guanosine-triphosphate and Plasmacytoma

Inosine monophosphate dehydrogenase (IMPDH) inhibitors have been used to induce leukemia blast cell differentiation but have not been tested in multiple myeloma for activity. Currently, available IMPDH inhibitor, mycophenolate mofetil (MMF), which is known as an immunosuppressant, was shown to induce apoptosis in myeloma cell lines. On the basis of our preclinical studies, we designed a clinical study to test our hypothesis that MMF has antimyeloma activity.. A Phase I MMF dose escalation study was conducted in relapsed and refractory myeloma patients who had documented disease progression by myeloma markers or bone marrow plasmacytosis to determine the maximum tolerated dose, toxicities, and efficacy of the drug. To assess the activity of IMPDH inhibition in the myeloma cells of patients, we measured intracellular nucleotide triphosphate levels by high-performance liquid chromatography-based analysis and examined the correlation with clinical response.. Among the 11 study patients, MMF was generally well tolerated and was administered up to a maximum dose of 5 g/day. The most common toxicity was grade 1 fatigue (n = 4, 36%). One patient had a partial response (3 g/day), four patients had stable disease, and six patients had progression of disease. There was a statistically significant difference in the intracellular dGTP level changes between the stable disease/partial response group versus progression of disease.. MMF at 1 to 5 g/day daily dose is well tolerated by patients with relapsed and refractory multiple myeloma patients. Positive correlation between clinical response and depletion of intracellular dGTP level was shown. Future drug development to target this enzyme maybe useful in treating myelomas.

Topics: Aged; Bone Marrow; Chromatography, High Pressure Liquid; Disease Progression; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Guanosine Triphosphate; Humans; Immunosuppressive Agents; IMP Dehydrogenase; Male; Maximum Tolerated Dose; Middle Aged; Multiple Myeloma; Mycophenolic Acid; Plasmacytoma; Salvage Therapy

A protein kinase activity with high specificity for histone H1 was isolated from mouse plasmacytoma, Morris hepatoma and normal mouse liver and compared by ion exchange chromatography after DEAE-cellulose, hydroxylapatite and Sephadex G-200 chromatography. This cAMP-independent histone H1 kinase is not affected by the heat-stable cAMP-dependent protein kinase inhibitor. It has the following particular properties: it prefers GTP to ATP as substrate and was found to be present with a great activity only in neoplastic tissues. No phosphatase activity was detected in the partially purified histone H1 kinase fraction from normal and neoplastic cells. These results suggest either an increase amount of histone H1 kinase and/or of its activator in neoplastic cells, or the presence of a strong inhibitor in normal cells. This histone H1 kinase appears to be analogous to the chromatin bound kinase which phosphorylates histone H1 at the NH2 and COOH terminal regions. We might suggest an implication of this kinase in the regulation of cell division.

Topics: Adenosine Triphosphate; Animals; Cyclic AMP; Guanosine Triphosphate; Histones; Liver Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Microsomes, Liver; Phosphorylation; Plasmacytoma; Protamine Kinase; Protein Kinases; Rats; Substrate Specificity

RNA capping by partially purified HeLa cell GTP:RNA guanylyltransferase has been shown to occur in the following sequence of two partial reactions involving a covalent protein-guanylate intermediate: (i) E(P68) + GTP in equilibrium E(P68-GMP) + PPi (ii) E(P68-GMP) + ppRNA in equilibrium GpppRNA + E(P68) Initially, the enzyme reacts with GTP in the absence of an RNA cap acceptor to form a covalent protein-guanylate complex. This complex consists of a GMP residue linked via a phosphoamide bond to a Mr = 68,000 protein. The enzyme then transfers the guanylate residue from the Mr = 68,000 polypeptide to the 5' end of diphosphate-terminated poly(a) to yield the capped derivative GpppA(pA)n. Both partial reactions have been shown to be reversible. In the reverse of Reaction i, E(P68--GMP) reacts with PPi to regenerate GTP. In the reverse of Reaction ii, the enzyme catalyzes the transfer of the 5'-GMP from capped RNA to the Mr = 68,000 protein to form protein-guanylate complex. A divalent cation is required for both partial reactions. The Mr = 68,000 protein is presumed to be a subunit of the HeLa guanylyltransferase. This interpretation is consistent with the sedimentation coefficient of 4.2 S of the native enzyme. Preliminary studies of RNA guanylyltransferase from mouse myeloma tumors suggest a similar mechanism of transguanylylation involving a Mr = 68,000 protein-guanylate complex. These data, in conjunction with previous studies of vaccinia virus guanylyltransferase (Shuman, S., and Hurwitz, J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 187-191) suggests that covalent GMP-enzyme intermediates may be a general feature of the RNA capping reaction.

Topics: Animals; Cations, Divalent; Cell Line; Guanosine Triphosphate; HeLa Cells; Humans; Kinetics; Mice; Neoplasms, Experimental; Nucleotidyltransferases; Plasmacytoma; Protein Binding; RNA Caps

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)-cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle-associated cAMP-independent protein kinase(s) using [gamma-32P]ATP and [gamma-32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP-independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Guanosine Triphosphate; Mice; Neoplasms, Experimental; Nucleoproteins; Phosphorylation; Plasmacytoma; Polyribosomes; Protein Kinases; Ribonucleoproteins; Substrate Specificity

Topics: Adenosine Triphosphate; Animals; Cell Line; Chromatography, Ion Exchange; DNA Nucleotidyltransferases; Guanosine Triphosphate; Mice; Mice, Inbred BALB C; Plasmacytoma; Retroviridae; RNA-Directed DNA Polymerase; Templates, Genetic; Thymine Nucleotides; Thymus Gland; Tritium

The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41 (mouse plasmocytoma) immunoglobulin light chain, purified to a single peak as shown by polyacrylamide gel electrophoresis, was used to synthesize complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. DNA synthesis is entirely dependent on added RNA template and oligo(dT) primer. Both the size and the concentration of the primer affect the reaction. The product behaves similarly to DNA during centrifugation in cesium sulfate density gradients. It is shown by hybridization that the DNA made is complementary to the purified template, light-chain mRNA. The high specific activity of the complementary DNA should make it suitable for gene-dosage experiments. According to alkaline sucrose gradient analyses, some complete complementary DNA transcripts of the 14S mRNA seem to be made. Oligo(dG) can also function as a primer for DNA synthesis, possibly by annealing to an internal cluster of cytidines in the mRNA, that correspond to the bases coding for amino-acids 119 and 120 of the MOPC 41 light chain.

Topics: Animals; Avian Leukosis Virus; Cells, Cultured; Centrifugation, Density Gradient; Cytosine Nucleotides; DNA; Guanosine Triphosphate; Immunoglobulins; Mice; Nucleic Acid Hybridization; Oligonucleotides; Plasmacytoma; RNA-Directed DNA Polymerase; RNA, Messenger; Templates, Genetic; Thymine Nucleotides; Transcription, Genetic; Tritium

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Carbon Isotopes; Chromatography, Paper; Glycine; Guanine Nucleotides; Guanosine Triphosphate; Liver; Mice; Mice, Inbred Strains; NAD; NADP; Neoplasm Transplantation; Nucleotides; Phosphates; Phosphorus Isotopes; Plasmacytoma; Purine Nucleotides; Transplantation, Homologous; Uracil Nucleotides