guanosine-triphosphate and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

T-cell lymphoid blastic phase (BP) transformation is rare in chronic myelogenous leukemia (CML). 2-amino-9-beta-D-arabinosyl-6-methoxy-9H-guanine (GW506U78), a prodrug of arabinosylguanine (ara-G), is effective in T-cell leukemias.. The authors present a case of a 48-year-old male with Philadelphia chromosome (Ph) positive CML and T-cell lymphoid BP after 17 months in the chronic phase.. Plasma pharmacokinetic studies after an infusion of GW506U78 at a dose of 40 mg/kg showed GW506U78 concentrations of 60 microM, and a peak ara-G concentration of 260 microM in the plasma. Cellular ara-G triphosphate (ara-GTP) concentration in the peripheral blood T-lymphoblasts was 80 microM at the end of GW506U78 infusion and reached a maximum of 150 microM. The patient achieved a complete response that lasted 13 months. Severe neurotoxicity related to GW506U78 was observed.. GW506U78 showed antileukemic activity against Ph positive T-cell BP CML. Neurotoxicity was dose-limiting in this patient. Treatment with GW506U78 and modulation of ara-GTP concentrations are therapeutic strategies that require further exploration in T-cell malignancies. Investigation of other dosing schedules may limit neurotoxicity.

Topics: Antineoplastic Agents; Arabinonucleosides; Arabinonucleotides; Blast Crisis; Guanosine Triphosphate; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Remission Induction; T-Lymphocytes

A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate.. Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days.. Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27).. Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Arabinonucleosides; Arabinonucleotides; Biomarkers; Female; Guanosine Triphosphate; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Prolymphocytic; Male; Middle Aged; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Treatment Outcome; Vidarabine

The increased activity in cancer cells of inosine 5'-monophosphate dehydrogenase (IMP DH, EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, was suggested as a sensitive target for chemotherapy. Tiazofurin (NSC 286193), through its conversion to the active metabolite, thiazole-4-carboxamide adenine dinucleotide (TAD), is a strong inhibitor of IMP DH. In our clinical trial, tiazofurin caused return to the chronic phase in patients with chronic granulocytic leukemia in blast crisis (Tricot, G.; Jayaram, H.N.; Weber, G.; Hoffman, R. Tiazofurin: Biological effects and clinical uses. Int. J. Cell Cloning 8:161-170; 1990). In K562 human leukemic cells, tiazofurin down-regulated the expression of c-Ki-ras and c-myc oncogenes, which was followed by induced differentiation. We now report down-regulation by tiazofurin of the c-Ki-ras oncogene in a patient with chronic granulocytic leukemia in blast crisis. A single tiazofurin infusion (2,200 mg/m2) on days one and two decreased IMP dehydrogenase activity (the apparent t1/2 was 30 min), GTP concentration (the apparent t1/2 was 6 hr), and expression of ras (the apparent t1/2 was 8 hr) and c-myc (the apparent t1/2 was 38.5 hr) oncogenes in the leukemic cells. No further tiazofurin was given, because on days three and four the chemotherapeutic impact became evident in a tumor-lysis syndrome and the blast cells were cleared from the periphery by day five. The decrease in IMP DH activity, GTP concentration, and expression of c-Ki-ras oncogene were early markers of the successful chemotherapeutic impact of tiazofurin in a patient with chronic granulocytic leukemia in blast crisis.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Blast Crisis; Blotting, Northern; Down-Regulation; Gene Expression Regulation; Genes, ras; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Pilot Projects; Ribavirin; RNA; Uric Acid

It is well established that Foxo3a is a fundamental module of signal transduction pathways regulating erythropoiesis; however, precise mechanism which regulates its physiological function still remains unclear. Here, our results revealed that the nuclear localization and stability of Foxo3a were modulated by the physical interaction of PKC and p38 signaling elements and that direct interactions led to phosphorylation of threonine residue(s) in Foxo3a. In addition, our findings revealed that the sequential activity of Foxo3a by guanosine 5'-triphosphate can impede cellular proliferation and suppress p73 expression as oncoprotein in K562 cells; thus identifying Foxo3a as a tumor suppressor in these p53 null cells. However, down-regulation of Foxo3a-dependent p73 expression causes cell differentiation along the erythroid lineage. Collectively, our findings suggest that restoration of Foxo3a function by pharmacological agents under the influence of specific activated protein kinases might constitute a potential therapeutic strategy for combating the CML disease.

Topics: Active Transport, Cell Nucleus; Cell Differentiation; Cell Nucleus; DNA-Binding Proteins; Down-Regulation; Erythroid Cells; Forkhead Box Protein O3; Forkhead Transcription Factors; Guanosine Triphosphate; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Nuclear Proteins; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase C; Signal Transduction; Tumor Protein p73; Tumor Suppressor Proteins

Topics: Antineoplastic Agents; Benzamides; Cell Differentiation; Cellular Senescence; Guanosine Triphosphate; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Models, Biological; Neoplastic Stem Cells; Piperazines; Pyrimidines

The Ras signal transduction pathway is often deregulated in human myeloid leukaemia. For example, activating point mutations in RAS genes are found in some patients with juvenile chronic myelogenous leukaemia (JCML), while other patients with JCML show loss of the neurofibromatosis type 1 (NF1) gene, a Ras GTPase activating protein. By generating mice whose haematopoietic system is reconsituted with Nf1 deficient haematopoietic stem cells we show that Nf1 gene loss, by itself, is sufficient to produce the myeloproliferative symptoms associated with human JCML. We also provide evidence to indicate that Nf1 gene loss induces myeloproliferative disease through a Ras-mediated hypersensitivity to granulocyte/macrophage-colony stimulating factor (GM-CSF). Finally, we describe a genetic screen for identifying genes that cooperate with Nf1 gene loss during progression to acute myeloid leukaemia.

Topics: Acute Disease; Animals; Bone Marrow; Cell Differentiation; Cell Line, Transformed; Crosses, Genetic; Disease Models, Animal; Disease Progression; Female; Genes, Neurofibromatosis 1; Granulocyte-Macrophage Colony-Stimulating Factor; Guanosine Triphosphate; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Liver; Male; Mice; Mice, Inbred C57BL; ras Proteins; Signal Transduction

Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.

Topics: Animals; Cell Division; Cells, Cultured; Child; Genes, Neurofibromatosis 1; Granulocyte-Macrophage Colony-Stimulating Factor; GTPase-Activating Proteins; Guanosine Triphosphate; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Neurofibromatosis 1; Neurofibromin 1; Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction

Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling.

Topics: Binding, Competitive; Cloning, Molecular; Escherichia coli; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutagenesis, Insertional; Oncogene Proteins; Oncogenes; Protein Biosynthesis; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcr; rac GTP-Binding Proteins; Recombinant Fusion Proteins

In the present work, anti-G protein antibodies were introduced inside streptolysin permeabilized O interleukin-2-activated natural killer (IANK) cells. Successful entry of the antibodies was determined by flow cytometry and fluorescence microscopy. Permeabilized cells showed typical large granular lymphocyte morphology and remained functional, significantly lysing both NK-sensitive K562 cells and NK-resistant/IANK-sensitive RAJI target cells. This method was utilized to study the effect of anti-G protein antibodies on the functional activities of IANK cells. Anti-Gs antibody inhibited IANK cell killing of RAJI but not of K562 target cells. Further analysis showed that K562 and RAJI cells enhance the binding of guanosine 5'-O-(thiotriphosphate) to IANK cell membranes, and increase the hydrolysis of [32P]GTP in these membranes. Immunoblot analysis showed that K562 and RAJI cells induce the release of alpha o, but not alpha i, alpha s, or alpha q.11 from IANK cell membranes. Cumulatively, these data indicate that putative receptors recognizing K562 or RAJI target cells are coupled to Go in IANK cells, however, only Gs seems to be coupled to receptors recognizing RAJI target cells. Our results point out the importance of Gs protein as a mediator of cellular cytotoxicity of the anti-tumor effector cells.

Topics: Animals; Antibodies; Bacterial Proteins; Burkitt Lymphoma; Cell Line; Cell Membrane Permeability; Cytotoxicity, Immunologic; Flow Cytometry; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Immunoglobulin G; Interleukin-2; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Rabbits; Streptolysins; Tumor Cells, Cultured

The ras oncogene product (p21ras, Ras) is a GTP-binding protein and is thought to transduce signals regulating cellular proliferation and differentiation. The active form Ras-GTP is inactivated by hydrolyzing bound GTP to GDP. Tiazofurin, a specific inhibitor of IMP dehydrogenase, decreased cellular GTP pools and downregulated c-ras gene expression, leading to differentiation (Olah, E. et al., Proc. Natl. Acad. Sci. USA 85: 6533-6537, 1988; Weber et al., Cancer Commun. 3:61-66, 1991). To clarify the link between the action of tiazofurin on metabolic alterations and the induction of differentiation, we examined the effect of tiazofurin on the ratio of active Ras-GTP to total Ras in K562 cells in culture. Cells were labeled for 6 h with [32P]Pi in phosphate-free RPMI 1640. Tiazofurin (100 or 200 microM) was added to cells, and samples were taken at 0, 2, 4, 6 and 12 h of incubation. Cell lysates were immunoprecipitated with monoclonal anti-p21 antibody (Y13-259), then developed on thin layer chromatography. GTP and GDP bound to Ras were visualized by autoradiography. Tiazofurin treatment decreased Ras-GTP concentration in a time- and dose-dependent fashion. In the untreated K562 cells the Ras-GTP concentration was 26.3 +/- 1.4, and tiazofurin (200 microM) decreased it at 6 h to 16.6 +/- 2.9 and at 12 h to 10.6 +/- 2.1%. Inhibition of the GTP salvage pathway with hypoxanthine (100 or 200 microM) enhanced the tiazofurin-induced decrease of Ras-GTP, whereas addition of guanosine (100 microM) prevented the Ras-GTP decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antimetabolites, Antineoplastic; Cell Differentiation; Guanosine; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Oncogene Protein p21(ras); Protein Binding; Ribavirin; Tumor Cells, Cultured

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.

Topics: Antimetabolites, Antineoplastic; Cell Cycle; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Ribavirin; Tumor Cells, Cultured

The effects of 4-carbamoylimidazolium 5-olate (SM-108), an antipurine compound, on a human leukemia cell line, K562, were studied. Treatment with SM-108 induced erythroid differentiation of K562 cells. During a 6-day culture with 100 microM SM-108, the cell number decreased to 37% of the control number, 77% of the cells became benzidine-positive, and the hemoglobin content increased from 2.1 +/- 0.2 to 10.6 +/- 1.3 pg/cell. Cell differentiation was associated with reduction of IMP dehydrogenase activity and intracellular GTP content to 25 and 36%, respectively, of the control values within 1.5 hr. The differentiation and decrease in the GTP pool induced by SM-108 were blocked by the presence of 25 microM guanine or guanosine. SM-108 also induced erythroid differentiation of K562 subline cells transfected with pMSG (K562/pMSG), which have an additional salvage pathway for GMP production from xanthine. The addition of 100 microM xanthine prevented erythroid differentiation of this subline and restored the GTP pool. These findings suggest that the induction of erythroid differentiation of K562 cells by SM-108 may be due to an early decrease in IMP dehydrogenase activity and a subsequent decrease in GTP content in the cells. Thus, purine metabolism may have an important role in SM-108-induced differentiation.

Topics: Cell Differentiation; Cell Line; Erythrocytes; Guanosine Triphosphate; Humans; Imidazoles; IMP Dehydrogenase; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Purines; Tumor Stem Cell Assay; Xanthine; Xanthines

Topics: Adult; Aged; Allopurinol; Antineoplastic Agents; Female; Genes, myc; Genes, ras; Guanosine Triphosphate; Humans; IMP Dehydrogenase; In Vitro Techniques; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Ribavirin; Tumor Cells, Cultured