J. P. R. M. van Laarhoven

Purine metabolism in relation to leukemia and lymphoid cell differentiation

Abstract A number of inborn errors of purine metabolism have been associated with immunodeficiency diseases. From studies to the possible mechanism(s) leading to the defects in the immune system, it appeared that the accumulation of deoxyATP and deoxyGTP and the subsequent inhibition of ribonucleotide reductase played an important role. The inhibition of methylation pathways through the accumulation of s -adenosylmethionine seems to be a second valid concept. The amount to which certain subtypes of lymphoid cells were affected by the enzyme deficiencies was strongly related to the enzymatic make-up of the cells. Lymphoid cells from different maturation stages could be affected in a specific way, depending on the different enzyme activities of these cells. Studies on human lymphoblastic leukemias showed that, related to the immunological subtype, the different leukemias could be characterized by a different enzymatic make-up. In this paper we discuss the possibilities for a specific enzyme directed chemotherapy, directed against specific subtypes of human lymphoblastic leukemias. Experimental evidence indicates that for example the adenosine deaminase inhibitor 2′-deoxycoformycin can be used as a specific drug against acute lymphoblastic leukemia with the T cell phenotype.

Enzymes of Purine Interconversions in Subfractions of Lymphocytes

Previously described micromethods for the determination of purine interconversion enzyme activities in lymphocytes (1) enable us to analyse purine metabolism systematically in lymphocyte subfractions using a relatively small number of cells (500–5000). A relation between purine interconversion defects and immune dysfunctions has been established (2–4). The mechanism by which adenosine deaminase (ADA) deficiency leads to impairment of the B and T cell and purine nucleoside Phosphorylase (PNP) deficiency leads to T cell dysfunction is not yet completely understood. A better understanding of purine interconversions in B and T cell subfractions might help to obtain a better view on B or T cell specificity in these immune diseases. One of the possibilities to achieve this might be a systematic enzymological analysis of purine metabolism in T and non-T lymphocytes. Nine purine enzyme activities were measured in T and non-T lymphocyte subpopulations using 500–5000 cells per assay.

A Simple and Sensitive Radiochemical Assay for Plasma Guanase

A simple and sensitive radiochemical micro-assay has been developed for the determination of plasma guanase activity. The method is based upon the measurement of the conversion of 14C-labeled guanine to xanthine, catalysed by the enzyme guanase (guanine aminohydrolase;EC 3.5.4.3). Using this method, the catalytic activity in the plasma of adult healthy controls was 0.040 +/- 0.09 nmol/h.mg protein (x +/- s). In children under 5 years of age higher levels of enzyme activity were demonstrated. In adult patients with liver disease plasma guanase activities were found to be 3 to 7-fold increased as compared to the normal adult mean value.

Purine Interconversion Pathways in T, B, Tγ and T-Tγ Cells from Human Peripheral Blood

In the last decade it has become clear, that a number of immunological diseases are related to imbalances of purine metabolism in lymphocyte subpopulations(1,2,3). Information regarding the enzymatic make-up and metabolic characteristics of lymphocyte subpopulations, both in healthy individuals and in patients, might contribute to a better diagnosis of immunological diseases and be of importance for the development of immuno-regulatory drugs. Not only T and B cell sub-populations, but also T and B cell subsets should be included in such studies.

Purine Metabolism in Human Thymocyte Subsets: Relevance for Lymphocytic Differentiation

The maturation pathway of the T lymphoid system, from (T) lymphopoietic precursor cell in the bone marrow via immature T lymphocytes in the thymus to mature immunocompetent T cells in peripheral organs and blood, is nowadays well established. Stages in T cell differentiation can be distinguished both by expression of cell surface and cytoplasmic differentiation markers (e.g., cell surface phenotyping with monoclonal antibodies to T lymphocyte (sub)populations1) and by functional capacity (e.g. differential responsiveness to mitogens like phytohaemagglutinin (PHA))2.

Micromethods for the measurement of purine enzymes in lymphocytes.

The involvement of purine interconversion enzyme defects in impairment of the immune system is now well documented (1–3). Although it has been suggested that deoxypurine nucleotides might be the toxic metabolites in these immune diseases (4–5), the mechanism which leads to dysfunctions of T or B cells, or both of them, is still not completely elucidated. A better understanding of purine interconversions in B and T cell subfractions might help to obtain a better view on B or T cell specificity in these immune diseases. One of the possibilities to achieve this might be a systematic analysis of purine metabolism in T and non-T lymphocytes.

DIFFERENTIAL IN VITRO EFFECTS OF ADENOSINE AND DEOXYADENOSINE ON HUMAN T- AND B-LYMPHOCYTE FUNCTION

Human peripheral blood lymphocytes were stimulated with different mitogens (PHA, PWM, ConA and SpA) known to activate different subpopulations. 3H-thymidine and 3H-leucine incorporation were studied in the presence and absence of adenosine, deoxyadenosine and the adenosine deaminase inhibitor EHNA. In addition, the effects on in vitro IgG secretion induced by PWM or SpA have been studied.

Purine metabolizing enzyme activities in lymphoblastic leukemia.

Studies on patients with immunological dysfunctions have revealed that genetically determined deficiencies of certain enzymes of purine metabolism are intimately related to lymphocytic malfunction(1,2). This as a consequence of a differentiation and/or maturation block originating from the enzyme deficiencies (3). Since lymphoblastic leukemia may be considered as a disorder of lymphocyte differentiation and maturation (4), it is of interest to study systematically purine metabolism in lymphoid cells from patients with acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).

Inhibition of 3H-Thymidine Incorporation by Adenosine and Deoxyadenosine in Human Peripheral Lymphocytes and Malignant Lymphoid Cell Lines

The purine converting enzyme adenosine deaminase (ADA, E.C. 3.5.4.4.) catalyzes the conversion of adenosine and deoxyadenosine into inosine and deoxyinosine respectively. The inherited deficiency of ADA is associated with an impairment of both cellular and humoral immunity1,2. Although all cells of patients with ADA deficiency lack a functional ADA enzyme, the expression of the deficiency seems to be restricted mainly to the immune system. This selective effect of ADA deficiency on lymphoid cells has drawn attention to the possibility of using ADA inhibitors as potential selective chemotherapeutic agents in treatment of lymphoproliferative disorders3.

Purine metabolism in normal and pathologic lymphoid cell differentiation

Intact purine metabolism is pivotal for proper functioning of (lymphoid) cells. During the generation of immunocompetent lymphocytes of T- or B-cell lineage, and during subsequent differentiation into effector cells, cell divisions occur which require DNA synthesis. Studies of the causal relationship between deficiency of certain purine nucleotide metabolizing enzymes and deficiencies in immune function, revealed several privileged pathways in purine metabolism (Fig. 1). These pathways are different for T- and B-cells in different stages of maturation, and are related to purine enzyme activities [1, 2]. Assessment of purine nucleotide metabolizing enzymes in lymphoreticular malignancies [3, 4] showed enzyme activities that could be related to normal lymphoid cell differentiation. In addition, the purine enzyme make-up of lymphoid cells may be applied in malignant cell typing and permits estimation of the effectiveness of enzyme-directed chemotherapy. This survey focuses on these different aspects.