guanosine-triphosphate and Multiple-Myeloma

The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various individual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.

Topics: Animals; Bone and Bones; Bone Neoplasms; Bone Resorption; Diphosphonates; Guanosine Triphosphate; Humans; Models, Biological; Models, Chemical; Multiple Myeloma; Neoplasm Metastasis; Nitrogen; Osteoclasts; Osteocytes; Osteoporosis; Protein Processing, Post-Translational; T-Lymphocytes; Treatment Outcome

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

Multiple myeloma (MM) is an incurable plasma cell malignancy. To investigate biochemical lesions associated with MM, we constructed an expression cDNA library from the OPM-2 human myeloma line. A highly transforming H-Ras mutant was identified by transfection analysis using NIH 3T3 cells. DNA sequencing demonstrated a single-point mutation at position 117 located in the guanine nucleotide-binding site resulting in a lysine-to-glutamic acid substitution. This mutant, H-Ras (K117E), was found to be constitutively activated in terms of GTP binding. We compared the biological effects of H-Ras (K117E) and H-Ras (G12V) in 32D murine hematopoietic progenitor cells. Whereas both Ras proteins are constitutively activated, 32D cells expressing H-Ras (G12V) are still dependent on IL-3 for survival and proliferation while cells carrying H-Ras (K117E) become IL-3 independent. Similar experiments conducted with the B9 line, an IL-6-dependent hybridoma, also demonstrated that B9/H-Ras (K117E) became IL-6-independent. Expression of H-Ras (K117E) in the human IL-6-dependent ANBL-6 myeloma line resulted in enhanced proliferation at suboptimal concentrations of IL-6. These observations suggest that H-Ras mutations at the binding site for the GTP nucleotide ring structure may also represent activating lesions and have additional biological effects when compared to previously described Ras mutants.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; Genes, ras; Growth Substances; Guanosine Triphosphate; Humans; Mice; Multiple Myeloma; Mutation; Tumor Cells, Cultured

Interferons (IFNs) are a family of hormone-like secretory proteins with multiple phenotypical changes, including gene expression and morphological alterations. Earlier studies have shown that IFN-activated Tyk2 kinase physical associates with p95Vav (Vav), a proto-oncogene gene product expressed in hematopoietic cells. Since Tyk2 is a cytoplasmic kinase and Vav is believed to be localized in the nuclear compartment, here we explored the possibility of Vav redistribution in IFN-alpha-activated cells, using the U266 human myeloma cell line as a model system. Using biochemical assays and in situ confocal microscopy, we demonstrate that IFN-alpha treatment triggers a rapid (10 min) translocation of Vav from the nuclear compartment to the cytoplasm. In addition, we also show the existence of IFN-alpha-induced physical interaction between Vav and Ku80, Ku80, and Tyk2, and among Vav, Ku80, and Tyk2 in the cytoplasmic compartment of IFN-stimulated cells. The observed IFN-alpha-induced association among Vav, Ku80, and Tyk2 was dependent on cellular tyrosine kinase activity. Since recently Vav has been shown to promote the GDP/GTP exchange activity of the cytoskeleton signaling molecule small GTPase Rac1 and activates its downstream signaling, our present findings raise the possibility of involvement of the small GTPase in IFN signaling leading to its biological effects, including cytoskeleton reorganization.

Topics: Antigens, Nuclear; Cell Cycle Proteins; Cell Nucleus; Culture Media, Serum-Free; Cytoplasm; DNA Helicases; DNA-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Interferon-alpha; Ku Autoantigen; Multiple Myeloma; Nuclear Envelope; Nuclear Proteins; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; Signal Transduction; Transcription Factors; Tumor Cells, Cultured; TYK2 Kinase

The transcription of U1 RNA genes was studied in isolated nuclei from mouse myeloma cells. Using a cloned U1b gene as a probe, we showed that isolated nuclei synthesize both U1b and U1a RNA. The U1 RNAs were initiated in vitro, as measured by incorporation of adenosine 5'-O-(2-thiotriphosphate) into U1 RNA. There was transcription of the 3'-flanking region but no transcription of regions directly 5' to the U1 genes. In addition to U1 RNAs of the correct length which were released from the nuclei, there were larger RNAs, presumably resulting from transcription into the 3'-flanking region, which were retained in the nuclei. Chase experiments showed that these longer transcripts were not precursors to mature U1 RNA, a finding consistent with the idea that 3'-end formation is coincident with transcription. During the chase, there was maturation of the 3' ends of U1a and U1b RNAs from slightly longer precursors. In addition to accurate transcription of U1 RNA, there was also synthesis of U2 and U3 RNA. All three of these RNAs were transcribed by RNA polymerase II, as measured by their sensitivity to alpha-amanitin.

Topics: Adenosine Triphosphate; Amanitins; Animals; Cell Nucleus; Cell-Free System; DNA, Recombinant; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Mice; Multiple Myeloma; Nucleic Acid Hybridization; RNA, Small Nuclear; Thionucleotides; Transcription, Genetic; Tumor Cells, Cultured

Precursor RNA substrates for splicing reaction were synthesized in vitro from a plasmid DNA in which the early region 2 gene of adenovirus 2 was fused to an efficient bacteriophage promoter (Salmonella phage 6). Pre-mRNA splicing activity from nuclear extracts of MOPC-315 mouse myeloma cells was partially purified 108-fold by three chromatographic steps. The in vitro splicing reaction catalyzed by the partially purified fractions was efficient (60-80% substrate conversion) and accurate at the nucleotide level. The reaction occurred with crude or purified fractions without any detectable lag and nucleotides (ATP or GTP) were absolutely required. Monoclonal anti-Sm antibodies that quantitatively immunoprecipitate U1 small nuclear ribonucleoprotein particles totally inhibited the splicing activity of the purified fractions, indicating that U1 small nuclear RNPs had co-purified with the activity and were absolutely required for the splicing reaction.

Topics: Adenosine Triphosphate; Adenoviridae; Animals; Base Sequence; Cell Line; DNA, Viral; Electrophoresis, Polyacrylamide Gel; Guanosine Triphosphate; Mice; Multiple Myeloma; Nucleic Acid Precursors; Plasmids; Ribonucleoproteins; Ribonucleoproteins, Small Nuclear; RNA Precursors; RNA Splicing; RNA, Messenger

Isolated nuclei incorporate few methyl groups into RNA when they are incubated with S-adenosyl[methyl-3H3]methionine and four ribotriphosphates. When the nuclei were supplemented with a soluble total cell protein extract, the incorporation of methyl groups into RNA was stimulated 3-6-fold. All classes of RNA were methylated. Methylation of the 2'-OH of ribose and the bases of ribosomal RNA occurred predominantly on endogenous ribosomal RNA precursors, with a minority (20%) occurring on the newly synthesized rRNA precursor. Methylation of the tRNA precursor occurred on both endogenous (40%) and newly synthesized (60%) molecules. The methylation of adenosine in hnRNA occurred predominantly on molecules transcribed in vitro and was sensitive to 1 microgram/mL alpha-amanitin. A final site of methylation was the 7 position of guanosine of the cap structure. About 10% of the RNA polymerase II transcripts were capped in vitro. Capping was blocked 90% by 1 microgram/mL alpha-amanitin and was independent of the presence of the cell protein extract.

Topics: Animals; Cell Line; Cell Nucleus; Cell-Free System; Guanosine Triphosphate; Methylation; Mice; Molecular Weight; Multiple Myeloma; RNA; RNA Caps; RNA, Ribosomal; RNA, Transfer

The effects of the newly synthesized compound 9-beta-D-arabinofuranosylguanine 5'-triphosphate (ara-GTP) on the activity of DNA polymerases from mouse cells and oncornavirus were compared with those of 9-beta-D-arabinofuranosyladenine 5'-triphosphate. Ara-GTP did not replace deoxyguanosine 5'- triphosphate as substrate for these DNA polymerases but inhibited the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. DNA polymerase alpha was more sensitive than DNA polymerases beta and gamma and viral DNA polymerase to inhibition by ara-GTP. The inhibitions by ara-GTP and 9-beta-D-arabinofuranosyladenine 5'-triphosphate were due to competition or partial competition 5'-triphosphate were due to competition or partial competition with deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied depending on the combination of inhibitor, substrate(s), and enzyme species.

Topics: Animals; Arabinonucleotides; Cells, Cultured; Deoxyribonucleotides; Guanosine Triphosphate; Kinetics; Mice; Multiple Myeloma; Neoplasms, Experimental; Nucleic Acid Synthesis Inhibitors; Rats; Rauscher Virus; Substrate Specificity; Vidarabine Phosphate

Nuclei isolated from mouse myeloma cells grown in tissue culture are capable of synthesizing RNA for prolonged periods of time. Addition of cytoplasmic extracts to the system stimulates slightly the rate and prolongs the time of synthesis. As judges by sedimentation in SDS and in formamide gradients, the size of the RNA synthesized is heterogeneous from smaller than 10S to larger than 45S, thus resembling in vivo made RNA. The characteristics of some of the RNA are in keeping with those expected to be for mRNA. Fifty percent of the RNA synthesis is sensitive to alpha-amanitin. After an incubation of two hours in the absence of alpha-amanitin about 10 percent of the newly synthesized RNA is found outside of the nuclei; it sediments with a broad distribution at 18S. A considerable fraction of the RNA that is released from nuclei in vitro can promote the formation of polyribosomes, and contains molecules that are polyadenylated and "capped".

Topics: Amanitins; Animals; Cell Nucleus; Cells, Cultured; Cytoplasm; Guanosine Triphosphate; Kinetics; Mice; Molecular Weight; Multiple Myeloma; Poly A; Polyribosomes; Reticulocytes; Ribonucleases; RNA, Messenger; RNA, Neoplasm; Uridine

Topics: Animals; Cell Fractionation; Cell Line; Cell Nucleus; Centrifugation, Density Gradient; DNA; DNA-Directed RNA Polymerases; Guanosine Triphosphate; Mice; Molecular Weight; Multiple Myeloma; Mycotoxins; Nucleic Acid Hybridization; Nucleoproteins; Phosphorus Radioisotopes; RNA, Neoplasm; RNA, Ribosomal; RNA, Transfer; Transcription, Genetic; Tritium; Xenopus

Topics: Adenosine Triphosphate; Ammonium Sulfate; Animals; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Cytosine Nucleotides; DNA Nucleotidyltransferases; DNA, Neoplasm; Glycerol; Guanosine Triphosphate; Manganese; Methods; Mice; Mice, Inbred BALB C; Molecular Weight; Multiple Myeloma; Neoplasms, Experimental; Polynucleotides; Templates, Genetic; Thymine Nucleotides; Tritium