Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a cytotoxic T lymphocyte-mediated cytotoxicity assay in which the release of a cytoplasmic enzyme, adenosine deaminase (ADA), instead of the widely used radioactive chromium is a measure of target lysis. In this enzyme-release assay the target is a mastocytoma P815-derived cell line, noted P815 ADA++, isolated by applying a selection procedure devised to specifically amplify the ADA gene. Gene amplification in P815 ADA++ was indeed demonstrated. Routine measurement of ADA activity from numerous supernatants is performed using a specific and sensitive colorimetric assay. The use of 96-well microtiter plates as well as of an automatic Multiscan spectrophotometer makes this measurement rapid and convenient. We show that this ADA-release assay is significantly more sensitive than the classical chromium-release test because of its consistently lower (5 to 10-fold) spontaneous release in 4 h, short-term cytotoxicity experiments. We also found that it is especially suited for the rapid detection, by visual screening, of rare, active killer clones among large, heterogeneous cytotoxic T lymphocyte populations. The assay could easily be adapted to other tumor targets (EL4, YAC-1, K562) of common use in studies involving immune lysis; indeed, the procedure of amplifying the ADA gene used in the isolation of the P815 ADA++ hyperactive line may be generally applied to these targets.
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PMID:Use of a P815-derived line with an amplified adenosine deaminase gene: an improved target for cellular cytotoxicity. 393 81

The growth of mouse mastocytoma P-815 cells in culture (37 degrees, 42 hr) was inhibited by exogenous adenosine (0.2 to 1.0 mM) and more effectively by AMP (0.01 to 0.1 mM), but not by adenine. The inhibited growth (a 25% inhibition by 0.5 mM adenosine and a 80% inhibition by 0.25 mM AMP) was restored to a near control level by the addition of uridine (0.5 mM) to the medium. The pretreatment (37 degrees, 3 hr) of the cells with adenosine or AMP caused a 60% inhibition of incorporation (37 degrees, 2 hr) of [U-14C]aspartate into uracil nucleotides, accumulating 14C-orotate and orotidine. Both dipyridamole, an inhibitor of adenosine uptake, and exogenous adenosine deaminase suppressed the growth inhibition induced by not only adenosine but also AMP. 2-Chloroadenosine, which is resistant to the action of adenosine deaminase, was a more potent growth inhibitor, while 3'AMP and 2'-AMP, which are not hydrolyzed to adenosine by membrane 5'-nucleotidase, were ineffective. Adenosine 5'-sulfate and other 5'-substituted adenosines were also ineffective. These observations indicate that AMP inhibits the growth of mastocytoma P-815 cells as a result of its continuous conversion to adenosine and a constant exposure of the cells to a low concentration of adenosine which readily permeates the cell membrane. In addition, adenosine, AMP and their agarose-linked forms rapidly (37 degrees, 20 min) elevated cellular levels of cAMP. This effect was not suppressed by dipyridamole. Apparently adenosine and AMP also act extracellularly for growth inhibition by regulating cAMP levels.
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PMID:Effect of adenosine and adenosine 5'-monophosphate on cell division of cultured mastocytoma P-815 cells. 625 14

Adenosine, a purine nucleoside found at high levels in solid tumors, is able to suppress the recognition/adhesion and effector phases of killer lymphocyte-mediated tumor cell destruction. Here, we demonstrate that adenosine, at concentrations that are typically present in the extracellular fluid of solid tumors, exerts a profound inhibitory effect on the induction of mouse cytotoxic T cells, without substantially affecting T-cell viability. T-cell proliferation in response to mitogenic anti-CD3 antibody was impaired in the presence of 10 microM adenosine (plus coformycin to inhibit endogenous adenosine deaminase). Antigen-specific T-cell proliferation was similarly inhibited by adenosine. Anti-CD3-activated killer T (AK-T) cells induced in the presence of adenosine exhibited reduced major histocompatibility complex-unrestricted cytotoxicity against P815 mastocytoma cells in JAM and (51)Cr-release assays. Diminished tumoricidal activity correlated with reduced expression of mRNAs coding for granzyme B, perforin, Fas ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), as well as with diminished Nalpha-CBZ-L-lysine thiobenzylester (BLT) esterase activity. Interleukin-2 and interferon-gamma synthesis by AK-T cells was also inhibited by adenosine. AK-T cells express mRNA coding for A(2A), A(2B) and A(3) receptors, but little or no mRNA coding for A(1) receptors. The inhibitory effect of adenosine on AK-T cell proliferation was blocked by an A(3) receptor antagonist (MRS1191) but not by an A(2) receptor antagonist (3,7-dimethyl-1-propargylxanthine [DMPX]). The A(3) receptor agonists (N(6)-2-(4-aminophenyl)ethyladenosine [APNEA] and N(6)-benzyl-5'-N-ethylcarboxamidoadenosine [N(6)-benzyl-NECA]) also inhibited AK-T cell proliferation. Adenosine, therefore, acts through an A(3) receptor to prevent AK-T cell induction. Tumor-associated adenosine may act through the same mechanism to impair the development of tumor-reactive T cells in cancer patients.
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PMID:Adenosine acts through an A3 receptor to prevent the induction of murine anti-CD3-activated killer T cells. 1199 7