Inhibition of nuclear envelope nucleoside triphosphatase-regulated nucleocytoplasmic messenger RNA translocation by 9-beta-D-arabinofuranosyladenine 5'-triphosphate in rodent cells.
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Nucleocytoplasmic translocation of polyadenylated messenger RNA is an energy-dependent process which is regulated by a nuclear envelope nucleoside triphosphatase; this enzyme was found to be stimulated by the 3'-terminal polyadenylic acid [poly(A)] tail of messenger RNA (Bernd, A., Schroder, H. C., Zahn, R. K., and Muller, W. E. G. Eur. J. Biochem., 129: 43-49, 1982). RNA efflux from isolated mouse lymphoma (L5178Y) cell nuclei is strongly reduced if 9-beta-D-arabinofuranosyladenine 5'-triphosphate (ara-ATP) is present in the transport medium. Half-maximal inhibition of RNA efflux occurs with 120 microM ara-ATP. Most likely, the inhibitory effect of ara-ATP is caused by inhibition of nuclear envelope nucleoside triphosphatase; this enzyme was found to be highly sensitive to inhibition by this antibiotic. The inhibition type of the nucleoside triphosphatase of rat liver nuclear ghosts is competitive with respect to ATP; the Ki:Km ratio was determined to be 0.27. Besides nucleoside triphosphatase, nuclear envelopes contain a protein phosphokinase modulating the affinity of pore complex laminae to poly(A). This enzyme was also found to be strongly inhibited by ara-ATP in a competitive way with respect to ATP (Ki:Km, 0.056) and could therefore also contribute to the overall inhibition of RNA transport. The polyadenylation of endogenous RNA by poly(A) polymerase(s) in intact rat liver nuclei as well as in nuclear matrices isolated from the same source was found to be markedly suppressed in the presence of ara-ATP. The inhibitions of both poly(A) polymerase activities (contained in whole nuclei or nuclear matrix bound) are of the competitive type with respect to ATP. In in vitro assays, nuclear envelope nucleoside triphosphatase is inhibited by microtubule protein. Of the 2 ATP-dependent enzyme activities associated with microtubule protein (cyclic adenosine 3':5'-monophosphate-dependent protein kinase and adenosine triphosphatase), only the kinase was slightly affected by ara-ATP. Cellular uptake of adenosine 5'-monophosphate and perhaps 9-beta-D-arabinofuranosyladenine 5'-monophosphate (ara-AMP) is facilitated by a cellular membrane-bound 5'-nucleotidase. Our studies revealed that neither cleavage of ara-AMP nor inhibition of the enzyme activity by ara-AMP occurs. 9-beta-D-Arabinofuranosyladenine and ara-AMP represent neither direct mutagens nor premutagens as determined by the Salmonella-mammalian microsome mutagenicity test. (+info)
Phase I clinical investigation of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (NSC 312887), a new purine antimetabolite.
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9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-monophosphate (NSC 312887) is a new purine antimetabolite that has been evaluated in a Phase I clinical trial. The schedule of administration consisted of a single i.v. infusion over a period of 30 min once each day for 5 consecutive days, repeated at 4-week intervals. Thirteen patients received 30 courses of the drug in a dose range of 18 to 40 mg/sq m/day. Granulocytopenia and thrombocytopenia were dose limiting. Repeated courses produced similar degrees of granulocytopenia, but in 7 of 7 patients receiving 2 or more courses, the degree of thrombocytopenia was less severe during the first than during subsequent courses. Myelosuppression in humans was more severe than predicted from the mouse model. Lymphopenia was profound at all dose levels, but reversed within 3 weeks. Somnolence occurred during infusion in 8 of 13 patients, but quickly cleared after the infusion was completed. The infused drug was rapidly dephosphorylated in plasma and then cleared so there was no cumulation of drug in plasma when it was rapidly infused once each day in these doses. Phase II studies of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate are planned at a starting dose of 18 mg/ sq m/day for patients with prior chemotherapy or radiotherapy and 25 mg/sq m/day for those without prior therapy, as a single dose on each of 5 consecutive days repeated at 21- to 28-day intervals. (+info)
Effects on human peripheral lymphocytes of in vivo administration of 9-beta-D-arabinofuranosyl-2-fluoroadenine-5'-monophosphate (NSC 312887), a new purine antimetabolite.
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9-beta-D-Arabinofuranosyl-2-fluoroadenine-5'-monophosphate (NSC 312887) (2-F-ara-AMP) is a new purine antimetabolite with documented preclinical activity against a number of animal tumors. Data from in vitro studies and preclinical animal toxicology trials indicated that 2-F-ara-AMP might be lymphocytotoxic. We studied effects of 2-F-ara-AMP on peripheral lymphocytes of patients receiving the agent in a Phase I clinical trial. Eleven patients received 13 courses given by i.v. bolus daily for 5 days. Mononuclear cells were isolated, and lymphocyte subsets were quantitated by immunofluorescence and flow cytometry 1 day before treatment and 4 hr after the final infusion. Lymphocytopenia developed rapidly (median time to nadir, 6 days) and was reversible. Standard leukocyte counts, differential counts, and percentages of isolated mononuclear cells reactive with monoclonal antibodies were used to calculate numbers of peripheral cells in each major lymphocyte subpopulation. Total T-lymphocyte counts fell during all treatment courses, with calculated mean absolute T-cell counts decreasing by 90%. Decreases were observed in all major T-lymphocyte subsets. By contrast, calculated B-lymphocyte counts decreased an average of 50% and were noted to increase during two treatment cycles. We also compared in vitro recoveries of cells from each major lymphocyte subpopulation before and after administration of 2-F-ara-AMP. Recoveries of total mononuclear cells, total T-cells, and non-T-, non-B-cells all were reduced substantially by 2-F-ara-AMP, but B-cell recovery was not reduced. These in vivo data, the first in human subjects, are in agreement with in vitro studies of halopurine nucleotide analogues which have demonstrated that T-cells are more sensitive than are B-cells to the cytotoxic effects of these compounds. (+info)
Selective penetration and pharmacological activity of lactosaminated albumin conjugates of adenine arabinoside 5-monophosphate (ara-AMP) in mouse liver.
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With the aim of improving the chemotherapeutic index of adenine arabinoside 5-monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes where it is digested in lysosomes. In mice, the L-[3H]SA-ara-AMP conjugates, intravenously injected, selectively penetrated the liver, only small quantities were taken up by cells of spleen, bone marrow, intestine, and brain. After administration of the conjugate to mice with Ectromelia virus hepatitis, ara-AMP was selectively concentrated in liver in a pharmacologically active form. If L-SA-ara-AMP conjugates behave in man as in mouse, their administration to patients with chronic hepatitis B should result in a selective concentration of ara-AMP in liver with a more efficient inhibition of virus replication accompanied by lower toxicity for other tissues. (+info)
Comparison of properties of woodchuck hepatitis virus and human hepatitis B virus endogenous DNA polymerases.
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The principal properties of the DNA polymerases of woodchuck hepatitis virus and human hepatitis B virus were compared. The enzymes of both viruses exhibited optimal activities in the same range of pH, ionic strength, and MgCl2 concentration. Like human hepatitis B virus DNA polymerase, the woodchuck hepatitis virus DNA polymerase was strongly inhibited by phosphonoformic acid but not by phosphonoacetic acid and aphidicolin. Similar inhibition patterns for both enzymes were observed with arabinofuranosyl nucleotides (9-beta-D-arabinofuranosyladenine-5'-triphosphate, 1-beta-D-arabinofuranosylcytosine-5'-triphosphate, 1-beta-D-arabinofuranosylthymine-5'-triphosphate) and dideoxythymidine triphosphate, whereas no effect was obtained with corresponding nucleosides. The therapeutic significance of these results and the relevance of the woodchuck as an experimental animal model for the study of human hepatitis B virus infections are discussed. (+info)
Effect of arabinofuranosylthymine on the replication of Epstein-Barr virus and relationship with a new induced thymidine kinase activity.
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1-beta-D-Arabinofuranosylthymine (araT) is a selective inhibitor of Epstein-Barr virus replication induced in both thymidine kinase (TK)-negative (TK-) and TK+ variants of the lymphoid cell line P3HR-I. This analog has no effect on the growth of noninduced cells (T. Ooka and A. Calender, Virology 104:219-223, 1980). The synthesis of early antigens is not affected by the analog, whereas that of late viral capsid antigens is completely inhibited, as demonstrated by the indirect immunofluorescence technique; kinetic reassociation experiments have also shown that araT strongly inhibits replication of viral DNA. Phosphorylation of the tritiated form of the analog ([3H]araT) was analyzed by thin-layer chromatography in cultures of control and induced cells, and the results demonstrated that only induced cells can convert the analog to the triphosphate form. These results indicate that the selective effect of araT in induced cells is probably related to a new virally induced TK activity. Preliminary characterization of this new activity has shown that it is able to phosphorylate the analog specifically, whereas cellular TKs cannot. araTTP, a final phosphorylation product of araT, is a potent inhibitor of Epstein-Barr virus-specific DNA polymerase, suggesting a possible inhibitory action of this product on Epstein-Barr virus replication. (+info)
Conformational analysis of oligoarabinonucleotides. An NMR and CD study.
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A 500 and 300 MHz proton NMR study of the series of oligoarabinonucleotides 5'aAMP, 3'aAMP, aA-aA, (aA-)2aA and (aA-)3aA is presented. In addition, circular dichroism is used to study the stacking behaviour of aA-aA. The complete 1H-NMR spectral assignment of the compounds (except the tetramer) is given. Proton-proton and proton-phosphorus coupling constants, obtained by computer simulation of the high-field region of the spectra, yield information on the conformation of the arabinose rings (N- or S-type) and on the intramolecular stacking properties of the dimer and the trimer. The monomers 5'aAMP and 3'aAMP exhibit a preference for N- and S-type sugar conformation, respectively. It is shown that the dimer aA-aA at low temperature prefers a mixed stacked state of the type aA(S)-aA(N). In the trimer the aA(2)-aA(3) fragment exhibits a conformation similar to that found in the dimer, whereas the aA(1) residue prefers to adopt S-type sugar and has some tendency to stack upon residue aA(2). (+info)
Quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in the leukemic cells from bone marrow and peripheral blood of patients receiving 1-beta-D-arabinofuranosylcytosine therapy.
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A method for the detection and quantitation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the active metabolite cells and leukemic cells of the peripheral blood from patients receiving ara-C therapy is described. ara-CTP is separated from normal cellular nucleotides by high-pressure liquid chromatography and is quantitated by its absorbance of ultraviolet light at 280 nm with a lower limit of sensitivity of 25 pmol/2 x 10(7) cell equivalents. During separate courses of continuous infusion of different therapeutic doses of ara-C, ara-CTP accumulated in the leukemic bone marrow cells of a patient with acute myelogenous leukemia in proportion to the dose of ara-C. Continuous infusion of ara-C (90 mg/sq m/day) resulted in plateau levels of ara-CPT in peripheral blast cells after 24 hr (115 pmol/1 x 10(7) cell equivalents). A priming dose of ara-C(125 to 250 mg/sq m) followed by a 1-hr infusion of an equal dose of ara-C to patients with acute myelogenous leukemia facilitated the determination of ara-CTP retention in bone marrow and peripheral blood leukemic cells in vivo. This procedure should be useful for extended studies of the biochemical pharmacology of ara-CTP in vivo. (+info)