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Molecular Biology of the Cell Jan 1993Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The...
Purine analogues are protein kinase inhibitors, and they block with varying potency and specificity certain of the biological actions of nerve growth factor (NGF). The analogue 6-thioguanine (6-TG) has been shown to inhibit with high specificity protein kinase N (PKN), a serine/threonine protein kinase activated by NGF in several cellular systems. In the present work, immunoprecipitates of p75 NGF receptors from PC12 cells (+/-NGF treatment) were assayed for protein kinase activity using the substrate myelin basic protein under phosphorylating conditions optimal for PKN and in the presence or absence of purine analogues. An NGF-inducible activity was detected, and approximately 80% was inhibited by purine analogues. This activity was maximally stimulated by NGF within 5-10 min, partially decreased by 60 min, and returned to basal levels after 15 h of NGF treatment. The analogue 6-TG inhibited the NGF-inducible p75-associated kinase activity with an IC50 in the range of 15-35 microM. In mutant PC12 nnr-5 cells that lack the Trk NGF receptor, the purine-analogue-sensitive p75-associated kinase activity was not inducible by NFG. In normal PC12 cells, cyclic AMP analogues and epidermal growth factor failed to induce the same activity. Application of either 2-aminopurine or 6-TG to intact cells only slightly inhibit the NGF-dependent induction of the purine-analogue-inhibited p75-associated kinase activity. This activity shares many similarities but also displays some significant differences with cytosolic PKN. Our findings therefore indicate the association of a purine-analogue-sensitive protein kinase with p75 NGF receptors.
Topics: 2-Aminopurine; Animals; Cell Line; Methylthioinosine; Myelin Basic Protein; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Nerve Growth Factor; Thioguanine
PubMed: 7680248
DOI: 10.1091/mbc.4.1.71 -
Journal of Chromatography Nov 1992A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human...
High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a single sample.
A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 x 10(8) red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-methylmercaptopurine metabolites, respectively.
Topics: Chromatography, High Pressure Liquid; Erythrocytes; Humans; Hydrolysis; Inosine Monophosphate; Mercaptopurine; Methylthioinosine; Thionucleotides
PubMed: 1484095
DOI: 10.1016/0378-4347(92)80347-s -
Cancer Research Aug 1992This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus...
This report describes a highly active chemotherapeutic drug combination, consisting of N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide plus 5-fluorouracil, in CD8F1 mice bearing spontaneous, autochthonous, breast tumors or first-passage advanced transplants of these spontaneous tumors. The combination and sequence of administration of these drugs were selected on the basis of known potentiating biochemical interactions. High performance liquid chromatography and nuclear magnetic resonance spectroscopy measurements of biochemical changes resulting from treatment with N-(phosphonacetyl)-L-aspartate plus 6-methylmercaptopurine riboside plus 6-aminonicotinamide indicated a severe depletion of cellular energy levels in the treated tumors. 6-Aminonicotinamide produced a severe block of the pentose shunt, and 5-fluorouracil severely inhibited both thymidylate synthase and thymidine kinase in the treated tumors. This quadruple drug combination, administered on a 10-11-day schedule, produced an impressive partial tumor regression rate of 67% of large, spontaneous, autochthonous, murine breast tumors and a tumor regression rate of 74% of first-passage transplants of the spontaneous breast tumors.
Topics: 6-Aminonicotinamide; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Female; Fluorouracil; Mammary Neoplasms, Experimental; Methylthioinosine; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Orotic Acid; Phosphonoacetic Acid; Phosphoribosyl Pyrophosphate
PubMed: 1379119
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1992The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and...
The pathway for de novo biosynthesis of purine nucleotides contains two one-carbon transfer reactions catalyzed by glycinamide ribotide (GAR) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylases in which N10-formyltetrahydrofolate is the one-carbon donor. We have found that the antifolates methotrexate (MTX) and piritrexim (PTX) completely block the de novo purine pathway in mouse L1210 leukemia cells growing in culture but with only minor accumulations of GAR and AICAR to less than 5% of the polyphosphate derivatives of N-formylglycinamide ribotide (FGAR) which accumulate when the pathway is blocked completely by azaserine. This azaserine-induced accumulation of FGAR polyphosphates is completely abolished by MTX, indicating that inhibition of the pathway is at or before GAR transformylase (reaction 3; Lyons, S. D., and Christopherson, R. I. (1991) Biochem. Int. 24, 187-197). Three h after the addition of MTX (0.1 microM), cellular 5-phosphoribosyl-1-pyrophosphate has accumulated 3.4-fold while 6-methyl-mercaptopurine riboside (25 microM) induces a 6.3-fold accumulation. These data suggest that amido phosphoribosyltransferase catalyzing reaction 1 of the pathway is the primary site of inhibition. In support of this conclusion, we have found that dihydrofolate-Glu5, which accumulates in MTX-treated cells, is a noncompetitive inhibitor of amido phosphoribosyltransferase with a dissociation constant of 3.41 +/- 0.08 microM for interaction with the enzyme-glutamine complex in vitro. Folate-Glu5, MTX-Glu5, PTX, dihydrotriazine benzenesulfonyl fluoride, and AICAR also inhibit amido phosphoribosyltransferase.
Topics: Amidophosphoribosyltransferase; Aminoimidazole Carboxamide; Animals; Azaserine; Folic Acid Antagonists; Leukemia, Experimental; Methotrexate; Methylthioinosine; Mice; Purines; Pyrimidines; Ribonucleotides; Tumor Cells, Cultured
PubMed: 1597445
DOI: No ID Found -
The Journal of Biological Chemistry Mar 1992Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal...
Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity.
Human B lymphoblast lines severely deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT) were selected for resistance to 6-thioguanine from cloned normal and phosphoribosylpyrophosphate (PP-Rib-P) synthetase-superactive cell lines and were compared with their respective parental cell lines with regard to growth and PP-Rib-P and purine nucleotide metabolism. During blockade of purine synthesis de novo with 6-methylthioinosine or aminopterin, inhibition of growth of all HGPRT-deficient cell lines was refractory to addition of Ade at concentrations which restored substantial growth to parental cell lines. Ade-resistant inhibition of growth of parental lines by 6-methylthioinosine, however, occurred during Ado deaminase inhibition. Insufficient generation of IMP (and ultimately guanylates) to support growth of lymphoblasts lacking HGPRT activity and blocked in purine synthesis de novo best explained these findings, implying that a major route of interconversion of AMP to IMP involves the reaction sequence: AMP----Ado----Ino----Hyp----IMP. PP-Rib-P generation and purine nucleoside triphosphate pools were unchanged by introduction of HGPRT deficiency into normal lymphoblast lines, in agreement with the view that accelerated purine synthesis de novo in this deficiency results from increased availability of PP-Rib-P for the pathway. Cell lines with dual enzyme defects did not differ from PP-Rib-P synthetase-superactive parental lines in rates of PP-Rib-P and purine synthesis despite 5-6-fold increases in PP-Rib-P concentrations, excretion of nearly 50% of newly synthesized purines, and diminished GTP concentrations. Fixed rates of purine synthesis de novo in PP-Rib-P synthetase-superactive cells appeared to reflect saturation of the rate-limiting amidophosphoribosyltransferase reaction for PP-Rib-P. In combination with accelerated purine excretion, increased channeling of newly formed purines into adenylates, and impaired conversion of AMP to IMP, fixed rates of purine synthesis de novo may condition cell lines with defects in HGPRT and PP-Rib-P synthetase to depletion of GTP with consequent growth retardation.
Topics: Aminopterin; B-Lymphocytes; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Methylthioinosine; Purine Nucleotides; Ribose-Phosphate Pyrophosphokinase
PubMed: 1311306
DOI: No ID Found -
Journal of Neurochemistry Feb 1992Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma...
Protein kinase N (PKN) is a soluble, apparently novel serine protein kinase that is activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma cells as well as in several nonneuronal cell lines. Purine analogs, such as 6-thioguanine and 2-aminopurine, have been found to inhibit PKN in vitro. When applied to intact cells, these compounds suppress certain biological responses to NGF, but not others, a findings suggesting the presence of multiple pathways in the NGF mechanism. We report here that 6-methylmercaptopurine riboside (6-MMPR) inhibits NGF-stimulated PKN activity in vitro with an apparent Ki of approximately 5 nM. This is approximately 1,000-fold lower than the Ki of the most potent purine inhibitor of PKN. Compounds similar to 6-MMPR, but lacking the methyl or riboside groups, were much less potent as PKN inhibitors. A survey of six additional purified protein kinases shows no inhibitory effect of 6-MMPR, thus indicating a good degree of specificity of this compound for PKN. In contrast to NGF-stimulated PKN, a PKN-like activity stimulated in PC12 cells in response to activation of cyclic AMP-dependent protein kinase was nearly insensitive to 6-MMPR. Application of 6-MMPR to intact PC12 cells resulted in blockade of several responses to NGF (neurite regeneration and ornithine decarboxylase induction) but not of several others (rapid enhancement of tyrosine hydroxylase phosphorylation and PKN activation). These findings suggest that 6-MMPR is a potent and selective agent for characterizing PKN in vitro and for assessing its potential role in the multiple pathways of the NGF mechanism of action.
Topics: Animals; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Induction; Methylthioinosine; Nerve Growth Factors; Ornithine Decarboxylase; PC12 Cells; Phosphotransferases; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases
PubMed: 1309569
DOI: 10.1111/j.1471-4159.1992.tb09774.x -
Journal of the National Cancer Institute Sep 1991Preclinical and clinical studies demonstrate that the selective antitumor activity of fluorouracil (5-FU) is enhanced by agents which perturb certain intracellular... (Clinical Trial)
Clinical Trial
Phase I trial of fluorouracil modulation by N-phosphonacetyl-L-aspartate and 6-methylmercaptopurine riboside: optimization of 6-methylmercaptopurine riboside dose and schedule through biochemical analysis of sequential tumor biopsy specimens.
Preclinical and clinical studies demonstrate that the selective antitumor activity of fluorouracil (5-FU) is enhanced by agents which perturb certain intracellular nucleotide pools. We previously demonstrated that the combination of N-phosphonacetyl-L-aspartate (PALA), which depletes pyrimidine nucleotide pools, and 5-FU yielded a 43% response rate among 37 assessable patients with colorectal carcinoma. In preclinical tumor models, 6-methylmercaptopurine riboside (MMPR), an inhibitor of purine synthesis, elevates phosphoribosylpyrophosphate (PRPP) pools and promotes the anabolism of 5-FU to fluorinated nucleotides. In vivo, the addition of MMPR enhances the therapeutic efficacy of PALA-5-FU. In a phase I trial, we sought to determine the optimal dose and schedule of MMPR in combination with PALA (250 mg/m2 on day 1) and 5-FU (1300 mg/m2 by 24-hour infusion on day 2). MMPR (75-225 mg/m2) was given intravenously on day 1 to 27 patients with solid tumors (15 colorectal, seven breast, five other). Toxic effects were mild to moderate and included leukopenia, mucositis, nausea, or rash. Two of seven patients given MMPR at 225 mg/m2 had grade 3 diarrhea. PRPP was measured using a [14C]orotic acid 14CO2 release assay in tumor biopsy specimens obtained before and 12 hours and 24 hours after MMPR doses were given to 20 patients. The addition of MMPR elevated PRPP pools in human solid tumors. At 12 hours after treatment, two (50%) of four patients showed a twofold or greater elevation of PRPP at the MMPR dose level of 75 mg/m2; a similar elevation was observed in five (71%) of seven patients given 150 mg/m2 MMPR and in three (43%) of seven patients given 225 mg/m2 MMPR. At 24 hours after treatment, results for the respective dose levels of MMPR were two (33%) of six patients, one (20%) of five patients, and four (57%) of seven patients. Administration of the two highest MMPR dose levels appeared to result in a greater increase in tumor PRPP levels. However, toxicity was greater at the 225 mg/m2 dose level; therefore, the 150 mg/m2 dose level was favored. Tumor levels of PRPP decreased between 12 hours and 24 hours in nine (56%) of 16 patients. This time course indicates that MMPR should be administered at the beginning of the 24-hour infusion of 5-FU.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Biopsy; Dose-Response Relationship, Drug; Drug Administration Schedule; Drug Evaluation; Drug Interactions; Female; Fluorouracil; Humans; Male; Methylthioinosine; Middle Aged; Neoplasms; Phosphonoacetic Acid; Phosphoribosyl Pyrophosphate
PubMed: 1714507
DOI: 10.1093/jnci/83.17.1235 -
Journal of Chromatography Mar 1991An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of methylmercaptopurine riboside (MMPR) in human plasma and urine...
An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of methylmercaptopurine riboside (MMPR) in human plasma and urine is reported. Plasma samples were prepared for analysis by addition of internal standard (6-dimethylaminopurine 9-riboside) followed by extraction using disposable C18 cartridges. Urine samples were filtered through a 0.22-micron membrane prior to HPLC separation. The column effluent was monitored at 289 nm and quantitation performed using peak heights. The linear range for MMPR determination was from 10 to 500 ng/ml in plasma and from 0.25 to 50 micrograms/ml in urine. The reported method is convenient, sensitive, and reproducible, illustrating its usefulness for application in pharmacokinetic studies.
Topics: Chromatography, High Pressure Liquid; Humans; Methylthioinosine
PubMed: 1860926
DOI: 10.1016/0378-4347(91)80095-t -
Advances in Experimental Medicine and... 1989
Topics: B-Lymphocytes; Cell Line; Dose-Response Relationship, Drug; Erythrocytes; Humans; Inosine; Methylthioinosine; Phosphotransferases; Purine Nucleotides; Pyrimidine Nucleosides; Pyrimidine Nucleotides; Ribose-Phosphate Pyrophosphokinase
PubMed: 2558549
DOI: 10.1007/978-1-4684-5676-9_2 -
Advances in Experimental Medicine and... 1989
Comparative Study
Topics: B-Lymphocytes; Cell Division; Cell Line; Humans; Hypoxanthine Phosphoribosyltransferase; Methylthioinosine; Pentosephosphates; Phosphoribosyl Pyrophosphate; Phosphotransferases; Purine Nucleotides; Purines; Ribose-Phosphate Pyrophosphokinase; Thioguanine
PubMed: 2481968
DOI: 10.1007/978-1-4684-5676-9_3