Randall C. Willis

A filtration assay specific for the determination of small quantities of l-glutamine

The l-glutamine-binding protein released from Escherichia coli by osmotic shock was used to determine the acid-extractable glutamine content of biological samples. With saturating concentrations of l[14C]glutamine, nonradioactive glutamine from samples competes with l[14C]glutamine in the formation of binding protein-ligand complex to give diminution of the protein-bound radioactivity which is collected on nitrocellulose membranes. The extent of the diminution of bound radioactivity by isotope dilution permits calculation of glutamine content of the sample. With total assay volumes as small as 50 μl, as little as 20 pmol can be measured with an error of <15% among triplicate samples. Of the naturally occurring amino acids only l-glutamine is measured. The assay is optimal over a pH range of 6.5–7.5 in 0.1 m potassium phosphate buffer and is unaffected by salt concentrations of 1 m.

Increases in Purine Excretion and Rate of Synthesis by Drugs Inhibiting IMP Dehydrogenase or Adenylosuccinate Synthetase Activities

Hershfield reported an assay system devoid of hypoxanthine for evaluating the rate of purine synthesis de novo based on rates of [14C]-formate incorporation into intracellular purine components of lymphoblasts and into purines excreted into the culture medium. Using this system normal and in vitro selected, biochemically-characterized HPRT* deficient lines were found to have similar rates of total purine synthesis; however, these cell lines differed markedly in the excretion of newly-formed purine into the medium (1,2).

Nucleoside and nucleotide modulation of genetic expression―A new approach to chemotherapy

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I). Examples of nucleosides and nucleotides which appear to exert their therapeutic effects via G protein control of cellular proliferation resulting in differentiation are tiazofurin, selenazofurin, and 8-chloro-cAMP which have been synthesized and studied in our laboratories. The clinical application of these nucleosides in cancer treatment is presently underway and offers a viable alternative to chemotherapy with highly cytotoxic agents. The use of these derivatives result in down-regulation of the G protein regulatory pathways responsible for rapid cell division. Alternatively, a series of guanosine analogs prepared in our laboratories, 8-bromoguanosine, 8-mercaptoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, all activate various aspects of the immune response by up-regulation of G protein regulatory pathways in various lymphocyte derived cells. Guanosine-like nucleosides which function in this manner could have major clinical application as antitumor, antiviral and antimetastatic agents providing the desired specificity can be achieved. Specific immune enhancement of the aged might be an attainable goal if suitable orally active guanosine derivatives with high specificity can be achieved. The G protein regulatory pathways for modulation of genetic expression in specific cell types provide a major modern approach to new chemotherapeutic agents.

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.

Zinc uptake by cultured human lymphoblasts

At physiological plasma concentrations (12–20 μM), zinc uptake by cultured human B lymphoblasts is biphasic with an early, rapid, saturable phase, followed by a slower, unsaturable phase. The saturable component is specific for zinc and cadmium, with aKm of 1–2 μM and aVmax of 3.0 pmol/min/106 cells. This early phase does not require metabolic energy derived directly from ATP or the presence of sulfhydryl groups. At higher zinc concentrations (>30 μM), zinc uptake is more complex, although biphasic kinetics are also evident.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Potent and specific inhibitors of mammalian phosphoribosylpyrophosphate (PRPP) synthetase

The monophosphates of the exocyclic amino ribonucleosides, 4-amino- and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine, are potent and specific inhibitors of human erythrocyte and B-lymphoblast PRPP synthetase. The inhibition by MRPP monophosphate is competitive (Ki = 35 microM with the PRPP synthetase cofactor, Pi (Km = 2 mM). The nucleosides are phosphorylated to the active metabolite by adenosine kinase and these nucleoside monophosphates accumulate in the cell. beta-ARPP is a substrate, albeit poor, for adenosine deaminase and solutions of the beta-anomer of this nucleoside and its monophosphate anomerize over time to give alpha- and beta-mixtures. beta-MRPP is more resistant to adenosine deaminase and anomerization of the nucleoside and its monophosphate is negligible. The effect of treatment of cells with the nucleosides is a time-dependent and nearly universal reduction in the nucleotide content which appears to result from a reduction in the availability of PRPP for dependent metabolic pathways. In studies with the WI-L2 lymphoblasts, some of these pathways, de novo and salvage (hypoxanthine and guanine) synthesis of purine nucleotides, are more sensitive to a restriction of PRPP availability than others, i.e. de novo pyrimidine synthesis. The nucleosides have shown promise as therapeutic agents in a mouse leukemia evaluation system but may also have future use in unravelling the complex regulation of PRPP synthetase and the dependent nucleotide synthesis pathways.

The mechanism of inhibition and “reversal” of mitogen-induced lymphocyte activation in a model of purine-nucleoside phosphorylase deficiency

Purine-nucleoside phosphorylase (PNP) is a purine degradative enzyme that catalyzes the phosphorolysis of (deoxy) inosine or (deoxy) guanosine to their respective bases and (deoxy) ribose 1-phosphate. A severe T-cell immune deficiency syndrome with hypouricemia is associated with impaired PNP function. To study the biochemical basis for this syndrome we created an in vitro model of PNP deficiency in mitogen (phytohemagglutinin)-stimulated normal human peripheral blood lymphocytes using guanosine to competitively inhibit deoxyguanosine phosphorolysis. Guanosine-induced guanine toxicity was reversed by adenine. Under these conditions, deoxyguanosine (5-45 microM) diminished mitogen stimulation to 30% of control while increasing the deoxyguanosine triphosphate pool (dGTP) by over 20-fold. Deoxycytidine reversed deoxyguanosine toxicity with a diminution of dGTP accumulation, but no significant change in the deoxycytidine triphosphate pool. Thymidine reversed the deoxyguanosine toxicity, repleted the thymidine triphosphate (dTTP) pool, and caused an even further increase in the accumulation of dGTP. These data support a model of lymphotoxicity in PNP deficiency based on dGTP accumulation with inhibition of ribonucleotide reductase and depletion of the thymidine triphosphate pool. Thymidine triphosphate depletion is reversed by either deoxycytidine or thymidine; however, the former diminishes dGTP accumulation (probably by competition for phosphorylation) and the latter potentiates dGTP accumulation (probably through feedback augmentation of guanosine diphosphate (GDP) reduction by ribonucleotide reductase secondary to an increased dTTP pool).

Interactions between Energy Metabolism and Adenine Nucleotide Metabolism in Human Lymphoblasts

Adenine nucleotides play an important role in the transfer of chemical energy for metabolic processes. In addition, adenine nucleotide degradation can be a major source of purine bases formed during certain kinds of metabolic stress. These two properties of adenine nucleotides may be related. Fructose and 2-deoxyglucose can produce elevated levels of purines in vivo (1), in perfused organs (2), and in cultured cells (3,4). The mechanism of nucleotide degradation caused by fructose or 2-deoxyglucose involves the utilization of ATP to form a slowly metabolized hexose phosphate which accumulates and decreases the intracellular concentration of inorganic phosphate (1–4). AMP deaminase, which is normally inhibited by phosphate, becomes more active when the phosphate concentration decreases and adenine nucleotides are broken down by the reactions: AMP → IMP + NH3; IMP → inosine + PO 4 2- . There also exists another pathway for adenine nucleotide degradation catalyzed by the enzymes purine 5′-nucleotidase and adenosine deaminase: AMP → adenosine + PO 4 2- ; adenosine → inosine + NH3.

834 STABILITY OF GENE TRANSFER IN LESCH NYHAN CELLS

One theoretical criterion for effective and efficient gene therapy for human genetic disease is stability of a functional gene transferred into defective cells. We are studying the deficiency of hypoxanthine guanine phosphoribosyl transferase (HPRT) in the Lesch Nyhan disease as a model system for the development of methods for gene therapy, and have used retroviral vectors to introduce a functional wild-type human HPRT gene into cultured cells derived from Lesch Nyhan patients. Recipient cells express the foreign gene to a variable extent and demonstrate partial phenotypic reversion of several parameters of aberrant purine biosynthesis characteristic of enzyme-deficient cells. Cells grown without selection and revertants to the HPRT-negative phenotype show the continued presence, loss or rearrangement of the foreign gene and of sequences derived from the vector. The introduction of the HPRT gene into random cellular sites by means of some retroviral vectors therefore clearly leads to variability not only in the degree of gene expression but also in its stability, and we conclude that in the presence or absence of selection pressure, the expression and stability of the newly introduced gene depend on the site of integration into the host cell genome.