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Biomedical subjects

D S Shewach

Publications and source records attributed to D S Shewach.

At least 19 recordsLinked to original sources
PubMed1992

Inhibition of adenosine deaminase by azapurine ribonucleosides.

We have synthesized several 8-azapurine nucleosides as inhibitors of adenosine deaminase. The presence of a nitrogen on the imidazole ring decreased the Ki value for nebularine by 100-fold but did not lower the Ki value for coformycin. Evaluation of these compounds in a MOLT-4 growth assay revealed that 2-azacoformycin was as effective as 2'-deoxycoformycin in potentiating growth inhibition by 2'-deoxyadenosine. The azapurine nucleosides merit further study as antitumor agents.

Adenosine Deaminase Inhibitors↗
PubMed1992

Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene.

We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.

Base Sequence↗
PubMed1992

Decrease in TTP pools mediated by 5-bromo-2'-deoxyuridine exposure in a human glioblastoma cell line.

The antitumor and radiosensitizing properties of 5-bromo-2'-deoxyuridine (BUdR) appear to be due, in part, to its incorporation into cellular DNA. To optimize conditions for incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdUMP) into DNA, we investigated the metabolism of BUdR to its DNA precursor form, the 5'-triphosphate BrdUTP, in the U251 human glioblastoma cell line. The results demonstrated that BrdUTP accumulated rapidly in this cell line, achieving steady-state values within 2 hr of drug addition. The level of BrdUTP accumulation was proportional to the amount of exogenous BUdR up to a concentration of 100 microM, without apparent saturation. Exposure of glioblastoma cells to BUdR was associated with substantial selective decreases in both the cellular dCTP and TTP pools, the extent of which was dependent on the exogenous BUdR concentration. In the absence of exogenous BUdR, BrdUTP was eliminated rapidly from cells with an initial half-life of approximately 15 min. As the cellular BrdUTP level declined, the dCTP and TTP levels increased to control values. Incorporation of BrdUMP into DNA appeared linear with time as long as the cellular BrdUTP level remained constant. This incorporation was not enhanced by the addition of 5-fluoro-2'-deoxyuridine (FUdR), a potent inhibitor of thymidylate synthetase, which at a concentration of 10 nM had no effect on TTP pools in this cell line. Thus, the decrease in cellular TTP pools mediated by BrdUTP allows the halogenated pyrimidine to enhance its own incorporation into DNA.

Antineoplastic Agents↗
PubMed1992

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy.

The active 5'-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5'-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5'-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.

Adult↗
PubMed1992

Quantitation of deoxyribonucleoside 5'-triphosphates by a sequential boronate and anion-exchange high-pressure liquid chromatographic procedure.

A rapid method for the quantitative determination of cellular deoxyribonucleoside 5'-triphosphates is described. Cell extracts are first separated by boronate chromatography at pH 8.9, which removes 99% of the ribonucleoside triphosphate (rNTPs) from the deoxyribonucleoside triphosphates (dNTPs). The resulting dNTP fraction is analyzed by gradient high-pressure liquid chromatography utilizing a strong anion-exchange column, which can separate minor rNTP peaks from the corresponding dNTPs. This sequential procedure, which requires less than 1 h per sample for both chromatographic steps, results in the quantitative recovery of greater than 98% of the dNTPs from cell extracts. Nucleotide analogs, such as 1-beta-D-arabinofuranosylcytosine-5'-triphosphate and 5-bromo-2'-deoxyuridine-5'-triphosphate, can also be quantitated efficiently by this method.

Boronic Acids↗
PubMed1992

Nucleotide specificity of human deoxycytidine kinase.

The ability of deoxycytidine kinase (dCK) to phosphorylate 2'-deoxycytidine (dCyd) and its analogs in the presence of eight nucleoside triphosphates (NTPs), simulating the cellular milieu, was investigated. Using highly purified dCK from MOLT-4 T lymphoblasts, Km and Vmax values were determined for the phosphorylation of dCyd in the presence of cellular concentrations of the eight endogenous NTPs. The results demonstrated that the efficiency of dCyd phosphorylation was greatest in the presence of all eight nucleotides, relative to ATP alone, according to relative Vmax/Km values. UTP was a better phosphate donor than ATP but was less efficient than the NTP mixture. The greater efficacy of the NTP mixture, compared with ATP alone, was due in large part to the presence of UTP, although the results suggested that the presence of other nucleotide(s) also enhanced dCyd phosphorylation. Previous results demonstrated that dCTP was a potent competitive or noncompetitive (with respect to dCyd) inhibitor of dCK, with a Ki value of approximately 1 microM. In contrast, the results presented here demonstrated that, in the presence of either the NTP mixture or UTP, inhibition of dCK was uncompetitive with respect to dCyd, with a Ki value of approximately 60 microM. Furthermore, the results demonstrated that the clinically relevant nucleoside analogs 1-beta-D-arabinofuranosylcytosine, 2',2'-difluoro-2'-deoxycytidine (dFdC), and 9-beta-D-arabinofuranosyl-2-fluoroadenine also preferred UTP or the NTP mixture, compared with ATP alone, as a phosphate donor. Of the three nucleoside analogs tested, dFdC was the most efficient dCK substrate. These data indicate that the preferred phosphate donor for dCK is UTP or a combination of UTP and another nucleotide. Furthermore, the dCTP concentration in intact cells, which is typically 10-20 microM, is not sufficient to cause substantial inhibition of dCK, due to the presence of UTP. Strategies to increase cellular dCK activity should focus on optimizing UTP concentrations.

Adenosine Triphosphate↗
PubMed1991

Cloning and expression of human deoxycytidine kinase cDNA.

Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.

Amino Acid Sequence↗
PubMed1991

The effects of leucovorin and dipyridamole on fluoropyrimidine-induced radiosensitization.

The biomodulators leucovorin and dipyridamole potentiate the cytotoxicity of 5-fluorodeoxyuridine (FdUrd) and 5-fluorouracil (5-FU), respectively. It was hypothesized that these biomodulators would increase fluoropyrimidine-mediated radiosensitization. This hypothesis was tested using cultured HT29 human colon cancer cells. As was predicted, leucovorin increased both FdUrd-mediated cytotoxicity and radiosensitization. The increase in radiation sensitivity was associated with a decrease in the repair of radiation-induced DNA double strand breaks (DSB's). Dipyridamole potentiated the cytotoxicity produced by 5-FU. However, dipyridamole appeared to confer slight protection from irradiation, thus decreasing 5-FU-mediated radiosensitization. This demonstrates that the simple fact that a biomodulator can increase fluoropyrimidine-induced cytotoxicity does not guarantee a corresponding increase in radiation sensitivity. Clinical trials combining fluoropyrimidines and their biomodulators will need to take these potentially complex interactions into account.

Adenocarcinoma↗
PubMed1991

The effect of fluorodeoxyuridine on sublethal damage repair in human colon cancer cells.

Although 5-fluoro-2'-deoxyuridine (FdUrd) has been combined with hyperfractionated radiation therapy in clinical trials, the optimal method of delivering radiation therapy is not yet known. To determine the importance of the time interval between fractions on the survival of tumor cells exposed to FdUrd, we studied the effect of FdUrd on sublethal damage repair in HT29 human colon cancer cells in culture. Cells were exposed to clinically achievable concentrations of FdUrd (10-100 nM) for 14 hr followed by either single dose (8-12 Gy) or split dose (4-6 Gy x 2) external cobalt irradiation. The interval between radiation fractions was varied from 0.5 to 6 hr. FdUrd impaired sublethal damage repair in a dose dependent fashion. FdUrd had no effect on the induction of DNA double strand breaks (DSB's), but significantly reduced the rate of the repair of DNA DSB's. Exposure to 100 nM FdUrd decreased intracellular TTP pools but elevated dATP pools. These findings suggest that FdUrd may decrease sublethal damage repair by perturbing nucleotide triphosphate pools, which leads to a decrease in the ability of the cell to repair DNA DSB's. Furthermore, they suggest that hyperfractionated irradiation will be superior to once daily treatment when combined with regional delivery of FdUrd.

Cobalt Radioisotopes↗
PubMed1990

Fluorodeoxyuridine-induced radiosensitization and inhibition of DNA double strand break repair in human colon cancer cells.

The halogenated pyrimidine, fluorodeoxyuridine (FdUrd), has been used in combination with radiation for the treatment of human neoplasms. In an attempt to improve the clinical use of this combination, FdUrd-radiation interactions were studied in vitro using human HT29 colon cancer cells. It was found that FdUrd produced radiosensitization at clinically achievable (1-100 nM) concentrations. Sensitization depended critically on the timing of exposure. When cells were irradiated after a 12-hr exposure to 100 nM FdUrd, marked sensitization was produced (mean inactivation dose (MID) = 2.01 +/- 0.01, compared to control of 4.35 +/- 0.16, p less than .01). No radiosensitization occurred when cells were irradiated 4 hr prior to incubation (MID = 3.95 +/- 0.05, p greater than 0.4). Radiosensitization appeared to result from an inhibition of thymidylate synthase since concentrations of FdUrd which produced radiosensitization depleted intracellular TTP pools and blocked the incorporation of deoxyuridine into DNA. Furthermore, radiosensitization was completely inhibited by co-incubation with thymidine. FdUrd also decreased the repair, but not the formation, of radiation-induced DNA double strand breaks (DSB's). These data are consistent with the hypothesis that FdUrd produces radiosensitization by depleting thymidine pools which leads to a decreased rate of DNA DSB repair. Furthermore, they suggest that in clinical trials FdUrd should be infused at least 8 hr before irradiation.

Cell Survival↗
PubMed1989

Differential metabolism of 9-beta-D-arabinofuranosylguanine in human leukemic cells.

9-beta-D-Arabinofuranosylguanine (araG) is a nucleoside analogue that elicits cytotoxicity through the intracellular accumulation of its 5'-triphosphate, araGTP, araG is selectively toxic to cultured T-lymphoblasts due to their ability to accumulate higher levels of the cytotoxic metabolite, araGTP, relative to B- and null lymphoblastoid cells. In an effort to determine whether this selectivity may occur in leukemic cells in vivo, we have investigated the metabolism of araG in MOLT-4 T-lymphoblasts. MGL-8 B-lymphoblasts, HL-60 promyelocytes, and HUT-102 mature T-cells and compared it to that in freshly isolated leukemic cells from patients. MOLT-4 T-lymphoblasts were 50- to 380-fold more sensitive to growth inhibition with araG and accumulated 80-fold higher levels of araGTP than any of the other cell lines studied. Incubation of peripheral blood from patients with leukemia with araG for 4 h demonstrated that T-acute lymphocytic leukemia cells accumulated significantly higher median levels of araGTP than did acute myelogenous leukemia or chronic lymphocytic leukemia cells (187 versus 72 and 31 pmol of araGTP per 10(7) cells, respectively), araGTP accumulation was not dependent on the rate of degradation of araG during the incubation. In contrast, araG did not exhibit similar selective growth inhibition, nor did the accumulation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the freshly isolated leukemic cells differ significantly among T-acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-T-, non-B-cell acute lymphocytic leukemia cells. These results demonstrate that the selective metabolism of araG observed in cultured cell lines was representative of the metabolism in freshly isolated leukemic cells. Furthermore, degradation of araG did not limit the accumulation of araGTP in the leukemic cells. These results indicate that araG may be valuable as a selectively acting chemotherapeutic agent in T-lymphoblastic malignancies.

Arabinofuranosylcytosine Triphosphate↗
PubMed1989

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase.

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.

Adenosine Triphosphate↗
PubMed1989

Human T-lymphoblast deoxycytidine kinase: purification and properties.

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.

Cell Line↗
PubMed1986

Human adenine phosphoribosyltransferase. Complete amino acid sequence of the erythrocyte enzyme.

We defined the amino acid sequence of adenine phosphoribosyltransferase isolated from human erythrocytes. Peptide fragments formed by cleavage at arginine, lysine, glutamic acid, and methionine were purified by high pressure liquid chromatography and sequenced by manual Edman degradation. The complete primary structure of human adenine phosphoribosyltransferase was established by sequence analysis of 19 peptide fragments. Presumed homology between the human and rodent enzymes was used to order fragments that had inadequate overlapping sequences. The enzyme has 179 residues with a calculated subunit molecular weight of 19,481. Mass spectrometry indicated that the NH2-terminal residue is acetylated. Human adenine phosphoribosyltransferase has sequence homology with xanthine-guanine phosphoribosyltransferase from Escherichia coli in 110-amino acid region encompassing the NH2-terminal section of the enzyme.

Adenine Phosphoribosyltransferase↗
PubMed1986

Cellular retention of 9-beta-D-arabinofuranosyladenine 5'-triphosphate and the pattern of recovery of DNA synthesis in Chinese hamster ovary cells.

The factors associated with the recovery of DNA synthesis in Chinese hamster ovary cells after inhibition with 9-beta-D-arabinofuranosyladenine have been investigated. Maintenance of cellular concentrations of the active metabolite 9-beta-D-arabinofuranosyladenine 5'-triphosphate was dependent upon a constant exogenous source of 9-beta-D-arabinofuranosyladenine, without which the triphosphate rapidly degraded. DNA synthesis was inhibited maximally at cellular concentrations of 9-beta-D-arabinofuranosyladenine 5'-triphosphate in excess of 100 microM, but it recovered substantially as the triphosphate concentrations declined below 30 microM. Autoradiographic studies indicated that 9-beta-D-arabinofuranosyladenine had a synchronizing effect on the cells and that recovery of DNA synthesis occurred initially at rates much reduced from those seen in uninhibited cells. A greater portion of an inhibited population recovered DNA synthesis than was able to retain clonogenic potential. These results are consistent with a mechanism of toxicity that postulates recovery of DNA synthesis as a requisite for the loss of viability.

Animals↗
PubMed1986

Potentiation of 2'-deoxyguanosine cytotoxicity by a novel inhibitor of purine nucleoside phosphorylase, 8-amino-9-benzylguanine.

We have synthesized and evaluated a series of 9-substituted analogues of 8-aminoguanine, a known inhibitor of human purine nucleoside phosphorylase (PNP) activity. The ability of these agents to inhibit PNP has been investigated. All compounds were found to act as competitive (with inosine) inhibitors of PNP, with Ki values ranging from 0.2 to 290 microM. The most potent of these analogues, 8-amino-9-benzylguanine; exhibited a Ki value that was 4-fold lower than that determined for the parent base, 8-aminoguanine. As a metabolically stable compound in human blood, 8-amino-9-benzylguanine was more effective than 8-aminoguanine at potentiating the toxicity of 2'-deoxyguanosine to MOLT-4 T-lymphoblasts in culture. 8-Amino-9-benzylguanine is the most potent base or nucleoside inhibitor of human PNP reported to date, and it is a promising lead compound in the development of more effective PNP inhibitors.

Antimetabolites, Antineoplastic↗