Purine metabolism in adenosine deaminase deficiency.

Purine and pyrimidine metabolites were measured in erythrocytes, plasma, and urine of a 5-month-old infant with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. Adenosine and adenine were measured using newly devised ion exchange separation techniques and a sensitive fluorescence assay. Plasma adenosine levels were increased, whereas adenosine was normal in erythrocytes and not detectable in urine. Increased amounts of adenine were found in erythrocytes and urine as well as in the plasma. Erythrocyte adenosine 5'-monophosphate and adenosine diphosphate concentrations were normal, but adenosine triphosphate content was greatly elevated. Because of the possibility of pyrimidine starvation, pyrimidine nucleotides (pyrimidine coenzymes) in erythrocytes and orotic acid in urine were measured. Pyrimidine nucleotide concentrations were normal, while orotic acid was not detected. These studies suggest that the immune deficiency associated with adenosine deaminase deficiency may be related to increased amounts of adenine, adenosine, or adenine nucleotides.     

Coordinate regulation of mRNAs encoding adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyltransferase by phorbol esters in human thymocytes.

Incubation of human thymocytes in the presence of phorbol esters caused a reversible decrease in the mRNAs encoding terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) and adenosine deaminase (ADA; EC 3.5.4.4) and an increase in the mRNA encoding purine nucleoside phosphorylase (PNP; EC 2.4.2.1). The effect of phorbol esters on TdT and ADA mRNA levels can be attributed to an apparent decrease in the stability of the mRNAs. The changes in ADA, TdT, and PNP mRNAs closely simulate changes in the activities of these enzymes that occur during T-cell differentiation in vivo, suggesting a role for protein kinase C activation in the regulation of the expression of these genes during intrathymic T-cell differentiation. A role for these purine degradation enzymes in the regulation of intracellular pools of the deoxynucleotide substrates of TdT is discussed.     

Elevated adenosine deaminase and purine nucleoside phosphorylase activity in peripheral blood null lymphocytes from patients with acquired immune deficiency syndrome

The purine metabolic enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) are important in lymphocyte differentiation, and genetic deficiencies of either enzyme have been associated with hereditary immunodeficiency states. Both ADA and PNP activity were measured in null cell-enriched and T cell-enriched peripheral blood lymphocytes from 16 patients with the acquired immune deficiency syndrome (AIDS), seven patients with the AIDS-related symptom complex (ARC), and seven asymptomatic homosexuals. ADA activity in nmol/10(6) lymphocytes/h was significantly elevated in null lymphocytes from AIDS (161 +/- 12) as compared with 23 healthy heterosexual controls (127 +/- 8;P less than .025). PNP activity was also significantly increased in null lymphocytes from AIDS patients (96 +/- 10;P less than .005) as well as those from ARC patients (84 +/- 11:P less than .025) relative to controls (61 +/- 5). No significant differences in enzyme activity were noted in T cell-enriched cells in any group. Along with elevated enzyme activity, AIDS patients had small yet significant increases in the percentages of HLA-DR (P less than .025), terminal deoxynucleotidyl transferase (TdT) (P less than .0001), and peanut agglutinin receptor (P less than .0001) positive lymphocytes in the null fraction compared with controls. TdT-positive cells appeared morphologically as large lymphoblasts with irregular nuclei. The data imply that the cellular immune deficiency in AIDS is not a result of deficiencies in lymphocyte ADA or PNP activity, but is more likely associated with an increase in an immature and/or activated lymphocyte subset.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: Decreased purine nucleoside phosphorylase and adenosine deaminase activity

Summary Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.This work was supported in part by the Austrian Research Fund (Project No. 3796).     

Substrate inhibition of adenosine phosphorylation in adenosine deaminase deficiency and adenosine-mediated inhibition of PP-ribose-P dependent nucleotide synthesis in hypoxanthine phosphoribosyltransferase deficient erythrocytes

Summary The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 µmol L^–1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at 30 µmol L^–1 adenosine. Adenosine, 1 µmol L^–1, also caused a 60% reduction in PP-ribose-P concentration in ADA deficient erythrocytes. For HPRT deficient erythrocytes in which ADA activity was blocked by coformycin, 10 µmol L^–1 inosine stimulated PP-ribose-P dependent nucleotide synthesis from adenine, whereas, 10 µmol L^–1 adenosine inhibited nucleotide synthesis. These observations suggest that adenosine phosphorylation and PP-ribose-P dependent nucleotide synthesis are inhibited under conditions in which adenosine accumulates, such as in hereditary or pharmacologically induced ADA deficiency.     

Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: Possible role of nucleoside kinase(s)

Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).     

Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.

Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 mumol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 mumol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.     

Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase.

An inherited deficiency of adenosine deaminase (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes severe combined immunodeficiency disease in humans. A similar deficiency in purine nucleoside phosphorylase (Puo phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) engenders a selective cellular immune deficit. To elucidate the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting human lymphocytes of the Ado deaminase substrates deoxyadenosine and adenosine and the Puo phosphorylase substrate deoxyguanosine. When Ado deaminase was inhibited, micromolar concentrations of deoxyadenosine progressively killed nondividing helper and suppressor-cytotoxic T cells, but not B cells. The toxicity required phosphorylation, with subsequent dATP formation. The deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, and adenine arabinonucleoside also killed resting T cells. Cell death was unrelated to inhibition of adenosylhomocysteinase (EC 3.3.1.1) but was preceded by a gradual decline in ATP levels. As much as 1 mM deoxyguanosine did not impair resting lymphocyte viability, despite the synthesis of dGTP. The combination of 200 microM adenosine plus 500 microM homocysteine thiolactone killed dividing lymphocytes but had no discernible toxic effect toward resting T cells, which accumulated adenosylhomocysteine over a 4-hr period but thereafter excreted the nucleoside into the culture medium. The different clinical syndromes associated with genetic deficiencies of Ado deaminase and Puo phosphorylase may be explained by the ability of dATP to kill mature resting T lymphocytes by depleting ATP levels.     

Combined assay of adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in the early clinical evaluation of B-chronic lymphocytic leukemia

The levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), lactic dehydrogenase (LDH), and LDH isoenzyme patterns (LD1 to LD5) have been measured in lymphocyte extract from 28 patients with B-chronic lymphocytic leukemia (B-CLL). The activities of ADA, PNP, and LDH have been correlated with two morphological groups of B-CLL classified according to the percentage of large, nongranular, atypical lymphocytes (AL) in peripheral blood: "typical" B-CLL (less than 10% of AL, 21 cases) and "atypical" B-CLL (10-25% of AL, seven cases). Patients with atypical B-CLL had significantly (P less than 0.001) higher activities of ADA (0.46 +/- 0.17 U/10(9) cells), PNP (1.74 +/- 1.0 U/10(9) cells), and LDH (48.3 +/- 9.7 U/10(9) cells) than patients with typical B-CLL (ADA, 0.29 +/- 0.1 U/10(9) cells; PNP, 0.58 +/- 0.23 U/10(9) cells; and LDH, 29 +/- 10 U/10(9) cells). In addition, the "treatment-free period" was also significantly (P less than 0.025) shorter in the group of atypical B-CLL compared with the typical B-CLL group. No clear-cut statistical differences in lymphocyte surface markers or in several other prognostic factors between the two subgroups of B-CLL were found. The present study supports the idea that in B-CLL the simultaneous determination of ADA, PNP, and LDH might be helpful in better understanding the pathophysiology, prognosis, and natural history of the disease.     

Purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) activities examined cytochemically in unfixed lymphocytes of patients with lymphoproliferative disorders

New techniques have been devised for the cytochemical demonstration of purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) activities in unfixed human lymphocytes. A suspension of living lymphocytes is mixed with agarose sol containing the reagents for the detection of PNP or ADA activity on a glass slide. The mixture solidifies, is incubated, and then dried for lightmicroscopic observation. Reactive cells are recognized by the diffusely deposited granules of formazan, the end-product of the cytochemical reaction, and are divided into three groups of the cell with the low, middle, and high enzyme activity by the number of the granule. In healthy adults, the mean percentages of PNP- and ADA-positive cells were more than 90% in unfractionated lymphocytes, T-cell fractions, and complement- receptor cell fractions and cells with middle PNP and ADA activities were predominant. The PNP and ADA staining was observed in lymphoid cells of patients with lymphoproliferative disorders. A decrease in the percentage of PNP-positive cells concomitant with a relative increase of cells with the low enzyme activity was observed in the lymphocytes of nine patients with chronic lymphocytic leukemia (CLL). Similar findings were obtained in the ADA staining of the lymphocytes of five patients with B-cell CLL.     

Selective killing of human malignant cell lines deficient in methylthioadenosine phosphorylase, a purine metabolic enzyme.

Seven out of 31 (23%) human malignant tumor cell lines had no detectable methylthioadenosine phosphorylase activity (less than 0.001 nmol/min per mg of protein), assayed with 5'-chloroadenosine as substrate. The enzyme-deficient cell lines were derived from five leukemias, one melanoma, and one breast cancer. None of 16 cell lines of nonmalignant origin, derived from lymphocytes, fibroblasts, and epithelial cells, lacked the enzyme (range, 0.156-1.447 nmol/min per mg of protein). As detected by autoradiography, intact enzyme-positive cell lines normal immature bone marrow cells, and four specimens of malignant tumor cells incorporated the adenine moiety of 5'-chloroadenosine into nucleic acids; however, no enzyme-deficient cell lines used 5'-chloroadenosine. When both types of cell lines were cultured in a medium containing 0.4 microM methotrexate, 16 microM uridine, and 16 microM thymidine (or 10 microM azaserine alone), no cells grew. If methylthioadenosine was added to the same medium, only enzyme-positive cells increased in number; most enzyme-deficient cells were dead after 3 days. Thus, human malignant tumor cell lines naturally deficient in methylthioadenosine phosphorylase could be selectively killed when de novo purine synthesis was inhibited and methylthioadenosine was the only exogenous source of purines.     

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.     

Arachidonate metabolism in cultured fibroblasts derived from normal and infarcted canine heart

Metabolites of arachidonic acid (eicosanoids) may have an important role in the healing process after myocardial infarction. We examined the ability of cardiac fibroblasts from normal and from healing infarcted ventricle to metabolize arachidonate. We induced myocardial infarction in dogs and then allowed them to recover for 1 week, at which time they were killed, and the heart was removed. Fibroblasts were harvested from normal and from the healing, infarcted areas of the left ventricle. The cells from each source were morphologically indistinguishable. There were 347 +/- 102-fold more fibroblasts cultured from the infarcted area than from the normal area. Interestingly, the infarct-derived cells had a slower doubling time (37.4 +/- 3.7 hours) than the normal cells (22.0 +/- 3.6 hours). The uptake of exogenous arachidonate and its distribution in complex lipids was the same in the cells from each area. When stimulated with the calcium ionophore, free exogenous arachidonate, bradykinin, or histamine the cells produced prostaglandin E2 and prostaglandin I2. In each case the infarct-derived cells produced from twofold to fivefold more prostaglandin than the normal cells. We also found that prostaglandin synthesis was highly dependent on the growth state of the cells with a marked decrease a confluence. Finally, in experiments designed to mimic the early state of infarction, we confirmed that isolated cardiac myocytes release arachidonate and showed that normal fibroblasts can incorporate it. The production of eicosanoids by cardiac fibroblasts may be substantial during the healing of myocardial infarction due to their dramatic proliferation and the increased prostaglandin production per cell.     

Purine synthesis de novo in cultured lymphoblast cells derived from patients with gout

Summary Rates of de novo purine synthesis in lymphoblast cell cultures derived from ten patients with gout were compared with those from control individuals. Since the rate of de novo purine synthesis was dependent on the growth rate of the culture, an assay procedure was developed to account for the variation in lymphoblast growth rates and to permit valid quantitative comparison between purine synthesis in each cell line. Clear differences were demonstrated between the rates of purine synthesis in cells from normal control subjects and those from patients with a deficiency of hypoxanthine-guanine phosphoribosyltransferase activity (HPRT-deficient). Lymphoblasts from the gouty patients showed purine synthesis either within the normal range or intermediate between this and the HPRT-deficient cells. In patients having normal renal function, de novo purine synthesis of lymphoblast cells correlated with the degree of urate production as reflected by the urinary excretion of urate over a 24 h period. Three patients, with demonstrable excessive production of urate in vivo, exhibited increased purine synthesis in lymphoblasts. This increased synthesis did not appear to result from any of the enzyme mutations currently recognized as responsible for abnormal purine metabolism.     

Abnormal copper metabolism in cultured fibroblasts from patients with Wilson's disease

Skin fibroblasts derived from patients with Wilson's disease have, under certain conditions, elevated concentrations of copper. However, the levels of intracellular copper varied from one experiment to another and the reason for this inconsistency has not yet been determined. 64Cu retention after 24 hours and its release in "chase" experiments was not abnormal, thus distinguishing these fibroblasts from Menkes' syndrome fibroblasts. The data provides evidence that the mutant gene responsible for Wilson's disease is expressed in fibroblasts under certain conditions.     

Abnormal copper metabolism in cultured fibroblasts from patients with Wilson's disease

Skin fibroblasts derived from patients with Wilson's disease have, under certain conditions, elevated concentrations of copper. However, the levels of intracellular copper varied from one experiment to another and the reason for this inconsistency has not yet been determined.⁶⁴Cu retention after 24 hours and its release in ‘chase’ experiments was not abnormal, thus distinguishing these fibroblasts from Menkes' syndrome fibroblasts. The data provides evidence that the mutant gene responsible for Wilson's disease is expressed in fibroblasts under certain conditions. This study was supported by a grant from the Australian National Health and Medical Research Council.     

Abnormal collagen metabolism in cultured skin fibroblasts from patients with Duchenne muscular dystrophy.

Total collagen synthesis is decreased by about 29% (P less than 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P less than 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.