guanosine-triphosphate and Leukemia--Lymphoid

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzamides; Cell Division; Cell Movement; Cell Transformation, Neoplastic; Erythroid Precursor Cells; Fusion Proteins, bcr-abl; GRB2 Adaptor Protein; Guanosine Triphosphate; Helminth Proteins; Humans; Imatinib Mesylate; Immunoblotting; Leukemia, Lymphoid; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Phosphorylation; Piperazines; Precipitin Tests; Proteins; Pyrimidines; Signal Transduction; Tyrosine

RP 56976 (taxotere), a new semisynthetic analogue of taxol, is a potentially important chemotherapeutic agent for the treatment of cancer. We report here that this drug is a potent inhibitor of cell replication and, like taxol, promotes the in vitro assembly of stable microtubules in the absence of guanosine triphosphate and induces microtubule-bundle formation in cells. Compared with taxol, RP 56976 is slightly more active as a promoter of tubulin polymerization. As an inhibitor of cell replication, RP 56976 is 2.5-fold more potent than taxol in J774.2 and P388 cells and at least 5-fold more potent in taxol-resistant cells.

Topics: Alkaloids; Animals; Cell Survival; Cells, Cultured; Cricetinae; Cricetulus; Docetaxel; Guanosine Triphosphate; Leukemia, Lymphoid; Microtubules; Paclitaxel; Structure-Activity Relationship; Taxoids; Tumor Cells, Cultured

The experimental antineoplastic agent triciribine (tricyclic nucleoside, TCN) is known to be activated to its phosphate TCN-P by adenosine kinase and to inhibit cell growth, purine nucleotide synthesis, and incorporation of amino acids into proteins. Our objective in this paper was to compare these effects in intact cells of a human cell line as a prerequisite to describing in a companion paper [Moore et al., Biochem. Pharmac. 38, 4045 (1989)] more detailed enzymic studies of their interrelationships. TCN treatment inhibited cloning of CCRF-CEM human leukemic lymphoblasts 50% at concentrations of 6, 30, and 90 microM with 8-day, 8-hr, and 2-hr exposures respectively. However, 6-20% of the cells survived exposure to 200 microM TCN for 24 hr. The intracellular formation of TCN-P from TCN was rapid, concentrative and essentially complete, but TCN-P did not exceed about 1.4 mM (1.4 nmol/10(6) cells) at 200 microM TCN. In cells exposed to 50 microM TCN for 1.25 to 24 hr, formate incorporation into ATP and GTP was inhibited the most rapidly and strongly; pools of ATP and GTP were decreased as much as 40% (as compared with controls); and incorporation of formate into RNA purines was inhibited as much as 65%. Incorporation of leucine into protein was more moderately inhibited up to 40%, apparently in proportion to the concentration of intracellular TCN-P, rather than of the TCN in the medium. These inhibitions occurred most rapidly during the first 2-4 hr and increased only gradually thereafter, whereas cloning ability was inhibited more slowly and uniformly over a longer time period. No one of these metabolic effects by itself showed a clear correlation with the loss of viability. The incorporation of formate into formylglycinamide ribotide (FGAR, when accumulated at a blockage by azaserine) was inhibited drastically by TCN. The rate of incorporation of hypoxanthine into ATP was increased by TCN, whereas incorporation into GTP was decreased. Thus, the principal sites of inhibition of purine synthesis by TCN-P were shown in these intact cells to be at a step prior to synthesis of FGAR in the de novo pathway and also at an additional site between IMP and GTP.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Cell Survival; Formates; Glycine; Guanosine Triphosphate; Humans; Hypoxanthine; Hypoxanthines; Kinetics; Leukemia, Lymphoid; Lymphocytes; Phosphorylation; Protein Biosynthesis; Purines; Ribonucleosides; Ribonucleotides; RNA; Tumor Cells, Cultured

In a previous report we demonstrated in mouse lymphoma (S-49) cells that DNA synthesis inhibition resulting from guanine starvation is associated with GTP rather than dGTP depletion. Since several effective anticancer drugs act via guanine depletion, it is important to test whether critical GTP depletion is unique to S-49 cells or also occurs in other cell lines. Mycophenolic acid-induced guanine starvation caused a drastic DNA synthesis inhibition in the human lymphoblastic T leukemia (CEM) and the mouse B leukemia (L1210) cell lines, which was again associated with GTP depletion rather than dGTP depletion. These results suggest that GTP depletion represents a common target of purine antimetabolites in mammalian cells.

Topics: Animals; Cell Division; Deoxyguanosine; DNA, Neoplasm; Guanosine; Guanosine Triphosphate; Humans; Leukemia L1210; Leukemia, Lymphoid; Mice; Mycophenolic Acid

The toxicity of the deoxyribonucleosides, 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine, for human T lymphoblasts is mediated by the accumulation of the corresponding deoxyribonucleoside triphosphate (dATP, dGTP, or dTTP, respectively). We have examined whether leukemic cells of non-T-cell origin are capable of accumulating deoxyribonucleotides in culture and whether this capability correlates with the activities of purine metabolizing enzymes in these cells. We have found that non-T, non-B acute lymphoblastic leukemia cells with low ecto-5'-nucleotidase and high adenosine deaminase activities increase their dATP pools by greater than tenfold when exposed to deoxyadenosine and an inhibitor of adenosine deaminase in culture. Cells from 2 of 9 patients with chronic lymphocytic leukemia and 4 of 11 patients with acute nonlymphoblastic leukemia achieved similar elevations in dATP, but there was no relationship between dATP accumulation and adenosine deaminase, purine nucleoside phosphorylase, or ecto-5'-nucleotidase activities. Treatment of four individuals with acute lymphoblastic leukemia with the adenosine deaminase inhibitor, 2'-deoxycoformycin, resulted in elevations in plasma deoxyadenosine concentrations and in increments in lymphoblast dATP levels that were similar to those measured in lymphoblasts cultured with deoxyadenosine and deoxycoformycin prior to treatment. In vitro incubations of leukemic cells with deoxyribonucleosides may provide a rational basis for the use of these compounds as chemotherapeutic agents.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Coformycin; Enzyme Inhibitors; Guanosine Triphosphate; Humans; Leukemia, Lymphoid; Lymphocytes; Pentostatin; Thymidine

Addition of 1 muM methotrexate to cultures of L5178Y cells results in an initial inhibition of thymidine, uridine, and leucine incorporation into acid-insoluble material followed, after about 10 hr, by a partial recovery in the extent of incorporation of these precursors. Acid-soluble adenosine triphosphate and guanosine triphosphate concentrations are greatly reduced initially, but guanosine triphosphate concentrations appear to recover partially by 10 hr. Acid-soluble uridine triphosphate and cytidine triphosphate concentrations initially increase after methotrexate treatment but then, with time, they too decline. Hypoxanthine and guanine are more effective than is adenine in overcoming the methotrexate-induced inhibition of thymidine incorporation. These results suggest that, in the presence of methotrexate, guanine nucleotides become limiting for nucleic acid synthesis before adenine nucleotides do. The block of purine de novo synthesis in L5178Y cells by methotrexate is almost complete and is not reversed with time. This suggests that the additional purine nucleotides that are available for nucleic acid synthesis 8 to 10 hr after addition of methotrexate are being derived from nucleic acid breakdown. Consistent with this is the observed reduction in the number of polyribosomes and hence, presumably in messenger RNA levels.

Topics: Adenine; Adenosine Triphosphate; Animals; Cell Line; Cytosine Nucleotides; Depression, Chemical; DNA; Guanine; Guanosine Triphosphate; Hypoxanthines; Leucine; Leukemia, Lymphoid; Methotrexate; Mice; Purines; RNA; Thymidine; Time Factors; Uracil Nucleotides; Uridine

Topics: Adenosine; Adenosine Triphosphate; Cell Line; Cells, Cultured; Chromatography; Cytosine Nucleotides; Deoxyribonucleotides; Deoxyuridine; DNA, Neoplasm; Guanosine Triphosphate; Humans; Kinetics; Leukemia, Lymphoid; Methotrexate; RNA, Neoplasm; Thymine Nucleotides; Tritium

Topics: Adenosine Monophosphate; Adenosine Triphosphate; Cytosine Nucleotides; Guanine Nucleotides; Guanosine Triphosphate; Humans; Leukemia, Lymphoid; Leukemia, Myeloid; Lymphocytes; NAD; NADP; Phosphorus; Ribonucleotides; Uracil Nucleotides

Evidence is presented that DNA polymerases from human leukemic cells are mutagenic. Nucleic acid-free extracts of acute lymphoblastic leukemic cells polymerized about 10-times more dCTP using poly(dA-dT).poly(dA-dT) as a template than did extracts from normal lymphocytes. Mutagenic DNA polymerases could perhaps play an important role in tumor progression.

Topics: Adenosine Triphosphate; Centrifugation, Density Gradient; Cytosine Nucleotides; DNA Nucleotidyltransferases; DNA Replication; DNA, Neoplasm; Guanosine Triphosphate; Humans; Leukemia, Lymphoid; Lymphocytes; Mutation; Phosphoric Diester Hydrolases; Phosphorus Isotopes; Polynucleotides; Templates, Genetic; Thymine Nucleotides; Tritium

Cells from a patient with childhood acute lymphoblastic leukemia contain an apparent DNA polymerase activity that was not found in any other cells except thymus cells. The enzyme has the properties of terminal transferase, an enzyme known to be found in thymocytes. The cells also contain the three major DNA polymerases found in growing cells. The results suggest that these tumor cells arose from a block in the differentiation of thymocytes. Terminal transferase may be a marker for the origin of leukemic cells.

Topics: Adenosine Triphosphate; Avian Leukosis Virus; Cell Line; Child; DNA Nucleotidyltransferases; Guanine Nucleotides; Guanosine Triphosphate; HeLa Cells; Humans; Infectious Mononucleosis; Leukemia, Lymphoid; Leukemia, Myeloid, Acute; Lymphocytes; Lymphoma, Non-Hodgkin; Male; Middle Aged; Moloney murine leukemia virus; Oligonucleotides; Polynucleotides; Templates, Genetic; Thymine Nucleotides; Thymus Gland; Tritium

Topics: Adenosine Triphosphate; Animals; Carbon Radioisotopes; Cell Division; Cells, Cultured; Chromatography; Cytosine Nucleotides; Guanosine Triphosphate; Leukemia, Experimental; Leukemia, Lymphoid; Mice; NAD; Purine Nucleotides; Uracil Nucleotides; Uridine Diphosphate Sugars

Topics: Adenosine Triphosphate; Avian Leukosis Virus; Centrifugation, Density Gradient; Chromatography; Chromatography, DEAE-Cellulose; Chromatography, Gel; Cytosine Nucleotides; DNA, Neoplasm; DNA, Viral; Guanosine Triphosphate; Humans; Leukemia, Lymphoid; Nucleic Acid Hybridization; Oligonucleotides; Polynucleotides; Ribonucleases; RNA-Directed DNA Polymerase; RNA, Viral; Templates, Genetic; Thymine Nucleotides; Tritium