Phosphorylation of Adenosine and Deoxyadenosine in Ehrlich Ascites Carcinoma Cells Resistant to 6-(Methylmercapto)purine Ribonucleoside

Lomax, Christopher A.; Henderson, J. Frank

doi:10.1139/o72-057

Cited by 20 publications

(8 citation statements)

References 5 publications

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“…We found, however, that such variants still convert adenosine to nucleotides at a significant rate, even though they fail to phosphorylate a number of adenosine analogs. Conversion of adenosine to nucleotides by AK-deficient cell variants has been observed by other investigators but attributed to the presence of multiple AKs or to deamination and subsequent incorporation via inosine-hypoxanthine-inosine monophosphate (IMP) (Bennet et al, 1966;Lomax and Henderson, 1972;Divekar and Hakala, 1971;McBurney and Whitmore, 1975;Debatisse and Buttin, 1977). In our study, however, the latter pathway was inoperative, either because of treatment of the cells with dCF or lack of hypoxanthine phosphoribosyltransferase (HPRT), or both, and no significant phosphorylation of either adenosine or adenosine analogs was detectable in cell-free extracts of the variants.…”

mentioning

confidence: 94%

Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Plagemann

Wohlhueter

1983

Journal Cellular Physiology

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Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5-diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild-type cells, both in intact cells and cell-free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine-hypoxanthine-inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2-deoxycoformycin. Cell-free extracts of the tubercidin-resistant variants, as well as of two adenosine-resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at less than 1% of the rate of extracts of wild-type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40-fold. Similarly, intact adenosine kinase-deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5-10% the rate observed with wild-type cells. Phosphorylation of adenosine and tubercidin in wild-type cells was inhibited by substrate at concentration above 5-10 microM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase-deficient cells increased linearly up to a concentration of 400 microM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild-type cells. Adenosine uptake by these kinase-deficient cells was inhibited by adenine and 5'-deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase-deficient cells via adenine. Indirect evidence implicates 5'-methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.

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confidence: 94%

Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Plagemann

Wohlhueter

1983

Journal Cellular Physiology

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“…Similarly, adenosine deriva tives with large alkyl groups at the ^-p o s i tion are not generally substrates for adeno sine kinase, while those with smaller alkyl groups are usually substrates [19]. The re sulting phosphorylated derivatives cause their cytotoxic effects by inhibiting purine synthesis de nova [10,20,21].…”

Section: Resultsmentioning

confidence: 99%

Cytotoxicity of N<sup>6</sup>-Cycloalkylated Adenine and Adenosine Analogs to Mouse Hepatoma Cells

Thedford¹,

Leyimu²,

Thornton³

et al. 1989

Pathobiology

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Cytotoxic effect(s) of N⁶-cycloalkylated adenine and adenosine derivatives, upon the viability of mouse hepatoma cells, were studied in vitro. N⁶-Cyclopropyl- and N⁶-cyclobutyladenine and adenosine derivatives (33 µg/ml; 24–48 h) exerted significant cytotoxic effects upon the cells. N⁶-Cyclopentyl- and N⁶-cyclohexyladenines exerted similar effects under different experimental conditions (133–166 µg/ml; 48–72 h), while no significant cytotoxic effect(s) were observed with the corresponding adenosine derivatives under these conditions. Observed physical changes in the treated cells included cell elongation, short stubby filaments, wide intracellular spaces and ruptured cell membranes. N⁶-Cycloal-kylated nucleosides were usually more cytotoxic than the cycloalkylated bases.

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“…Y.) for separation of adenine, adenosine, hypoxanthine, inosine, and nucleotides, as previously described (9,15). Due to the presence of purine nucleoside phosphorylase activity, the sum of radioactivity in inosine and hypoxanthine was used in determining Adenosine kinase was assayed in a reaction mixture containing 100 mM sodium phosphate (pH 5.8), 5 mM ATP, 1.0 mM MgC12, 40 AM [8-'C]adenosine (59 mCi/mmol), 1 ,g/ml coformycin, and extract.…”

Section: Methodsmentioning

confidence: 99%

“…The assay conditions were optimized with respect to ATP, MgC12, adenosine concentrations, and pH with human lymphocyte lysates. The use of erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) (16) and coformycin (9,15) in studying adenosine phosphorylation has previously been reported; these compounds prevent substrate depletion by inhibition of adenosine deaminase activity. Aliquots of formic acid-terminated assay mixtures were spotted on cellulose thin layers and developed as before (9) to measure nucleotide formation and monitor substrate deamination.…”

Section: Methodsmentioning

confidence: 99%

“…After centrifugation, 0.100 ml of the supernate was neutralized by transfer to a tube containing 5 ,ul of 4.2 M KOH on ice, and after a further 5 min the potassium perchlorate was removed by centrifugation and 25-,gl aliquots were spotted on cellulose for chromatography. The metabolism of [14C] adenosine was followed by chromatography of extracts to separate adenine, adenosine, hypoxanthine, inosine, and nucleotides as previously described (9,15). The phosphorolysis and possible phosphorylation of ["C]guanosine were followed by chromatographic separation of guanosine, guanine, and nucleotides, as described for the purine nucleoside phosphorylase assay.…”

Section: Methodsmentioning

confidence: 99%

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Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

Snyder¹,

Mendelsohn²,

Seegmiller³

1976

J. Clin. Invest.

140

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A B S T R A C T The association of a human genetic deficiency of adenosine deaminase activity with combined immunodeficiency prompted a study of the effects of adenosine and of inhibition of adenosine deaminase activity on human lymphocyte transformation and a detailed study of adenosine metabolism throughout phytohemagglutinin-induced blastogenesis. The adenosine deaminase inhibitor, coformycin, at a concentration that inhibited adenosine deaminase activity more than 95%, or 50 uM adenosine, did not prevent blastogenesis by criteria of morphology or thymidine incorporation into acid-precipitable material. The combination of coformycin and adenosine, however, substantially reduced both the viable cell count and the incorporation of thymidine into DNA in phytohemagglutinin-stimulated lymphocytes.Incubation of lymphocytes with phytohemagglutinin for 72 h produced a 12-fold increase in the rate of deamination and a 6-fold increase in phosphorylation of adenosine by intact lymphocytes. There was no change in the apparent affinity for adenosine with either deamination or phosphorylation. The increased rates of metabolism, apparent as early as 3 h after addition of mitogen, may be due to increased entry of the nucleoside into stimulated lymphocytes. Increased adenosine metabolism was not due to changes in total enzyme activity; after 72 h in culture, the ratios of specific activities in extracts of stimulated to unstimulated lymphocytes were essentially unchanged for adenosine kinase, 0.92, and decreased for adenosine deaminase, 0.44. As much as 38% of the initial lymphocyte adenosine deaminase activity accumulated extracellularly after a 72-h culture with phytohemag- glutinin. In phytohemagglutinin-stimulated lymphocytes, the principal route of adenosine metabolism was phosphorylation at less than 5 FM adenosine, and deamination at concentrations greater than 5 WM. In unstimulated lymphocytes, deamination was the principal route of adenosine metabolism over the range of adenosine concentrations studied (0.5-250 MM). These studies demonstrate the dependence of both the unstimulated and stimulated lymphocyte on adenosine deaminase activity for the metabolism of adenosine and may account for the observed sensitivity of mitogen-stimulated lymphocytes to the toxic effects of exogenously supplied adenosine in the presence of the adenosine deaminase inhibitor coformycin.A single case of immunodeficiency disease has been reported in association with purine nucleoside phosphorylase deficiency. The catabolism of guanosine was also found to be enhanced in stimulated normal lymphocytes; phosphorolysis of guanosine to guanine by intact lymphocytes increased six fold after 72-h culture with phytohemagglutinin. The specific activity of purine nucleoside phosphorylase in extracts, with guanosine as substrate, was essentially the same in stimulated and unstimulated lymphocytes after 72 h of culture.

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Phosphorylation of Adenosine and Deoxyadenosine in Ehrlich Ascites Carcinoma Cells Resistant to 6-(Methylmercapto)purine Ribonucleoside

Cited by 20 publications

References 5 publications

Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells

Cytotoxicity of N<sup>6</sup>-Cycloalkylated Adenine and Adenosine Analogs to Mouse Hepatoma Cells

Adenosine metabolism in phytohemagglutinin-stimulated human lymphocytes.

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