guanosine-triphosphate and Leukemia

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.

Topics: Animals; Brain Neoplasms; Cafe-au-Lait Spots; Cell Transformation, Neoplastic; Female; Genes, Dominant; Genes, Neurofibromatosis 1; Glioma; Guanosine Triphosphate; Humans; Hypertension; Learning Disabilities; Leukemia; Male; Mice; Mice, Knockout; Neurofibroma; Neurofibromatosis 1; Neurofibromin 1; Protein Structure, Tertiary; ras Proteins; Rhabdomyosarcoma; Scoliosis

The purpose of this review is to provide an overview of the literature linking Ras signaling pathways and leukemia and to discuss the biologic and potential therapeutic implications of these observations. A search of MEDLINE from 1966 to October 1998 was performed.. A wealth of data has been published on the role of Ras pathways in cancer. To be biologically active, Ras must move from the cytoplasm to the plasma membrane. Importantly, a posttranslational modification--addition of a farnesyl group to the Ras C-terminal cysteine--is a requisite for membrane localization of Ras. Farnesylation of Ras is catalyzed by an enzyme that is designated farnesyltransferase. Recently, several compounds have been developed that can inhibit farnesylation. Preclinical studies indicate that these molecules can suppress transformation and tumor growth in vitro and in animal models, with little toxicity to normal cells.. An increasing body of data suggests that disruption of Ras signaling pathways, either directly through mutations or indirectly through other genetic aberrations, is important in the pathogenesis of a wide variety of cancers. Molecules such as farnesyl transferase inhibitors that interfere with the function of Ras may be exploitable in leukemia (as well as in solid tumors) as novel antitumor agents.

Topics: Alkyl and Aryl Transferases; Animals; Antineoplastic Agents; Cell Membrane; Farnesyltranstransferase; Genes, ras; Genetic Therapy; Guanosine Triphosphate; Humans; Leukemia; Phosphatidylinositol 3-Kinases; ras Proteins; Signal Transduction

As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.

Topics: Alkyl and Aryl Transferases; Animals; Antigen Presentation; Antineoplastic Agents; Cell Adhesion Molecules; Cell Transformation, Neoplastic; Cytokines; Drug Design; Endopeptidases; Enzyme Activation; Farnesyltranstransferase; Fungal Proteins; Fusion Proteins, bcr-abl; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Immune System; Immunotherapy; Leukemia; Mice; Models, Biological; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neurofibromin 1; Oligonucleotides, Antisense; Proteins; Proto-Oncogene Proteins p21(ras); Reoviridae Infections; Repressor Proteins; Signal Transduction; SOS1 Protein; T-Lymphocyte Subsets

Topics: Antimetabolites, Antineoplastic; Deoxyguanine Nucleotides; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Neoplasms; Purines; Ribavirin; Signal Transduction

Topics: Animals; Antineoplastic Agents; Antiviral Agents; Cell Differentiation; Cell Division; Guanosine Triphosphate; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Leukemia

Recently, screens for mediators of resistance to FLT3 and ABL kinase inhibitors in leukemia resulted in the discovery of LZTR1 as an adapter of a Cullin-3 RING E3 ubiquitin ligase complex responsible for the degradation of RAS GTPases. In parallel, dysregulated LZTR1 expression via aberrant splicing and mutations was identified in clonal hematopoietic conditions. Here we identify that loss of LZTR1, or leukemia-associated mutants in the LZTR1 substrate and RAS GTPase RIT1 that escape degradation, drives hematopoietic stem cell (HSC) expansion and leukemia in vivo. Although RIT1 stabilization was sufficient to drive hematopoietic transformation, transformation mediated by LZTR1 loss required MRAS. Proteolysis targeting chimeras (PROTAC) against RAS or reduction of GTP-loaded RAS overcomes LZTR1 loss-mediated resistance to FLT3 inhibitors. These data reveal proteolysis of noncanonical RAS proteins as novel regulators of HSC self-renewal, define the function of RIT1 and LZTR1 mutations in leukemia, and identify means to overcome drug resistance due to LZTR1 downregulation.. Here we identify that impairing proteolysis of the noncanonical RAS GTPases RIT1 and MRAS via LZTR1 downregulation or leukemia-associated mutations stabilizing RIT1 enhances MAP kinase activation and drives leukemogenesis. Reducing the abundance of GTP-bound KRAS and NRAS overcomes the resistance to FLT3 kinase inhibitors associated with LZTR1 downregulation in leukemia. This article is highlighted in the In This Issue feature, p. 2221.

Topics: Cullin Proteins; Guanosine Triphosphate; Humans; Leukemia; Protein Kinase Inhibitors; Proteolysis; Proto-Oncogene Proteins p21(ras); ras Proteins; Transcription Factors

The Ras homologous (Rho) protein family of GTPases (RhoA, RhoB and RhoC) are the members of the Ras superfamily and regulate cellular processes such as cell migration, proliferation, polarization, adhesion, gene transcription and cytoskeletal structure. Rho GTPases function as molecular switches that cycle between GTP-bound (active state) and GDP-bound (inactive state) forms. Leukaemia-associated RhoGEF (LARG) is a guanine nucleotide exchange factor (GEF) that activates RhoA subfamily GTPases by promoting the exchange of GDP for GTP. LARG is selective for RhoA subfamily GTPases and is an essential regulator of cell migration and invasion. Here, we describe the mechanisms by which LARG is regulated to facilitate the understanding of how LARG mediates functions like cell motility and to provide insight for better therapeutic targeting of these functions.

Topics: Guanosine Triphosphate; Humans; Leukemia; ras Guanine Nucleotide Exchange Factors; ras Proteins; rho GTP-Binding Proteins; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

Topics: Active Transport, Cell Nucleus; Amino Acid Motifs; Antigens, CD34; Cell Nucleus; Exportin 1 Protein; Guanosine Triphosphate; HIV-1; Homeodomain Proteins; Humans; K562 Cells; Karyopherins; Leukemia; Mutation; NF-kappa B; NFATC Transcription Factors; Nuclear Pore Complex Proteins; Oncogene Proteins, Fusion; Promoter Regions, Genetic; ran GTP-Binding Protein; Receptors, Cytoplasmic and Nuclear; rev Gene Products, Human Immunodeficiency Virus; Transcription, Genetic

Topics: Animals; Antineoplastic Agents; Apoptosis; Chromatography, High Pressure Liquid; Clinical Trials as Topic; Down-Regulation; Guanosine Triphosphate; History, 20th Century; Humans; Ischemia; Kidney Neoplasms; Leukemia; Models, Biological; Neoplasms; Ribavirin; Signal Transduction; Time Factors; Tumor Cells, Cultured

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.

Topics: Animals; Antineoplastic Agents; Cell Division; Computer Simulation; Crystallography, X-Ray; Enzyme Inhibitors; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Molecular Structure; Neoplasms; Organoselenium Compounds; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

Purine nucleoside phosphorylase deficiency leads to a dGTP-mediated T-lymphopenia, suggesting that an analogue of deoxyguanosine would be selectively effective in T-cell disease. 9-beta-D-Arabinofuranosylguanine (ara-G) is relatively resistant to hydrolysis by purine nucleoside phosphorylase and selectively toxic to T cells, but its low solubility has prevented its use in the clinic. 2-Amino-6-methoxy-arabinofuranosylpurine (GW506U) serves as the water-soluble prodrug for ara-G. A Phase I trial in patients with refractory hematological malignancies demonstrated that the clinical responses to this agent were directly related to the peak levels of ara-G 5'-triphosphate (ara-GTP) in target cells. The aim of the present study was to develop and test strategies to increase intracellular accumulation of ara-GTP in primary human leukemia cells of myeloid and B-lymphoid origin. Three strategies were tested. First, incubations with 100 microM ara-G for 4 h produced a linear median accumulation rate of 19 microM/h (range, 2-45 microM/h; n = 15) in lymphoid leukemia cells and 16 microM/h (range, 0.5-41 microM/h; n = 11) in myeloid leukemia cells. Saturation of ara-GTP accumulation was achieved only after 6-8 h exposure in both lymphoid and myeloid leukemia cells, suggesting a rationale for prolonged infusion. Second, a dose-dependent increase in ara-GTP accumulation was observed with incubations of 10-300 microM ara-G for 3 h. Hence, dosing regimens that achieve high plasma levels of ara-G during therapy may increase cellular levels of ara-GTP. Finally, a biochemical modulation approach using in vitro incubation of leukemia cells with 10 microM 9-beta-D-arabinofuranosyl-2-fluoroadenine for 3 h, followed by either 50 or 100 microM ara-G for 4 h, resulted in a statistically significant median 1.3-fold (range, 1.1-9.0-fold; P = 0.034) and 1. 8-fold (range, 0.9-10.6 fold; P = 0.018) increase in ara-GTP compared to cells incubated with ara-G alone. Extension of these studies to ex vivo incubations confirmed our in vitro findings. These strategies will be used in the design of clinical protocols to increase ara-GTP accumulation in leukemia cells during therapy.

Topics: Antineoplastic Agents; Arabinonucleosides; Arabinonucleotides; Biotransformation; Guanosine Triphosphate; Humans; In Vitro Techniques; Kinetics; Leukemia; Leukemia, B-Cell; Leukemia, Myeloid

In dibutyryl cAMP-differentiated human leukemia (HL-60) cells, the potent histamine H1-receptor agonist, 2-(3-chlorophenyl)histamine, activates pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi-subfamily by a mechanism which is independent of known histamine receptor subtypes (Seifert et al. Mol Pharmacol 45: 578-586, 1994). In order to learn more about this G-protein activation, we studied the effects of histamine and various 2-substituted histamine derivatives in various cell types and on purified G-proteins. In HL-60 cells, histamine and 2-methylhistamine increased cytosolic Ca2+ concentration ([Ca2+]i) in a clemastine-sensitive manner. Phenyl- and thienyl-substituted histamines increased [Ca2+]i as well, but their effects were not inhibited by histamine receptor antagonists. 2-Substituted histamines activated high-affinity GTPase in HL-60 cell membranes in a PTX-sensitive manner, with the lipophilicity of substances increasing their effectiveness. Although HEL cells do not possess histamine receptors mediating rises in [Ca2+]i, 2-(3-bromophenyl)histamine increased [Ca2+]i in a PTX-sensitive manner. It also increased GTP hydrolysis by Gi-proteins in HEL cell membranes. All these stimulatory effects of 2-substituted histamine derivatives were seen at concentrations higher than those required for activation of H1-receptors. In various other cell types and membrane systems, 2-substituted histamine derivatives showed no or only weak stimulatory effects on G-proteins. 2-Substituted histamine derivatives activated GTP hydrolysis by purified bovine brain Gi/Go-proteins and by pure Gi2 (the major PTX-sensitive G-protein in HL-60 and HEL cells). Our data suggest the following: (1) histamine and 2-methylhistamine act as H1-receptor agonists in HL-60 cells; (2) incorporation of bulky and lipophilic groups results in loss of H1-agonistic activity of 2-substituted histamine derivatives in HL-60 cells but causes a receptor-independent G-protein-stimulatory activity; (3) the effects of 2-substituted histamine derivatives on G-proteins are cell-type specific.

Topics: Animals; Calcium; Cell Line; Cricetinae; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Histamine; Histamine Agonists; Humans; Leukemia; Leukemia, Erythroblastic, Acute; Rats; Receptors, Histamine

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Topics: Adenosine Triphosphate; Cell Survival; DNA Primase; DNA, Neoplasm; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia; Mycophenolic Acid; Nucleosomes; Ribonucleosides; RNA Nucleotidyltransferases; RNA, Neoplasm; Tumor Cells, Cultured

Dibutyryl cAMP-differentiated HL-60 human leukemia cells possess receptors for the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), C5a and leukotriene B4 (LTB4). We compared the effects of these chemoattractants in HL-60 membranes and in intact HL-60 cells. fMLP, C5a and LTB4 stimulated GTP hydrolysis and guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) binding in HL-60 membranes with similar effectiveness and in a pertussis toxin (PTX)-sensitive manner. They also stimulated photolabeling of the alpha-subunits of the guanine nucleotide-binding proteins (G-proteins), Gi2 and Gi3 with similar effectiveness. Chloride salts of monovalent cations differentially enhanced and inhibited chemoattractant-induced GTP hydrolyses. C5a was less effective than fMLP in enhancing cholera toxin-catalysed ADP-ribosylation of Gi alpha 2 and Gi alpha 3, and LTB4 was ineffective. fMLP was more effective than C5a and LTB4 in stimulating Ca2+ influx in HL-60 cells. C5a- and LTB4-induced rises in cytosolic Ca2+ concentration ([Ca2+]i) were PTX-sensitive, whereas the effect of fMLP was partially PTX-insensitive. LTB4-induced rises in [Ca2+]i were more sensitive towards homologous desensitization than those induced by C5a, and the effect of fMLP was resistant in this regard. C5a was considerably less effective than fMLP in activating superoxide anion formation and azurophilic granule release, and LTB4 was ineffective. Our data suggest that fMLP, C5a and LTB4 effectively activate the G-proteins, Gi2 and Gi3, in HL-60 cells and that fMLP may additionally activate PTX-insensitive G-proteins. fMLP, C5a and LTB4 are full, partial and incomplete secretagogues, respectively, and these differences may be due to differences in homologous receptor desensitization and qualitative Gi-protein activation.

Topics: Bucladesine; Calcium; Cell Differentiation; Complement C5a; Enzyme Activation; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Leukemia; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Signal Transduction; Tumor Cells, Cultured

Deoxynucleoside 5'-triphosphates (dNTPs) can be determined in cell extracts by high performance liquid chromatography after prior selective degradation of ribonucleoside 5'-triphosphates with sodium periodate and methylamine. When the method is used for the evaluation of deoxynucleoside triphosphates in 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)-containing cell extracts, an additional peak coeluting with thymidine triphosphate (dTTP) is observed. This peak is due to the formation of a carboxylic acid derivative of ara-CTP by periodate oxidation, and it can lead to considerable overestimation of dTTP. Formation of this peak can be avoided by using alkaline reaction conditions (pH 7.5) and by changing the sequence of addition of the reagents used in the periodation procedure. By employing this modified protocol, cellular dNTP and ara-CTP levels can be monitored in extracts of leukaemic blasts during cytosine arabinoside treatment in two separate HPLC runs.

Topics: Adenosine Triphosphate; Arabinofuranosylcytosine Triphosphate; Cell Line; Chromatography, High Pressure Liquid; Cytidine Triphosphate; Deoxyribonucleotides; Guanosine Triphosphate; Humans; Leukemia; Ribonucleotides; Thymine Nucleotides; Uridine Triphosphate

Tiazofurin is an oncolytic agent which has shown therapeutic activity in end-stage acute nonlymphocytic leukemia (ANLL) and blast crisis of chronic granulocytic leukemia (CGL-BC). Tiazofurin is anabolized to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), which inhibits IMP dehydrogenase activity, leading to reduction of guanylate pools and cessation of cancer cell proliferation. The concentration of TAD in neoplastic cells of patients treated with tiazofurin should be a good indicator of sensitivity to the drug and also might herald the emergence of drug-resistant cells. Therefore, the precise quantitation of TAD in cancer cells during tiazofurin treatment is essential. In this paper we report a highly sensitive method for the determination of TAD in biological samples. With this technique, in addition to TAD, thirteen other biologically relevant adenine, guanine, cytosine and uridine nucleotides can be separated and quantitated accurately. TAD standard was separated on a Waters Partisil 10-SAX column in a RCM-10 module using an ammonium phosphate buffer system. TAD eluted at 21 min with a limit of detection of 15 pmol and linearity up to 3 nmol. The coefficient of variation was 0.6 +/- 0.1% for retention time and 2 +/- 0.3% for TAD concentration. Recovery of TAD was 96% with reproducibility of 98%. To examine the applicability of this method to a clinical setting, blood samples were obtained from a patient with CGL-BC and leukocytes were separated on a Ficoll-Hypaq gradient, extracted with trichloroacetic acid, and an aliquot was analyzed on HPLC. The TAD peak was identified by comparing the retention time and spectral analysis of the standard. After the patient was treated with a 2200 mg/m2 (12.7 mM) dose of tiazofurin, the TAD concentrations in the mononuclear cells at 2, 6, and 24 hr were 23.1, 13.6, and 0.8 microM. TAD levels at 2, 6, and 24 hr after a tiazofurin dose of 3300 mg/m2 (21.1 mM) were 42.8, 26.1, and 1.4 microM respectively.

Topics: Adenine Nucleotides; Antineoplastic Agents; Chromatography, High Pressure Liquid; Guanosine Triphosphate; Humans; Leukemia; Monocytes; Reproducibility of Results; Ribavirin; Sensitivity and Specificity

The objective of this investigation was to evaluate the ability of arabinosyl nucleotides to modulate the cellular metabolism of different arabinosyl nucleosides in K562 cells. The maximum rate of accumulation of the respective 5'-triphosphate (TP) was observed in cells incubated with 10 microM arabinosylcytosine (ara-C), 10 microM arabinosylguanine (ara-G), 300 microM arabinosyl-2-fluoroadenine (F-ara-A), and greater than 1000 microM arabinosyladenine (ara-A). Cell extract fractionation studies demonstrated that ara-C and F-ara-A were phosphorylated by dCyd kinase, whereas ara-A was phosphorylated by dCyd kinase and Ado kinase; ara-G phosphorylation was attributed to dGuo kinase. When nucleoside kinase was rate limiting to arabinosyl nucleotide accumulation, cells preloaded with F-ara-ATP showed increased rates of ara-CTP and ara-GTP accumulation, whereas cells preloaded with ara-CTP had decreased rates of F-ara-ATP and ara-GTP accumulation. Preloading cells with ara-GTP had little effect on arabinosyl nucleoside triphosphate accumulation. F-ara-ATP accumulation was inhibited in cells containing all other arabinosyl nucleotides, whereas ara-ATP metabolism was not affected by preincubation with any other nucleoside. Cells incubated with ara-C and ara-G had a general rise in dNTP, whereas F-ara-A incubation was associated with a decrease in cellular dNTP. The differential effects of arabinosyl nucleotides and cellular metabolism of other arabinosyl nucleosides are due to phosphorylation by distinct nucleoside kinases that likely have characteristic sensitivities to cellular dNTP levels.

Topics: Arabinofuranosylcytosine Triphosphate; Arabinonucleosides; Arabinonucleotides; Cytarabine; Deoxyribonucleotides; Drug Interactions; Guanosine Triphosphate; Humans; Leukemia; Phosphorylation; Phosphotransferases; Tumor Cells, Cultured; Vidarabine; Vidarabine Phosphate

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.

Topics: Binding Sites; Cell Line; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Isoproterenol; Leukemia; Magnesium; Neomycin; Oligopeptides; Pertussis Toxin; Receptors, Adrenergic, beta; Receptors, Formyl Peptide; Receptors, Immunologic; Signal Transduction; Virulence Factors, Bordetella

The hypothesis was tested that the increased IMP dehydrogenase activity in human myelocytic leukemic cells, and along with it guanylate biosynthesis, might be a sensitive target to chemotherapy by tiazofurin. 1. IMP dehydrogenase activity in normal leukocytes was 3.1 +/- 0.5 (means +/- S.E.) nmol/hr/mg protein and in leukemic cells it was elevated 15- to 41-fold. The activity of guanine phosphoribosyltransferase in normal leukocytes was 389 +/- 27 nmol/hr/mg protein and in the leukemic cells it increased 2.8- to 6.8-fold. 2. IMP dehydrogenase was purified 4,900-fold to homogeneity from rat hepatoma 3924A with a yield of 30%. The kinetic properties of the hepatoma enzyme were similar to those of the enzyme in human myelocytic leukemic blast cells because of the similarity of the Km's for IMP (23 microM), NAD (44 and 65 microM); the Ki for TAD was 0.1 microM in both enzymes. 3. There was a selectivity of the in vitro response to tiazofurin in human normal and leukemic leukocytes. When labeled tiazofurin was incubated with leukocytes from normal, healthy volunteers and from leukemic patients, the leukemic leukocytes made 20- to 30-fold more TAD and the GTP content decreased as compared to normal leukocytes. This procedure proved to be a suitable predictive test in a clinical setting because patients with positive tests responded to tiazofurin whereas those with negative ones did not. 4. The National Cancer Institute approved a chemotherapeutic phase I/II trial which concentrates on treatment of refractory acute myelocytic leukemia. Tiazofurin is infused in a 60-minute period with a pump to insure uniform delivery. A novel aspect of the trial was that it was directed primarily by the biochemical impact of tiazofurin on IMP dehydrogenase activity and GTP concentration and the tiazofurin doses were to be adjusted accordingly. Patients received allopurinol as a routine precaution against possible accumulation of uric acid in the kidney. 5. In the first eight patients, there was one complete remission, two entered the chronic phase, two entered into partial remission, one did not respond, and two were not evaluable. In the five patients who responded, there was a rapid, profound decrease in IMP dehydrogenase activity of the blast cells and a gradual decline in GTP concentrations. The blast cell count followed the decrease in the GTP concentration. The white blood cell count was largely preserved. 6. Bone marrow aspirates and peripheral blood samples showed that w

Topics: Adenine; Aged; Allopurinol; Animals; Drug Evaluation; Female; Guanine; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia; Leukemia, Myeloid, Acute; Liver Neoplasms, Experimental; Male; Middle Aged; Rats; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

The cellular origin and the control of neopterin release associated with immune stimulation was studied in cell cultures. Using purified human mononuclear cells, the intracellular change in concentrations of GTP and pterins was measured under various kinds of stimulation. Three enzymes involved in tetrahydrobiopterin biosynthesis, i.e. GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, were also determined. Human macrophages stimulated with culture supernatant from activated T-lymphocytes were the main producers of neopterin. In these cells, GTP cyclohydrolase I activity was elevated due to high GTP levels and therefore neopterin accumulated. Human macrophages lack 6-pyruvoyl tetrahydropterin synthase activity. Exogenous tetrahydrobiopterin added to the culture medium of stimulated T cells and macrophages suppressed the elevation of GTP cyclohydrolase I activity and neopterin concentration, but not the elevation of intracellular GTP. Stimulation of macrophages with recombinant human interferon-gamma and neutralization of the effect of T cell supernatants by addition of a monoclonal antibody specific for human interferon-gamma showed that immune interferon induced the alterations in GTP cyclohydrolase I activity and neopterin concentration. In the human macrophage line U-937 and in the leukemia line HL-60, no GTP cyclohydrolase I activity or intracellular pterins were detected, but high levels of GTP. In mouse mononuclear cells, no neopterin was detected, but biopterin and pterin. After stimulation, biopterin was elevated in the same way as neopterin in human mononuclear cells. This is explained by the different regulation of the rate-limiting steps of tetrahydrobiopterin biosynthesis in man and in mouse. These results suggest that neopterin is an unspecific marker for the activation of the cellular immune system.

Topics: Alcohol Oxidoreductases; Animals; Cell Line; GTP Cyclohydrolase; Guanosine Triphosphate; Humans; Leukemia; Leukemia, Experimental; Macrophages; Monocytes; Phosphorus-Oxygen Lyases; Pterins; T-Lymphocytes

This study reports the selective sensitivity to tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide, NSC-286193) of human leukemic leukocytes as compared to normal ones in bone marrow and peripheral blood samples by comparing the production of the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), from labeled tiazofurin and the depression of GTP concentration. When labeled tiazofurin was incubated with leukocytes obtained from healthy volunteers or from leukemic patients (acute non-lymphocytic leukemia or acute lymphoblastic leukemia), the TAD production was 27.0 +/- 8.3, 551.3 +/- 71.8 and 755.9 +/- 94.1 pmoles/10(9) cells per hr, respectively. Thus, the leukemic cells produced over 20-fold higher concentrations of TAD than the normal leukocytes. Incubation with tiazofurin in leukemic leukocytes decreased the GTP pools (to 48-79%), whereas there was no change in the normal leukocytes. These results indicate a selectivity of response to tiazofurin in human normal and leukemic leukocytes. The procedure reported in this work may be suitable as a rapid predictive test for the sensitivity of leukemic leukocytes to tiazofurin. Such a diagnostic test should be helpful in identifying neoplastic cells sensitive to tiazofurin in the Phase II trials now being developed.

Topics: Adenine Nucleotides; Adult; Aged; Antineoplastic Agents; Female; Guanosine Triphosphate; Humans; Leukemia; Leukocytes; Male; Middle Aged; Ribavirin; Ribonucleosides

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.

Topics: Adenosine Triphosphate; Cell Line; Chromatography, High Pressure Liquid; Guanosine Triphosphate; Humans; Kinetics; Leukemia; Mercaptopurine; Neoplasms; Nucleotides; Phosphoribosyl Pyrophosphate

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.

Topics: Cell Line; Cytarabine; Deoxyguanosine; Drug Synergism; Guanosine Triphosphate; Humans; Leukemia; Phosphorylation

Deoxyribonucleic acid synthesis requires adequate cellular concentrations of the four deoxyribonucleoside triphosphates. Using a sensitive enzymic assay, we have measured the concentrations (pools) of these compounds in human bone marrow cells and in lymphocytes. The mean concentrations (pmol/10(6) cells) in normal human bone marrow cells were: deoxyadenosine triphosphate (dATP) 1.5; deoxyguanosine triphosphate (dGTP) 0.4; thymidine triphosphate (dTTP) 1.4 and deoxycytidine triphosphate (dCTP) 0.6; and in normal phytohaemagglutinin (PHA)-stimulated lymphocytes (72 h cultures); dATP 3.7; dGTP 1.9; dTTP 9.4 and dCTP 2.9. The deoxyribonucleoside triphosphate concentrations were increased approximately threefold in the nucleated marrow cells from patients with leukaemia and myeloproliferative diseases. PHA-stimulation of lymphocytes caused a marked increase of the deoxyribonucleoside triphosphate concentrations, particularly of dTTP, between 24 and 48 h of culture. In PHA-stimulated lymphocytes, the antifolate drugs methotrexate, pyrimethamine and trimethoprim, all produced a fall in dTTP and a rise in dATP concentrations within 1 h. These effects could be reversed by folinic acid. 5-Fluorouracil caused a fall in dTTP and in dCTP but no consistent changes in dATP; hydroxyurea caused a fall in dATP with a rise in dTTP. BCNU caused a significant fall in dATP and dCTP. Dibutyryl cyclic 3', 5' adenosine monophosphate and theophylline had no consistent effect on the deoxyribonucleoside triphosphate concentrations. 6-Mercaptopurine caused a fall in dATP and dGTP, the fall in dATP being marked after 4 h incubation. It is concluded that measurement of the deoxyribonucleoside triphosphates in human cells provides a new method of studying DNA synthesis in human disease states and of analysing the action of antimetabolite drugs on normal and diseased cells.

Topics: Adenosine Triphosphate; Antimetabolites; Antineoplastic Agents; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Cytosine Nucleotides; Deoxyribonucleotides; DNA; DNA, Neoplasm; Guanosine Triphosphate; Leukemia; Lymphocytes; Myeloproliferative Disorders; Thymidine; Thymine Nucleotides