Masaru Kubota

5′-Methylthioadenosine is the Major Source of Adenine in Human Cells

The thioether nucleoside, 5′-methylthioadenosine (MTA) (Figure 1) is a product of transpropylamine reactions which lead to the synthesis of spermidine and spermine (Figure 2)(1). These polyamines are ubiquitous in mammalian cells (2). Their synthesis, and concomitantly the production of MTA, increases during periods of rapid growth (3). MTA does not accumulate in mammalian cells. Rather, the nucleoside is cleaved by MTA Phosphorylase (5′-methylthiadenosine: orthophosphate methylthioribosyltransferase), to yield adenine and 5-methylthioribose 1-phosphate (Figure 2)(4).

Deoxynucleoside overproduction in deoxyadenosine-resistant, adenosine deaminase-deficient human histiocytic lymphoma cells

Deoxyadenosine toxicity toward lymphocytes may produce immune dysfunction in patients with adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) deficiency. The relationship between endogenous deoxynucleoside synthesis in adenosine deaminase-deficient cells and sensitivity to adenosine and deoxyadenosine toxicity is unclear. The human histiocytic lymphoma cell line (DHL-9) naturally lacks adenosine deaminase, and has minimal levels of thymidine kinase. Dividing DHL-9 cells excrete deoxyadenosine and thymidine into the extracellular space. The present experiments have analyzed nucleoside synthesis and excretion in a mutagenized clone of DHL-9 cells, selected for increased resistance to deoxyadenosine toxicity. The deoxyadenosine-resistant cells excreted both deoxyadenosine and thymidine at a 6-7-fold higher rate than wild-type lymphoma cells. The deoxyadenosine overproduction was accompanied by a reduced ability to form dATP from exogenous deoxyadenosine, and a 2.5-fold increase in ribonucleotide reductase activity. The pace of adenosine excretion, the growth rate, and the levels of multiple other enzymes involved in deoxyadenosine and adenosine metabolism were equivalent in the two cell types. These results suggest that the excretion of deoxyadenosine and thymidine, but not adenosine, is exquisitely sensitive to alterations in the rate of endogenous deoxynucleotide synthesis. Apparently, small changes in deoxynucleotide synthesis can significantly influence cellular sensitivity to deoxyadenosine toxicity.

Methylthioadenosine (MeSAdo) phosphorylase deficiency in malignancy.

The abnormal growth properties of cancer cells must be mediated finally by changes in specific metabolic pathways. In general, malignant transformation is associated with an increase in the activities of enzymes involved in DNA, RNA, and protein synthesis, and a concomitant decrease in the activities of enzymes that degrade cellular metabolites. However, no specific metabolic pathway has been found that distinguishes normal from malignant cells, or is common to all tumors.

5′-Methylthioadenosine Phosphorylase Deficiency in Malignant Cells: Recessive Expression of the Defective Phenotype in Intra-Species (Mouse X Mouse) Hybrids

In 1977, Toohey reported that some mouse cell lines lacked the recently described purine metabolic enzyme, 5′-methylthio-adenosine (MTA) Phosphorylase (5′-methylthioadenosine:orthophosphate methylthioribosyltransferase)(1). Subsequently, we found the same enzyme deficiency in seven out of thirty-one established human malignant cell lines. In contrast, none of sixteen cell lines of benign origin lacked the enzyme (2).

5'-deoxy-5'-methylthioadenosine phosphorylase deficiency in leukemia: genetics and biochemical aspects.

In mammalian cells, 5′-deoxy-5′-methylthioadenosine (MTA) derives from decarboxylated S-adenosyl methionine during spermidine and spermine synthesis.1 To a lesser extent, MTA Is also produced following the aminocarboxypropyl group transfer from S-adenosylmethionine to certain tRNA uridine residues2 (Fig. 1). Although MTA can inhibit polyamine amlnopropyl transferase reactions3, the thioether nucleoside does not accumulate In normal cells but Is rapidly cleaved to adenine and 5-methylthioribose 1-phosphate by the enzyme MTA Phosphorylase. As shown In Figure 1, MTA Phosphorylase is important not only for the balanced synthesis of polyamlnes, but also for the economic Intracellular salvage of adenine nucleotides and methionine. 4, 5 The enzyme is present In all normal tissues studied thus far. Recently, we have assigned the gene for MTA Phosphorylase to the 9pter→9q12 region of human chromosome 9 by analysis of mouse-human somatic cell hybrids.

Regulation of S-Adenosylmethionine and Methylthioadenosine Metabolism in Methylthioadenosine Phosphorylase Deficient Malignant Cells

The synthesis and metabolism of both polyamines and S-adenosyImethionine (Adomet) are important for cell growth regulation. However, our understanding of the regulation of polyamine and Adomet metabol ism in intact mammalian cells is incomplete. 5′-deoxy-5′-methylthioadenosine (abbreviated as MTA or MeSAdo) is the purine end product of the polyamine biosynthetic pathway. Polyamines are organic cations that all dividing cells produce in abundance. Their exact metabolic functions are not known. However, states of increased cellular proliferation, such as cancer, are uniformly associated with accelerated rates of polyamine synthesis (Pegg & McCann, 1982; Heby, et al., 1976.

S-Adenosylmethionine Metabolism as a Target for Adenosine Toxicity

Adenosine exerts marked cytostatic and cytotoxic actions to mammalian cells. The nucleoside has been reported to inhibit pyrimidine nucleotide synthesis, to foster cyclic AMP accumulation and to cause the accumulation of S-adenosylhomocysteine (AdoHcy).1,2 Adenosine kinase (EC 2.7.1.20) deficient mammalian cells do not phosphorylate adenosine, but adenosine still blocks their growth, and this Is not reversed by addition of uridine. 2,3 Thus, adenosine may exert toxicity at the nucleoside level. Also, some adenosine analogs are cytotoxic without being converted to nucleotides.4

Genetic Analysis of Deoxyadenosine Toxicity in Dividing Human Lymphoblasts

An Inherited deficiency of adenosine deaminase (ADA) Impairs specifically the development of the human lymphoid system (reviewed In 1). In ADA deficient children, plasma deoxyadenosine (dAdo) concentrations reach 1-2 μM. Micromolar concentrations of dAdo are toxic toward Immature human T-lymphocytes, and toward T-Iymphoblastoid cell lines grown In medium supplemented with deoxycoformycin, a tight binding ADA Inhibitor.1 Compared to other cell types, human T-lymphoblasts preferentially phosphorylate dAdo and accumulate dATP.

Infection and Immunity: Mechanisms of Immunodeficiency in Inborn Errors of Purine Metabolism

Much of our understanding of the cellular regulation of immune mechanisms has come from studies of those “experiments of nature” the immunodeficiency diseases. For many years the biochemical basis of these defects was unknown. Recently, however, an association has been made between inherited deficiencies of two enzymes of purine metabolism and immunodeficiency disease. A lack of adenosine deaminase (ADA) produces combined immunodeficiency syndrome [ 1 1 . The absence of purine nucleoside phosphorylase (PNP) leads to a selective cellular immune deficit [2] . Until this time no specific role for these enzymes in lymphocyte growth and differentiation had been suspected. Although ADA deficiency is a rare disorder, it accounts for approximately 25-33% of patients with the autosomal recessive form of severe combined immunodeficiency disease [ 3 I . ADA deficient children do not respond to mitogenic or antigenic challenge, and suffer from recunent bacterial, viral and fungal infections. Untreated, the disease is invariable progressive, and leads to death