Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chronic perturbations of intracellular deoxyribonucleoside triphosphate (dNTP) pools have been associated with a mutator phenotype and increased mutation rates at several genetic loci. We have examined the specific effects of transient pharmacological purine dNTP pool perturbations on mutations induced at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in a cultured human T-lymphoblast cell line. Incubation of CEM cells with 50 microM 2'-deoxyguanosine for 6 h increased intracellular dGTP levels 43-fold and induced a 40-fold increase in mutation frequency at the HPRT locus. Six-h incubations with 5, 10, and 20 microM 2'-deoxyadenosine increased dATP pools 4.8-, 8-, and 14.5-fold, respectively, with 59-, 34-, and 43-fold increases in HPRT mutant fractions. In contrast, 24-h incubations with hydroxyurea at concentrations which inhibited cell growth to similar extents did not induce HPRT mutations. Sequencing of HPRT complementary DNA derived from mutant cell lines revealed that the mutations induced by transient purine dNTP pool perturbations exhibited no significant misincorporation of the nucleotide in excess or next-nucleotide effect, and were similar in nature and location to spontaneous HPRT mutations. We conclude that mutations caused by transient purine dNTP pool elevations in these dividing cells are most likely induced by inhibition of DNA repair processes.
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PMID:Mutations induced at the hypoxanthine-guanine phosphoribosyltransferase locus of human T-lymphoblasts by perturbations of purine deoxyribonucleoside triphosphate pools. 236 32

Sezary's syndrome is a leukemic proliferation of OKT4+ lymphocytes. Sezary cells were isolated by differential centrifugation and fused to CEM.8azar.C, and HGPRTase-lacking clone of CEM. The hybrid cells were studied for their ability to produce soluble mediators of human monocyte cytotoxicity. The product of a single clone, FtF3, which bore the surface phenotype of Sezary cells, was characterized. Monocyte cytotoxicity-inducing factor (MCF) was found to be stable at pH 2 for 1 hr, unlike IFN-gamma, and was found to be more heat stable as well. Moreover, treatment of MCF with antisera to IFN-gamma, IFN-alpha or a combination of IFN-gamma and IFN-alpha failed to neutralize its biologic activity. MCF binds to matrix gel Red A. MCF eluted from this dye-ligand was found to have an apparent m.w. of 11,500 by gel filtration and 14,700 by SDS-polyacrylamide gel electrophoresis. MCF produced by hybridized Sezary cells appear to be neither IFN-gamma nor an altered molecular form of IFN-gamma, yet is a potent inducer of human monocyte cytotoxicity.
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PMID:Identification of a human monocyte cytotoxicity-inducing factor from T cell hybridomas produced from Sezary's cells. 308 3

In an attempt to elucidate the role of somatic mutation in the development of resistance to cancer chemotherapy, an assay was sought to measure the frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants in human tumors. Based on the same principle as [3H]thymidine/autoradiography, a method was developed to identify cell proliferation using the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd). BrdUrd incorporation into DNA was measured following the immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analog. The human leukemic cell line, CCRF-CEM, was used to investigate the conditions necessary for the stringent selection of HPRT- mutants using 6-thioguanine (6TG). The appropriate 6TG exposure necessary to inhibit BrdUrd incorporation in wild-type cells, while allowing proliferation of spontaneous HPRT- mutants, was greater than or equal to 30 microM 6TG for 72 h (10 microM BrdUrd added 24 h prior to harvest). BrdUrd did not affect the growth of HPRT- mutants in the presence of 6TG. BrdUrd-labeled 6TG-resistant cells were enumerated flow cytometrically using fluorescent microspheres as an internal standard and the nonparametric, Kolmogorov-Smirnov test was used for independent statistical analysis of the subpopulations of fluorescent, 6TG-resistant cells. Evidence that CCRF-CEM cells which incorporated BrdUrd in the presence of 6TG were, in fact, HPRT- mutants was sought. It was demonstrated that spontaneous 6TG-resistant cells from the CCRF-CEM population were reduced by growth in medium containing aminopterin. The mutant frequency in the CCRF-CEM cell line was found to be 4.28 x 10(-5) +/- 2.04 x 10(-5) using the BrdUrd/flow cytometric technique.
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PMID:Flow cytometric enumeration of drug-resistant tumor cells. 316 54

WI-L2 B lymphoblasts deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) excreted amounts of hypoxanthine two to three times larger than CEM T lymphoblasts deficient in HGPRT, despite similar growth rates. ATP consumption occurred at a higher rate in WI-L2 cells than in CEM cells when cultivated in a glucose-free buffer, because of higher RNA synthesis in WI-L2 cells. The introduction of actinomycin D and azaserine resulted in lower hypoxanthine excretion in WI-L2 cells than in CEM cells, not in parallel with changes of the adenylate pool size. When the energy charge was high, de novo purine synthesis was a major determinant for purine excretion. The adenylate pool ratio (AMP/ATP) change caused by the introduction of oligomycin was greater during ATP depletion and recovery in WI-L2 cells than in CEM cells. WI-L2 cells were observed to have AMP deaminase activity three to four times higher than CEM cells. The major component of AMP deaminase in these cells was liver type. The higher rate of RNA synthesis caused greater changes of (AMP/ATP) and required higher AMP deaminase activity for recovery. When the energy charge was low, AMP deaminase was a major determinant for purine excretion.
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PMID:Major determinants of purine excretion from human lymphoblasts. 343 82

6-Thio-3-deazaguanine (TDG), a relatively new purine antimetabolite, exhibits significant antitumor activity against a variety of experimental animal tumor models including C3H mammary adenocarcinoma, Lewis lung carcinoma, adenocarcinoma 755, and leukemias L1210 and P388. However, the drug was ineffective against 3-deazaguanine-resistant L1210 (both in vitro and in vivo) and CEM cells (in vitro). The resistant cells appear to lack HGPRTase activity because the extracts from these cell lines failed to convert hypoxanthine to IMP. These data indicate that TDG needs to be activated by hypoxanthine guanine phosphoribosyltransferase prior to its growth inhibitory effects. Cytotoxicity of TDG was completely reversed by hypoxanthine and inosine. TDG inhibited the synthesis of DNA and RNA equally and effectively, whereas the inhibition of protein synthesis required a prolonged drug exposure and appears to be a consequence of the inhibition of DNA and RNA synthesis. Data from these studies suggest that TDG is an effective antitumor agent, and its spectrum of antitumor activity and mechanism of action appears to be different from that of 3-deazaguanine.
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PMID:Antitumor activity and mechanism of action of 6-thio-3-deazaguanine. 381 77

To study human T cell suppression of immunoglobulin (Ig) synthesis with homogeneous populations of immunoregulatory cells, human suppressor T cell hybridomas were prepared by somatic cell fusion of concanavalin A-activated peripheral blood T cells with hypoxanthine-guanine phosphoribosyltransferase-(HGPRT, EC 2.4.2.8) deficient human leukemic CEM T cells. After selection in hypoxanthine-aminopterin-thymidine (HAT) medium and cloning by limiting cell dilution, two human T cell hybridomas were identified that produced 60 to 80% suppression of in vitro polyclonal immunoglobulin production when cocultured with pokeweed mitogen- (PWM) stimulated peripheral blood lymphocytes. Further, one of the suppressor T cell hybridomas constitutively secreted a soluble suppressor factor(s) (TsF) of m.w. 70,000 to 85,000 daltons, which produced reversible noncytotoxic inhibition of lectin-activated B cell Ig production. In contrast, this TsF did not inhibit lectin- or antigen-induced T cell proliferation, nor did it interfere with the generation or effector function of cytotoxic T cells. Additional studies indicated that this Tsf acts directly on B cells or monocytes rather than indirectly modulating the activity of immunoregulatory T cells. In summary, these studies suggest that techniques of somatic cell fusion may provide a valuable approach to further study human immunoregulatory cell-cell interactions as well as provide a source of sufficient quantities of important lymphokines for further purification and characterization.
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PMID:Production of human suppressor T cell hybridomas. 621 85

The exact source of de novo adenine produced by mammalian cells remain poorly understood, and this prompted the present study. Using a human lymphoblastoid cell line (WI-L2) deficient in adenine phosphoribosyltransferase (EC 2.4.2.7), we have quantitated the rate of adenine synthesis and the relative importance of the phosphorolysis of 5'-methylthioadenosine versus adenosine or 2'-deoxyadenosine in adenine generation. Dividing adenine phosphoribosyltransferase-deficient WI-L2 cells produced adenine at a rate of 0.27 nmol/mg protein/h. This represented approximately 10% of the rate of hypoxanthine production by WI-L2 cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) but was equivalent to the rate of 5'-methylthioadenosine synthesis by human lymphoblastoid CCRF-CEM deficient in 5'-methylthioadenosine, phosphorylase (5'-methylthioadenosine: orthophosphate methylthioribosyltransferase). Up to 97% of adenine, but not hypoxanthine, synthesis was inhibited dose-dependently by the S-adenosylmethionine decarboxylase-inhibitor methylglyoxal bis(guanylhydrazone) and also by spermidine and spermine, but was enhanced by putrescine. The addition of 2-fluoroadenine, a potent competitive inhibitor of methylthioadenosine phosphorylase (Ki = 0.43 microM) to adenine phosphoribosyl-transferase-deficient cells resulted in a progressive accumulation of 5'-methylthioadenosine in the culture medium, and up to an 85% decrease in adenine production at non-toxic concentrations. These results show that de novo adenine synthesis by dividing human cells is considerable, and that 85-97% derives from the cleavage of 5'-methylthioadenosine and hence from polyamine synthesis.
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PMID:Dependence of adenine production upon polyamine synthesis in cultured human lymphoblasts. 679 2

The thiopurines 6-thioguanine (6TG) and 6-mercaptopurine (6MP) are cytotoxic to proliferating cells by a mechanism involving incorporation into DNA via the purine salvage pathway, and resistance to these agents can be conferred by lack of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase. However, human and murine hypoxanthine-guanine phosphoribosyltransferase-deficient leukemia cell lines have been shown to respond to 6TG by growth arrest and differentiation by a mechanism apparently not involving incorporation of 6TG into DNA. If so, leukemia cells resistant to 6MP should still respond to 6TG by growth arrest via an undescribed epigenetic mechanism. To test this, polyclonal 6MP-resistant variants were produced from three human leukemia cell lines, HL-60, U937, and CCRF-CEM. Treatment of both sensitive and resistant cells with 6TG induced growth arrest. The effect of 6TG in the 6MP-sensitive HL-60 and U937 cells was associated with significant loss of viability and DNA fragmentation. In contrast, the 6TG-treated 6MP-resistant cells exhibited a slower decline in viability and no DNA fragmentation. To identify the mechanism by which 6TG may induce growth arrest, tRNA was isolated from 6MP-resistant cells cultured for 48 h with 6TG. 6TG was found to be incorporated into tRNAs normally containing queuine in the anticodon wobble position. These studies may provide a basis for the development of new therapeutic regimens for the treatment of leukemia.
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PMID:6-Thioguanine-induced growth arrest in 6-mercaptopurine-resistant human leukemia cells. 792 70

The induction of mutations at the Hprt locus and minisatellite sequences was studied in V79 cells, peripheral blood lymphocytes (PBL) and lymphoblastoid cells (CCRF-CEM) exposed to gamma rays. In V79 cells the Hprt mutant frequency increased with dose at least up to 6.0 Gy, whereas the number of HPRT mutant lymphocytes increased up to 3 Gy. Clones derived from single irradiated cells were screened for mutations at minisatellite sequences by DNA fingerprint analysis. In V79 cells, a dose-response curve for minisatellite alterations was obtained up to 4.5 Gy. In contrast, very few mutations at minisatellite sequences (2/137) were detected among clones isolated from PBL of two donors irradiated with 1-4 Gy. Similar results were observed in lymphoblastoid CCRF-CEM cells irradiated with 2-3 Gy (4 mutants/180 clones), suggesting that in human lymphoid cells minisatellite DNA is more stable than in other mammalian and human cell lines.
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PMID:Minisatellite and HPRT mutations in V79 and human cells irradiated with gamma rays. 1219 85

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) in cultured cells require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. (3)H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas (3)H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.
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PMID:Determinants of sensitivity of human T-cell leukemia CCRF-CEM cells to immucillin-H. 1827 55


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