phosphorus-radioisotopes and Leukemia--Erythroblastic--Acute

Topics: Anemia; Anemia, Sideroblastic; Animals; Chromosome Aberrations; Chromosome Disorders; Female; Granulocytes; Hematologic Diseases; Humans; Japan; Leukemia; Leukemia, Erythroblastic, Acute; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Nuclear Warfare; Phosphorus Radioisotopes; Polycythemia Vera; Precancerous Conditions; Radiation Injuries; Thrombocytopenia

Tissue transglutaminase (TGC, TG2, 80 kDa) is inactive in cross-linking reactions and is converted in vitro and in vivo to the TG (55 kDa) active isoform (Fraij in J Cell Biochem 112:2469-2489, 2011). Two isoforms of human TGC were cloned from human erythroleukemia (HEL) cells induced with retinoic acid (RA) and termed TGH, 63 kDa (Fraij et al. in J Biol Chem 267:22616-22673, 1992) and TGH2, 37 kDa. The purified TGC isoforms exhibited GTPase activity and TGH and TGH2 showed higher activities than the native TGC protein. In all normal cells examined, TGC was found in membrane fractions several fold higher than the supernatant fractions; however, in the natural tumor cell line HEL the TGC cellular distribution was reversed. Although TGC is the major enzyme in normal human erythrocytes, its expression level was significantly decreased in HEL cells. RA treatment induced a sevenfold increase in the level of TGC protein in HEL cells and was accompanied by its translocation to cell membranes. When isolated membrane and supernatant fractions from normal human foreskin (CF3), normal human embryonic lung (WI-38), and HEL cells treated with or without RA were incubated with [(32)P]-ATP at 37 °C for 1 h, more radio-labeled proteins were detected in the membrane fractions than the cytosolic fractions. More labeled protein bands were detected in RA treated HEL cells in comparison to control HEL cell extracts. Radio labeled proteins coimmunoprecipitated with the TGC isoforms in RA treated HEL membrane fractions thereby confirming that the radio-labeled material consists of endogenous proteins associated with TGC isoforms. Protein phosphorylation is related to the induction and translocation of the isoforms in RA treated cells. These results show that the TGC isoforms complexes with proteins in vivo and that the phosphorylation of these proteins is catalyzed directly by TGC kinase activity or indirectly by the TGC phosphorylation of other protein kinases.

Topics: Adenosine Triphosphate; Analysis of Variance; Blotting, Western; Cell Line; Cell Line, Tumor; Cell Membrane; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Isoenzymes; Leukemia, Erythroblastic, Acute; Phosphorus Radioisotopes; Phosphorylation; Protein Glutamine gamma Glutamyltransferase 2; RNA, Messenger; Tissue Distribution; Transglutaminases; Tretinoin

Protein phosphorylation is an early event that follows the interaction of erythropoietin (Epo) with its receptor, even though this receptor lacks a kinase domain. To further define the role of protein kinases in Epo-mediated signal transduction, the effect of Epo on serine-threonine kinase activity was examined in the Epo-dependent cell line, HCD-57, using a kinase renaturation assay. In HCD-57 cells synchronized in G0 phase by centrifugal elutriation, multiple serine-threonine kinases were constitutively active, and exposure to Epo was associated with an increase in the activity of kinases with apparent molecular masses of 170, 120, and 90-95 kD. Phosphoamino acid analysis established the covalent incorporation of 32P into serine and threonine for constitutively active kinases and into serine alone for the 90-95 kD kinase. Reelectrophoresis experiments established that 32P incorporation represented kinase autophosphorylation as opposed to protein substrate phosphorylation. Epo-associated serine kinase autophosphorylation was both hormone concentration and time dependent as well as restricted to the G0, G1, and S phases of the cell cycle. Cell fractionation studies localized the activity of the 90-95 kD serine kinase to the plasma membrane.

Topics: Animals; Cell Cycle; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Cells, Cultured

GATA-1 is a zinc finger DNA-binding protein thought to be involved in the expression of the vast majority of erythroid specific genes. We have examined the phosphorylation of GATA-1 in murine erythroleukemia (MEL) cells and have mapped the sites of phosphorylation by overexpression of GATA-1 in monkey kidney COS cells. We show that GATA-1 is phosphorylated on 6 serines within its amino terminus in uninduced MEL cells and that a 7th site, serine 310, becomes phosphorylated after MEL cells are induced to differentiate by exposure to dimethyl sulfoxide. This site lies near the carboxyl boundary of the DNA-binding domain in a conserved region of the protein believed to be involved in DNA bending. Detailed analyses indicate, however, that phosphorylation at this site, or the other sites identified, does not significantly influence DNA-binding affinity or specificity, DNA bending, or transcriptional transactivation by GATA-1.

Topics: 3T3 Cells; Amino Acid Sequence; Amino Acids; Animals; Autoradiography; Base Sequence; Binding Sites; Cell Differentiation; Cell Line; Chlorocebus aethiops; Conserved Sequence; Dimethyl Sulfoxide; DNA Primers; DNA Probes; DNA-Binding Proteins; Erythroid-Specific DNA-Binding Factors; GATA1 Transcription Factor; Gene Expression; Kidney; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Peptide Mapping; Phosphopeptides; Phosphorus Radioisotopes; Phosphorylation; Plasmids; Polymerase Chain Reaction; Transcription Factors; Transfection; Tumor Cells, Cultured; Zinc Fingers

Three polyphosphoinositides containing phosphate at the D-3 position of the inositol ring can be generated in vitro by phosphorylation of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate by a phosphatidylinositol-3-kinase (Auger, K. R., Serunian L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175. An alternative pathway for in vivo synthesis of one of these lipids was recently suggested: phosphatidylinositol 3,4-bisphosphate could be produced by phosphorylation of phosphatidylinositol 3-phosphate at the D-4 position of the inositol ring (Yamamoto, K., and Lapetina, E. G. (1990) Biochem. Biophys. Res. Commun. 168, 466-472). Here we demonstrate the presence of an enzyme in human platelets that phosphorylates [32P]phosphatidylinositol 3-phosphate to produce [32P]phosphatidylinositol 3,4-bisphosphate. This enzyme is Mg2(+)-dependent and its apparent molecular mass is approximately 150 kDa as estimated by sucrose gradient centrifugation and gel filtration chromatography. Unlike the major phosphatidylinositol-4-kinase in platelets that is stimulated by the detergent Nonidet P-40, the phosphatidylinositol-3-phosphate-4-kinase is inhibited by Nonidet P-40. Both activities are also differentiated by the action of adenosine. The discovery of this new enzyme raises the possibility that multiple pathways exists for generating D-3 phosphorylated phosphoinositides.

Topics: 1-Phosphatidylinositol 4-Kinase; Adenosine; Blood Platelets; Cell Line; Centrifugation, Zonal; Chromatography, High Pressure Liquid; Cytosol; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Phosphatidylinositol 3-Kinases; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphorus Radioisotopes; Phosphorylation; Phosphotransferases

Using as a hybridization probe cDNA 35s RNA from the Rauscher leukemia cells, a part of rRNA gene cluster from the gene library of mice C-1 erythroleukemia cells has been cloned. Fragment 6.7 kb recloned into pUC19 rRNA was used as a probe to analyse organization of rRNA genes of mice with RL. The observed amplification and rearrangement in genome DNA of spleen cells are determined by a new type of their rRNA genes rearrangement in the nontranscribed as well as in the transcribed part of rRNA.

Topics: Animals; Cloning, Molecular; DNA; DNA Probes; Gene Amplification; Gene Rearrangement; Genes; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Mice; Mice, Inbred AKR; Mice, Inbred BALB C; Nucleic Acid Hybridization; Phosphorus Radioisotopes; Poly A; Polymorphism, Restriction Fragment Length; Rauscher Virus; RNA Probes; RNA, Ribosomal

Lipid constituents were examined in the spleen of mice 10, 14 and 18 days after infection with Rauscher leukaemia virus. (i) The total lipid content, as related to the amount of protein, decreased with the development of the tumour. (ii) Spleen cells of control and infected mice synthesized the same lipids. (iii) In the infected spleen, free fatty acids increased by 80% and triglycerides decreased by 44% as compared to the control. The percentage distribution of phospholipid components showed a change in tumour cells: lecithin and phosphatidylethanolamine increased, lysolecithin and sphingomyelin decreased. (iv) After intraperitoneal injection of [32P] orthophosphate, in infected mice the incorporation of 32P into phosphatidylcholine and phosphatidylethanolamine increased, while 32P incorporation into lysophosphatidylcholine + sphingomyelin and phosphatidylinositol + phosphatidylserine fractions decreased.

Topics: Animals; Cholesterol; Chromatography, Thin Layer; Fatty Acids, Nonesterified; Glycerides; Injections, Intraperitoneal; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lipid Metabolism; Lysophosphatidylcholines; Mice; Mice, Inbred BALB C; Phosphates; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Phosphorus Radioisotopes; Rauscher Virus; Sphingomyelins; Spleen; Triglycerides