Hydrophilic-interaction liquid chromatography-tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV.

Stable isotope dilution mass spectrometric assay for PRPP using enzymatic procedures.

5-Phosphoribosyl-1-pyrophosphate (PRPP) is an important regulator of de novo purine synthesis. A method for the measurement of PRPP in erythrocytes was designed, which is based on the determination of [(13)C(5)]glutamate derived from [(13)C(5)]glutamine following the utilization of PRPP by the action of amidophosphoribosyltransferase. The present study describes a gas chromatographic-mass spectrometric method for determination of [(13)C(5)]glutamate using [(13)C(2)]glutamate as an internal standard. The methods involved purification by anion-exchange chromatography using a BondElut SAX and derivatization with isobutyl chlorocarbonate in water-methanol-pyridine. Quantitation was performed by selected ion monitoring of the protonated molecular ions in the chemical ionization mode. The intra-day reproducibility in the amounts of [(13)C(5)]glutamate determined was in good agreement with the actual amounts added in erythrocytes. A linear relationship was found between the amount of PRPP added and the amount of [(13)C(5)]glutamate formed from [(13)C(5)]glutamine using amidophosphoribosyltransferase.

Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates.

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102+/-11 microM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N(alpha)(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2-d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3-12-fold. These data indicate that LTX induces the most potent inhibition of APRT.

6-Mercaptopurine: cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts.

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of 5-fluorouracil metabolism in human colorectal cancer and colon mucosa.

The metabolism of 5-fluorouracil (5-FU) was studied in biopsy specimens of primary colorectal cancer and healthy colonic mucosa obtained from previously untreated patients immediately after surgical removal. The conversion of 5-FU to anabolites was measured under saturating substrate (5-FU) and cosubstrate concentrations. For all enzymes, the activity was about threefold higher in tumor tissue compared with healthy mucosa of the same patient. The activity of pyrimidine nucleoside phosphorylase with deoxyribose-1-phosphate (dRib-1-P) was about tenfold higher (about 130 and 1200 nmol/hr/mg protein in tumors) than with ribose-1-phosphate (Rib-1-P), both in tumor and mucosa. Synthesis of the active nucleotides (5-fluoro-uridine-5'-monophosphate [FUMP] and 5-fluoro-2'-deoxyuridine-5'-monophosphate [FdUMP]) was studied by adding physiologic concentrations of adenosine triphosphate (ATP) to the reaction mixture; the rate of FdUMP synthesis was 50% of that of FUMP (about 4 and 7 nmol/hr/mg protein in tumors). Direct synthesis of FUMP from 5-FU in the presence of 5-phosphoribosyl-1-pyrophosphate (PRPP) was about 2 nmol/hr/mg protein. With the natural substrate for this reaction, orotic acid, the activity was about 14-fold higher. To obtain insight into the recruitment of precursors for these cosubstrates, the authors also tested the enzyme activity of pyrimidine nucleoside phosphorylase with inosine and ribose-5-phosphate (Rib-5-P, as precursors for Rib-1-P) and deoxyinosine (as a precursor for dRib-1-P); enzyme activities were approximately 7%, 7%, and 3%, respectively, of that with the normal substrates, both in tumors and mucosa. However, when ATP and Rib-5-P were combined, the synthesis of FUMP was about 70% of that with PRPP, but only in tumors. In normal tissues no activity was detectable. These data suggest a preference of colon tumor over colon mucosa for the conversion of 5-FU to active nucleotides by a direct pathway; a selective antitumor effect of 5-FU may be related to this difference.

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.

Effect of L-histidinol on the metabolism of 5-fluorouracil in the BALB/c x DBA/8 F1 murine tumor system.

L-Histidinol, a structural analogue of histidine, which transiently inhibits proliferation, can protect cells from the toxic effects of proliferation-dependent chemotherapeutic agents such as 5-fluorouracil (FUra). In the BALB/c x DBA/8 F1 (hereafter called CD8F1) murine tumor system, L-histidinol protected mice from FUra-induced leukopenia, weight loss, and mortality; however, the therapeutic index was not improved since L-histidinol also protected the tumor against the toxic effects of FUra. In order to understand the mechanism of this protection, we examined the effects of L-histidinol on the metabolism of FUra. Results indicate that L-histidinol had no effect on the phosphoribosyl pyrophosphate levels in tumor, the activation of FUra to nucleotides or levels of free 5-fluorodeoxyuridine monophosphate in either tumor or bone marrow. L-Histidinol (7 mg/mouse, every 2 h for 5 doses) reduced RNA and DNA synthesis, as measured by 32P incorporation in vivo, by approximately one-half in tumor, and by 70% in bone marrow. This in turn resulted in reduced incorporation of FUra into RNA in both tumor and bone marrow. At 2 h, 4 h, and 24 h after FUra administration the level of FUra in RNA was 24-37% less in both tumor and bone marrow of mice that received L-histidinol with FUra. Using 32P as a monitor of overall RNA synthesis, the [3H]FUra/32P ratio remained unchanged, suggesting that the reduction of FUra incorporation into RNA was due to decreased RNA synthesis rather than a decrease in the number of FUra molecules per RNA chain. In contrast, L-histidinol had no effect on the in vivo inhibition of thymidylate synthetase by 5-fluorodeoxyuridine monophosphate as measured by the incorporation of [3H]-2'-deoxyuridine into DNA or on the percentages of thymidylate synthetase in the free versus 5-fluorodeoxyuridine monophosphate-bound state. We conclude that L-histidinol reduces FUra toxicity by reducing FUra incorporation into RNA. Since the major mechanism of action in the CD8F1 breast tumor system is the incorporation of FUra into RNA, the reduction in toxicity and antitumor activity observed when L-histidinol is combined with FUra is consistent with the observed reduction in tumor and bone marrow RNA containing incorporated FUra residues.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts.

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

Modified phosphoribosylpyrophosphate (PRPP) radioenzymatic assay: increased sensitivity, technical simplification and new applications.

Phosphoribosylpyrophosphate in amounts as low as 25 pmol could be reliably and economically measured with a CO2-releasing radioenzymatic assay when appropriate technical modifications were introduced. The concentration of commercially available phosphoribosylpyrophosphate used for reference standards was ascertained by a method based on the utilization of phosphoribosylpyrophosphate by hypoxanthine catalyzed by hypoxanthine phosphoribosyltransferase from red blood cell lysates. The addition of inorganic phosphate increased intracellular phosphoribosylpyrophosphate levels in HL-60 cell lysates and can be used to amplify low levels of phosphoribosylpyrophosphate. This phosphoribosylpyrophosphate assay amplified by inorganic phosphate has been developed to assay perturbations in the purine biosynthetic nucleotide pathway in response to various chemotherapeutic agents, such as anti-folates, or as a result of folate deficiency.

Effects of uridine diphosphoglucose (UDPG) infusion on 5-phosphoribosyl pyrophosphate (PRPP) levels of mouse tissues.

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate.

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.

Cellular pharmacokinetics of mercaptopurine in human neoplastic cells and cell lines.

The accumulation, metabolism, and retention of mercaptopurine (MP) was studied in four human neoplastic cell lines (three acute leukemia lines Molt-4, CCRF-CEM, and HL-60; and one Burkitt's lymphoma line, Wilson), each of which was sensitive to MP. Two cell lines resistant to MP (WilsonR and CCRF-CEMR) were also studied. The cell lines were incubated for 3 h in 10 microM [14C]MP and then placed in drug-free media for an additional 3 h. Cell samples were obtained at regular intervals, and the intracellular MP metabolites were measured in the acid-soluble fractions by anion-exchange high-pressure liquid chromatography. MP accumulated progressively within cells during the 3-h drug exposure period and declined rapidly when the cells were placed in drug-free media. Over 80% of the intracellular MP was present in the form of three nucleotide metabolites, MP ribose monophosphate, thioxanthosine monophosphate, and thioguanosine monophosphate. MP ribose monophosphate was found in greatest amount, accounting for 59-85% of the intracellular metabolite pool. Thioxanthosine monophosphate thioguanosine monophosphate were detected in lesser amounts. Study of leukemic cells obtained from patients demonstrated a similar pattern of MP accumulation, metabolism, and retention, although the overall amounts of the various metabolites formed were less. In contrast, there was essentially no MP nucleotide metabolite formation in the two MP-resistant cell lines. A more complete understanding of the cellular pharmacokinetics of MP in human neoplastic cells is likely to lead to a more rational use of the drug in the clinical setting.

Enhancement by uridine of the anabolism of 5-fluorouracil in mouse T-lymphoma (S-49) cells.

Uridine enhances the growth inhibition due to 5-fluorouracil (FUra) in a cultured mouse T-cell lymphoma (S-49). Using colony formation assays we found that cytotoxicity produced by 24-h continuous exposure to FUra (0.5 to 3.5 microM) was increased more than two-fold by simultaneous exposure to 10 microM uridine. Studies were undertaken to explain the mechanism by which uridine enhanced the cytotoxicity of FUra in S-49 cells. Uridine (10 microM) increased by about 50% both the anabolism of 1.0 microM [3H]FUra to acid-soluble metabolites and the incorporation of 1.0 microM [3H]-FUra into RNA. However, the incorporation of 1.0 microM [3H]FUra into these fractions was less than that seen with 2.4 microM [3H]-FUra, a dose which was equitoxic to 1.0 microM [3H]FUra plus 10 microM uridine. High-pressure liquid chromatography analysis of the acid-soluble metabolites of FUra did not show any selective change in specific FUra nucleotides, which could explain the increased cytotoxicity associated with 10 microM uridine. In addition, 5-fluoro-2'-deoxyuridine monophosphate levels and the amount of [3H]FUra which was incorporated into the alkali-stable, acid-insoluble fraction were not increased by uridine. Uridine (10 microM) inhibited de novo pyrimidine biosynthesis by 70%, while 5-phosphoribosyl-1-pyrophosphate levels were unchanged. Presumably, the inhibition of de novo pyrimidine biosynthesis decreased orotic acid levels and allowed more FUra to be anabolized to 5-fluorouridine monophosphate via orotate phosphoribosyl transferase. Furthermore, 2.4 microM FUra inhibited the incorporation of [3H]deoxyguanosine into DNA by 50% after 24 h of incubation. In contrast, 1.0 microM FUra plus 10 microM uridine did not inhibit the incorporation of [3H]deoxyguanosine into DNA. The data suggested that there was a qualitative difference in the mechanism by which 1.0 microM FUra plus 10 microM uridine killed S-49 cells as compared to 2.4 microM FUra alone, and that the enhancement by uridine of the cytotoxicity of FUra was due, in part, to the increased anabolism of FUra to ribonucleotides.

Partial phenotypic correction of human Lesch-Nyhan (hypoxanthine-guanine phosphoribosyltransferase-deficient) lymphoblasts with a transmissible retroviral vector.

A human Lesch-Nyhan (hereditary, severe hypoxanthine-guanine phosphoribosyltransferase (HPR transferase) deficiency) B-lymphoblast line was infected with an amphotropic retroviral vector containing human HPR transferase cDNA under transcriptional control of viral long terminal repeat sequences. Of 17 clones isolated, 12 integration groups were defined by analysis of restriction enzyme digests of their genomic DNA with HPR transferase and viral long terminal repeat probes. These groups had HPR transferase activity restored to levels of 4 to 23% of normal values. Aberrant metabolic parameters associated with severe deficiency of HPR transferase activity, i.e. elevated rates of purine excretion, increased accumulation of hypoxanthine, elevated 5-phosphoribosyl-1-pyrophosphate contents, altered nucleoside triphosphate pools, resistance to toxic effects of 6-thioguanine, were partially to nearly completely corrected; the degree of correction generally corresponded to the degree of restoration of HPR transferase activity. The integration of the HPR transferase gene was found to be variably stable during 9 months of culture of the virally transformed lymphoblasts under nonselective conditions. The HPR transferase gene-infected lines reverted to resistance to 20 microM 6-thioguanine, i.e. severe HPR transferase deficiency, at frequencies of 10(-6) to in excess of 10(-5) per generation. The reversions were accompanied by either a loss or rearrangement of the integrated HPR transferase sequences or by retention of the sequences in an unaltered form.

A simplified method for estimating 5-phosphoribosyl 1-pyrophosphate in mouse liver and spleen.

A simplified method for the estimation of 5-phosphoribosyl 1-pyrophosphate (PRPP) in mouse liver and spleen is described. The method uses the enzymic conversion of [8-14C] hypoxanthine to [8-14C] inosine 5'-monophosphate in the presence of PRPP and has the advantage over previously published methods in that the enzyme used in this assay, hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase), is present in the tissue being analyzed and does not require preparation. In addition, each assay uses as its internal standard PRPP, thus overcoming the problem of the chemical instability inherent in this molecule. The two-minute heat treatment used in the method was found to destroy most interfering enzymes and resulted in average recovery of PRPP of 56% and 85% for the liver and spleen, respectively. The counts obtained in the assay were about ten-fold above background (80 c.p.m) and allowed accurate measurement of the phosphoribose sugar. The levels of PRPP (+/- standard deviation) found for liver and spleen in twelve (12) separate Balb/C male mice were 6.3 +/- 1.8 and 10.8 +/- 5.0 nmoles/g wet weight tissue, respectively, and agree with the tissue concentrations already reported for this metabolite. A further advantage of this assay is that it gives an index of the HG-PRTase activity in the tissues.