ABSTRACT
Azathioprine and 6-mercaptopurine are antimetabolite thiopurine drugs that play important roles in the treatment of leukemia and in the management of conditions requiring immunosuppression, such as inflammatory bowel disease. The biochemical pharmacology of these drugs suggests that inhibition of purine nucleotide formation through the 6-thioguanine nucleotide metabolites is their key molecular mechanism. However, it is unclear how these metabolites suppress immunity. We hypothesized that azathioprine produces a selective inhibitory effect on activated but not quiescent T lymphocytes. We first established a model system of T lymphocyte culture with azathioprine that produced pharmacologically relevant concentrations of 6-thioguanine nucleotides. Using genome-wide expression profiling, we identified a group of azathioprine-regulated genes in quiescent and activated T lymphocytes. Several genes involved in immunity and inflammation were selectively down-regulated by azathioprine in stimulated but not quiescent cells. Quantitative reverse transcription-polymerase chain reaction for three of these genes, tumor necrosis factor-related apoptosis-inducing ligand, tumor necrosis factor receptor superfamily member 7, and alpha4-integrin, confirmed down-regulated expression of transcript levels. Tumor necrosis factor-related apoptosis-inducing ligand protein expression was further studied and found to be inhibited by azathioprine, 6-mercaptopurine, and 6-thioguanine, implying that the inhibitory effects of azathioprine on expression are mediated by 6-thioguanine nucleotides. These results therefore provide a previously unrecognized molecular mechanism for the immunosuppressive properties of thiopurine antimetabolite drugs.
Subject(s)
Azathioprine/pharmacology , Gene Expression/drug effects , Immunosuppressive Agents/pharmacology , Inflammation/metabolism , Lymphocyte Activation/drug effects , Mercaptopurine/pharmacology , T-Lymphocytes/metabolism , Annexin A5 , Apoptosis Regulatory Proteins , Cells, Cultured , Coloring Agents , Dose-Response Relationship, Drug , Enzyme Inhibitors , Enzyme-Linked Immunosorbent Assay , Gene Expression Profiling , Humans , Immunity/genetics , Inflammation/genetics , Interleukin-2/metabolism , Membrane Glycoproteins/metabolism , Oligonucleotide Array Sequence Analysis , Propidium , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/drug effects , TNF-Related Apoptosis-Inducing Ligand , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation/drug effectsABSTRACT
Mean corpuscular volume may correlate with erythrocyte 6-thioguanine nucleotide concentrations in patients treated with azathioprine and 6-mercaptourine. We conducted a study of 166 patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine to determine the relationship between mean corpuscular volume and erythrocyte 6-thioguanine nucleotide concentrations, disease activity as measured by the Inflammatory Bowel Disease Questionnaire (active disease <170, remission >170), and leukopenia. Blood was submitted for mean corpuscular volume, whole blood 6-thioguanine nucleotide concentration, and leukocyte count. The mean +/- SD mean corpuscular volume during treatment was 94.7 +/- 6.6 fL and the mean +/- SD change in mean corpuscular volume was 7.5 +/- 6.3 fL. There were significant correlations between mean corpuscular volume and erythrocyte 6-thioguanine nucleotide concentration (r(s) = 0.33, p < 0.001) and between change from baseline in mean corpuscular volume and erythrocyte 6-thioguanine nucleotide concentration (r(s) = 0.26, p = 0.001). There was no correlation between Inflammatory Bowel Disease Questionnaire scores and mean corpuscular volume values (r(s) = 0.01, p = 0.94). The mean corpuscular volume values in 55 patients with active disease and 111 patients in remission were similar (95.1 vs. 94.5 fL, p = 0.57). There was a weak negative correlation between the mean corpuscular volume and the leukocyte count, (r(s) = -0.18, p = 0.022). In patients with inflammatory bowel disease treated with azathioprine or 6-mercaptopurine, mean corpuscular volume and change from baseline in mean corpuscular volume correlated with erythrocyte 6-thioguanine nucleotide concentrations and negatively with leukocyte counts, but did not correlate with disease activity as measured by the Inflammatory Bowel Disease Questionnaire. Measurement of mean corpuscular volume is a simple and inexpensive alternative to measurement of 6-thioguanine nucleotide concentrations in patients treated with azathioprine or 6-mercaptopurine.
Subject(s)
Azathioprine/therapeutic use , Erythrocytes/physiology , Immunosuppressive Agents/therapeutic use , Inflammatory Bowel Diseases/drug therapy , Mercaptopurine/therapeutic use , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers/analysis , Endpoint Determination , Erythrocyte Indices , Female , Guanine Nucleotides/analysis , Humans , Inflammatory Bowel Diseases/blood , Leukocyte Count , Male , Middle Aged , Severity of Illness Index , Surveys and Questionnaires , Thionucleotides/analysis , Treatment OutcomeABSTRACT
The action of a general base is needed in two possible steps during the aldehyde dehydrogenase catalyzed oxidation of an aldehyde to an acid. The base is glutamate at position 268 in the cytosolic and mitochondrial class 1 and 2 enzyme. A chemical modification approach was undertaken to determine if the base were necessary in the initial attack of the nucleophilic cysteine (302) on the aldehyde as well as the attack by water on the acyl intermediate formed after the aldehyde is oxidized. A metabolite of disulfiram, S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), was used as the modifying agent. Three recombinantly expressed mutant forms of the human mitochondrial enzyme along with the native one were used. These were the E268Q mutant that was lacking the general base; the E487K Oriental variant of the enzyme and R475Q, a mutant possessing the residue that binds to E487. As expected, the E268Q mutant was inactivated very slowly compared with the native or other mutants that were inactivated more slowly than the native enzyme. The presence of NAD did not increase the rate of inactivation except with the R475Q mutant. It is concluded that it is necessary to activate the cysteine at the active site to make it a good nucleophile as well to activate water during the hydrolysis of the thio-acyl intermediate. Further, it is surmised that the reason some mutants have a lowered specific activity is that in those the general base is not capable of functioning as it does in the native enzyme.