dideoxynucleosides, using deoxycytidine kinase purified from human thymus extracts. Nucleosides with the 2'-deoxyribose moiety were activated 30 times faster than were 2',3'-dideoxynucleosides. The adenosine deaminase inhibitor, 2'-deoxycoformycin, showed an unexpected ability to inhibit purine and pyrimidine dideoxynucleoside phosphorylation; such inhibition was not competitive and was not observed when 2'-deoxycytidine was the substrate. 2'-Deoxycytidine, the natural substrate, inhibited dideoxynucleoside phosphorylation in a manner similar to that observed with 2'-deoxycoformycin. Thus, dideoxynucleosides are activated by deoxycytidine kinase through a different catalytic interaction than occurs in 5'-activation of 3'-hydroxynucleosides by this enzyme.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides.

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.

Adenosine Kinase↗
PubMed1986

Tiazofurin metabolism in human lymphoblastoid cells: evidence for phosphorylation by adenosine kinase and 5'-nucleotidase.

The exact route of metabolism of tiazofurin, a novel nucleoside with antitumor activity, is controversial. Using human cell lines severely deficient in salvage nucleotide enzymes, we were able to identify the route of activation in tiazofurin metabolism. With loss of adenosine kinase activity by mutation in two lymphoblastoid cell lines, CCRF-CEM and WI-L2, the growth sensitivity to tiazofurin decreased by 6- and 3-fold, respectively. In contrast, the mutant lines were about 3000- to 1500- and 16- to 4-fold more resistant to the structurally similar tiazofurin analogues pyrazofurin and ribavirin, respectively. Other mutants with defective deoxycytidine or uridine kinase activity showed normal sensitivity to all three analogues. Both cell lines with defective adenosine kinase activity accumulated about 50% wild-type levels of tiazofurin-5'-monophosphate and thiazole-4-carboxamide adenine dinucleotide analogue of tiazofurin at cytotoxic concentrations of the drug. Extracts of wild-type lymphoblasts catalyzed the phosphorylation of tiazofurin in the presence of adenosine 5'-triphosphate and Mg2+. Loss of adenosine kinase activity in the mutant extract eliminated this phosphorylating activity for tiazofurin consistent with the notion that adenosine kinase catalyzes phosphorylation of tiazofurin. However, an enzyme activity that catalyzed the phosphorylation of tiazofurin in the presence of inosine-5'-monophosphate as donor and Mg2+ was detected in the extracts of both wild-type cells and adenosine kinase-deficient mutants. The monophosphate donor specificity, divalent metal, high salt requirement, and nucleoside acceptor specificity of this enzyme activity paralleled that of a 5'-nucleotidase (EC 3.1.3.5) which catalyzes inosine phosphorylation. In addition, tiazofurin phosphorylation was competitively inhibited by inosine and the apparent Ki value was similar to the apparent Km value for inosine phosphorylation. These results indicate that two enzymes, adenosine kinase and a cytoplasmic 5'-nucleotidase, are functionally important anabolizing enzymes for tiazofurin in human cells.

5'-Nucleotidase↗
PubMed1985

Metabolism and selectivity of arabinonucleoside in human lymphoid cells.

The selective toxicity of purine deoxynucleosides against lymphoid cells appears to be mediated by a preferential accumulation of the corresponding triphosphates in these cells. We report a study of the metabolism and toxicity of arabinonucleosides of guanine and cytosine toward human T- and B-lymphoblastoid-cell lines. Both compounds inhibited the growth of T lymphoblasts at concentrations less than 2 microM. However, only ara-G exhibited a strong selectivity for T lymphocytes as indicated by a 100-fold greater toxicity to T than B cells. ara-G is not significantly degraded to guanine but is metabolized to the triphosphate. In common with the other arabinonucleoside, cytotoxicity by ara-G was associated with specific inhibition of DNA synthesis in cells. The capacity of T cells (CCRF-CEM) to accumulate ara-GTP was dependent primarily on deoxycytidine kinase. The level of intracellular ara-GTP accumulated after incubation with the corresponding nucleoside was 20- to 40-fold higher in T cells than either of two B-lymphoblast-cell lines, WI-L2 or PF-2S. The levels of phosphorylating activity for ara-C in extracts of T- and B-cell lines were approximately equal; in contrast, ara-G phosphorylating activity was four- to fivefold higher in B lymphoblasts. After removal of arabinonucleosides from the culture medium, ara-GTP levels in B lymphoblasts declined at a rate that was two to four times faster than that of ara-CTP. In marked contrast, no catabolism of the arabinonucleoside triphosphates was detected in T lymphoblasts. These results suggest that the selectivity of arabinonucleosides to human lymphoid cells of various phenotypes can be correlated with their nucleotide metabolism. The selectivity of ara-G for T and B cells can be correlated with their differential ability to catabolize ara-GTP.

Arabinonucleosides↗
PubMed1985

Metabolic basis of arabinonucleoside selectivity for human leukemic T- and B-lymphoblasts.

Purine analogues are potentially useful agents for selective chemotherapy of lymphoproliferative diseases. We compared the toxic effects of various arabinonucleosides against eight human T- and B-lymphoblastoid lines. The arabinosides of cytosine (ara-C), 2-fluoroadenine (F-ara-A), adenine (ara-A) and guanine (ara-G) all inhibited the growth of T-lymphoblasts at concentrations below 2 microM. Only ara-G showed strong selectivity for T-cells, as indicated by a 15- to 250-fold greater toxicity toward T-cell lines than B-cell lines. To investigate the biochemical basis for ara-G selectivity, we compared the metabolism of the arabinonucleosides in CCRF-CEM (T-) versus PF-2S (B-) lymphoblasts. Comparison of arabinonucleoside triphosphate accumulation indicated differences favoring selective ara-GTP formation in T-cells. In contrast, ara-C, ara-A, and F-ara-A formed almost corresponding amounts of their triphosphates in both cell types. Triphosphate accumulation correlated directly with inhibition of DNA synthesis in CCRF-CEM and PF-2S cells. PF-2S cells accumulated less than 20% ara-GTP from the nucleoside than did CCRF-CEM cells. Nucleoside kinase measurements showed no significant differences in arabinonucleoside phosphorylation that could account for the preferential ara-GTP accumulation in T-cells. After removal of arabinonucleoside-containing medium, ara-GTP levels in PF-2S cells declined with a half-life of 49 min whereas, in CCRF-CEM cells, the level of analogue triphosphate remained unchanged. Furthermore, the half-life of ara-CTP, ara-ATP, and F-ara-ATP in the B-cells was 3- to 5-fold longer than that of ara-GTP. These results indicate that ara-G is more selective than other known arabinonucleosides; such selectivity warrants further assessment of the therapeutic potential of this agent against T-cell malignancies and other lymphoid disorders.

Arabinofuranosylcytosine Triphosphate↗
PubMed1984

Selection of 9-beta-D-arabinofuranosyladenine-resistant human T-lymphoblasts with altered ribonucleotide reductase activity.

We sought to define the cellular activity that mediates resistance in human leukemic cells (CCRF-CEM) to the nucleoside 9-beta-D-arabinofuranosyladenine (ara-A). Stable mutants were obtained by continuous selection at ara-A concentrations of 1 or 2.5 microM in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin. Four clones selected for further investigation were 4- to 11-fold less sensitive to the cytotoxicity of ara-A than the parental CCRF-CEM line. These clones also showed cross-resistance to deoxyadenosine and thymidine, but normal sensitivity to arabinosylcytosine and adenosine, and increased sensitivity to the etoposide VP16-213. No change was found in the activity of kinases that phosphorylate ara-A and the various nucleosides that could account for the resistant phenotype in these mutant lines. Resistance was associated with a 2- to 8-fold increase in the level of all four deoxyribonucleoside triphosphates. The triphosphate pools in the mutants were resistant to the inhibition produced in wild-type cells by addition of deoxy-adenosine or thymidine, although significant activation in the deoxyguanosine triphosphate pool was obtained by higher concentrations of thymidine. An examination of ribonucleotide reductase in extracts of two of the mutants revealed a specific alteration in the normal sensitivity of the enzyme for deoxyadenosine triphosphate and adenosine triphosphate but not 9-beta-D-arabinofuranosyladenine 5'-triphosphate. When the level of ribonucleotide reductase activity was measured, it was found that the ara-A-resistant cells contained approximately twice the wild-type level of cytidine diphosphate reductase activity at physiological adenosine triphosphate level. This combination of increased enzyme activity and alteration in sensitivity to the nucleoside triphosphates could account for both the changes in deoxyribonucleotide pool sizes and the resistant phenotype of the presumed mutants.

Cell Line↗
PubMed1983

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells.

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.

Adenosine↗
PubMed1983

2'-O-nitro-1-beta-D-arabinofuranosylcytosine. A new derivative of 1-beta-D-arabinofuranosylcytosine that resists enzymatic deamination and has antileukemic activity.

To overcome the susceptibility of the anticancer drug 1-beta-D-arabinofuranosylcytosine (ara-C) to enzymatic deamination, and hence deactivation, we prepared the 2'-O-nitro-1-beta-D-arabinofuranosylcytosine (termed nitrara-C) and evaluated it for biological activity. Nitrara-C was resistant to enzymatic deamination and inhibited the proliferation of several strains of human leukemic T and B lymphoblasts grown in culture. Moreover, it substantially extended the life spans of mice with L1210 leukemia. Studies with ara-C-resistant human leukemic lymphoblasts deficient in deoxycytidine kinase activity disclosed that the inhibitory activity of the new compound depends on its phosphorylation.

Animals↗
PubMed1983

Effects of the epipodophyllotoxin VP-16-213 on cell cycle traverse, DNA synthesis, and DNA strand size in cultures of human leukemic lymphoblasts.

Serial studies of human leukemic lymphoblasts (CCRF-CEM line) cultured with 0.25 to 2.5 microM VP-16-213 for 0 to 6 hr indicated that the mechanism of cytotoxicity of this compound involves a primary effect on DNA. The most striking early change shown by flow cytometry in VP-16-213-treated cells was a delay in S-phase transit before arrest of cells in G2. Coinciding with this S-phase delay was a selective inhibition of thymidine incorporation into DNA as well as concentration-dependent scission of DNA strands. Using alkaline elution methods, we were able to detect DNA breakage at concentrations of VP-16-213 well below the level required to demonstrate kinetic effects or inhibition of DNA synthesis. These data suggest that DNA strand scission is the initial event in the sequence of kinetic and biosynthetic changes leading to growth inhibition and death of VP-16-213-treated cells. Inhibition of replicon initiation due to strand scission is a plausible explanation for the cytotoxic action of this podophyllotoxin derivative.

Cell Cycle↗
PubMed1982

Mode of action of 1-beta-D-arabinofuranosylcytosine in human lymphoblasts: differential effects of the drug on chromosomal replication.

This paper describes a novel biochemical effect of 1-beta-D-arabinofuranosylcytosine (Ara-C), a potent antitumor agent, on the process of DNA replication in cultures of human lymphoblasts (CCRF-CEM). By using short incubation periods of five minutes with 3H-thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients, it was found that the initial effect on DNA replication by the addition of 5 nM Ara-C is inhibition of initiation of new replicating units in DNA. A second effect of Ara-C, which is evident 30 minutes after drug addition, is a reduction in the process of elongation of these units. A model is presented to explain the mechanism by which Ara-C may cause a differential effect on DNA chain initiation and elongation in CCRF-CEM cells.

Cells, Cultured↗
PubMed1981

Identification of the mechanism of activation of 9-beta-D-arabinofuranosyladenine in human lymphoid cells using mutants deficient in nucleoside kinases.

The biochemical basis of cellular resistance to 9-beta-D-arabinofuranosyladenine (ara-A) and its natural purine derivative, deoxyadenosine, was investigated with two mutants of cultured human T-lymphoblastoid CCRF-CEM cells. One mutant that lacked deoxycytidine kinase activity, designated CEM/ara-C, retained about 10% of wild-type deoxyadenosine kinase and deoxyguanosine kinase activity each but maintained normal adenosine kinase or thymidine kinase activity. This suggested that in these human T-lymphoblastoid cells, as in other previously studied mammalian cells, deoxycytidine and purine deoxyribonucleosides are phosphorylated by the same enzyme. Despite this extensive reduction of purine nucleoside kinase activities, the cytotoxicity of ara-A or deoxyadenosine was not appreciably affected, decreasing by only 2.5- and 6-fold, respectively. A second mutant, isolated by selecting CEM/ara-C mutants that were resistant to ara-A, showed a 100-fold increase in resistance to ara-A cytotoxicity. This ara-A-resistant subline was deficient in the activities of two enzymes, deoxycytidine kinase and adenosine kinase, and showed a high degree of resistance to deoxyadenosine, adenosine, and pyrazofurin but not to pyrimidine analogs, such as 5-fluorodeoxyuridine or 5-fluorouridine. Further studies of ara-A and deoxyadenosine phosphorylation in wild-type and resistant cell lines disclosed that, although deoxycytidine kinase is the principal enzyme for their phosphorylation in vitro, their intracellular conversion to cytotoxic nucleotides depends on the joint action of deoxycytidine kinase and adenosine kinase rather than purine-specific deoxynucleoside kinase, as previously thought.

Adenosine Kinase↗
PubMed1980

Mode of action of 9-beta-D-arabinosyladenine and 1-beta-D-arabinosylcytosine on DNA synthesis in human lymphoblasts.

The effects of 9-beta-D-arabinosyladenine (AraAde), 1-beta-D-arabinosylcytosine (AraCyt) and 2'-deoxyadenosine on DNA replication in cultured human lymphoblasts (CCRF-CEM line) were studied by pulse-labeling cells with [3H]thymidine and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients. At doses that reduced the overall rate of DNA synthesis to 50--70% of control values, both AraAde and AraCyt profoundly inhibited the formation of new replicons, with secondary effects on chain elongation contributing to the total inhibition of DNA synthesis. In contrast, the suppression of DNA synthesis by 2'-deoxyadenosine stemmed mainly from an inhibition of chain elongation. These studies also disclosed that about 100 times more AraAde than AraCyt was required to produce a similar inhibition of DNA replication in CCRF-CEM cells. Determination of intracellular concentrations of the nucleoside triphosphates (AraCTP and AraATP) indicated to 90% inhibition of DNA synthesis was achieved at 1.6 and 25 pmol/1 . 10(6) cells, respectively. Studies with cell lysates revealed that the replicative machinery in CCRF-CEM cells is more sensitive to AraCTP than to AraATP. This finding contrasts with earlier research, in which the inhibtion of purified DNA polymerase by either AraATP of AraCTP yielded essentially the same Ki value. The difference in sensitivity of the cell lysate to these arabinonucleotides may reflect either a target enzyme other than DNA polymerase or, more plausibly, some subtle interaction of the polymerase with other components of the replicative process.

Cells, Cultured↗
PubMed1977

Inhibition of deoxyribonucleic acid chain initiation: a new mode of action for 1-beta-D-arabinofuranosylcytosine in human lymphoblasts.

A novel biochemical effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on DNA replication in cultured human lymphoblasts is described. By incubating the cells with [3H] thymidine for 5 min and analyzing the nascent DNA by velocity sedimentation in alkaline sucrose gradients, it was possible to discern the initial effect of a very low concentration of drug (5 nM) on DNA replication. During the first 30 min of incubation, ara-C inhibited the initiation of new replicating units of DNA but did not affect the elongation of previously initiated units. A later effect was the reduction of the rate of DNA chain elongation. A model, based on the incorporation of ara-C into nascent DNA, is presented to account for a differential effect of the drug on DNA chain initiation and elongation in mammalian cells.

Cell Line↗