Erik H. Willis

Sequential metabolism of 5′-isobutylthioadenosine by methylthioadenosine phosphorylase and purine-nucleoside phosphorylase in viable human cells

Abstract The exact route of metabolism of 5′-isobutylthioadenosine is controversial. Using human cell lines deficient in methylthioadenosine phosphorylase, purine-nucleoside phosphorylase, or adenosine deaminase, we have ascertained the relative roles of the three enzymes in isobutylthioadenosine metabolism. The results showed that viable human cells progressively converted isobutylthioadenosine to 5′-isobutylthioinosine via sequential metabolism by methylthioadenosine phosphorylase and purine nucleoside phosphorylase acting in opposite directions, rather than through direct deamination. An identical pathway converted 5′-methylthioadenosine to 5′-methylthioinosine.

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).

Selection and characterization of a murine lymphoid cell line partially deficient in S-adenosylhomocysteine hydrolase

The exact role of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) in mediating the toxic effects of adenosine toward mammalian cells has not been ascertained. The selection and characterization of S-adenosylhomocysteine hydrolase-deficient cell lines offers a biochemical genetic approach to this problem. In the present experiments, a mutant clone (Sahn 12) with 11-13% of wild-type S-adenosylhomocysteine hydrolase activity was selected from the murine T lymphoma cell line R 1.1 after mutagenesis and culture in adenosine, deoxycoformycin, uridine and homocysteine thiolactone-supplemented medium. In the presence of 0.5 mM homocysteine thiolactone and 10-200 microM adenosine, wild-type and mutant cells synthesized S-adenosylhomocysteine intracellularly at markedly different rates, and excreted the compound extracellularly. Thus, at time points up to 10 h, the S-adenosylhomocysteine hydrolase-deficient lymphoblasts required 5-10-fold higher concentrations of adenosine in the medium to achieve the same intracellular S-adenosylhomocysteine levels as wild-type cells. Similarly, the Sahn 12 lymphoblasts were 5-10-fold more resistant than R 1.1 cells to the toxic effects of adenosine plus homocysteine thiolactone. These results establish that (i) 11-13% of wild-type S-adenosylhomocysteine hydrolase activity is compatible with normal growth, (ii) in medium supplemented with both adenosine and homocysteine thiolactone, intracellular S-adenosylhomocysteine is synthesized by S-adenosylhomocysteine hydrolase, (iii) the net intracellular level of S-adenosylhomocysteine is determined by both the rate of S-adenosylhomocysteine synthesis and its rate of excretion, (iv) under such conditions the accumulation of S-adenosylhomocysteine is related to cytotoxicity, (v) in the absence of an exogenous homocysteine source, S-adenosylhomocysteine derives from endogenous sources, and the accumulation of S-adenosylhomocysteine is not the primary cause of adenosine induced cytotoxicity.

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).

Characterization of the Human Autoantibody Response to Poly(ADP.Ribose) Polymerase

Poly(ADP-ribose) polymerase is a DNA-binding protein whose catalytic activity is stimulated strongly by DNA containing strand breaks (1). Recently, it has been proposed that a major function of the enzyme is to inactivate and eliminate cells with damaged DNA (2). DNA-binding proteins are established targets of autoimmunity in patients with systemic autoimmune diseases (3). Considering the possible functions of poly(ADP-ribose) polymerase in cells with DNA strand breaks, and the ability of the enzyme to undergo DNA-dependent automodification, it was conceivable that poly(ADP-ribose) polymerase could represent a potential autoantigen. For these reasons, we systematically searched for autoantibodies to poly(ADP-ribose) polymerase in human sera. Here we report the presence of autoantibodies to the poly(ADP-ribose) polymerase protein in patients with rheumatic diseases.

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.

Selection of mutant murine lymphoid cells partially deficient in S-adenosylhomocysteine hydrolase

The thioether nucleoside S-adenosylhomocysteine (SAH) is a product and potent natural inhibitor of diverse transmethylation reactions necessary for cell growth and function (Mann et al, 1963; Zappia et al, 1969). SAH does not accumulate in normal cells, but rather is cleaved to adenosine and L-homocysteine by S-adenosylhomocysteine hydrolase (SAHase) (De la Haba et al, 1959; Walker et al, 1975).

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.

19 2-HALO-2|[prime]|,3|[prime]|-DIDEOXYADENOSINES: METABOLICALLY STABLE DIDEOXYNUCLEOSIDES WITH ACTIVITY AGAINST THE HUMAN IMMUNODEFICIENCY VIRUS (HIV)

2′,3′-dideoxyadenosine (ddA) has activity against the human immunodeficiency virus-1 (HIV), but is rapidly catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we developed a simple method to synthesize the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of ddA. The isolated 2-halo-ddA derivatives were not deaminated significantly by cultured T lymphoblasts, which converted the dideoxynucleosides to the respective 5′-monophosphate, 5′- diphosphate, and 5′- triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 μM), the 2-halo-ddA derivatives inhibited the cytopathic effects of HIV toward T lymphoblasts, and retarded viral replication. Experiments with a deoxycytidine kinase deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-halo-ddA derivatives. Thus, the 2-halo-ddA congeners, in contrast to ddA itself, are not degraded by T lymphocytes, and represent promising compounds for in vivo chemotherapy of HIV infection.

5|[prime]|-DEOXY|[ndash]|5|[prime]|-METHYLTHIOADENOSINE (MTA) PHOSPHORYLASE DEFICIENCY IN LEUKEMIA: GENETICS AND BIOCHEMICAL ASPECTS: 28

MTA is produced in eukaryotic cells during the synthesis of polyamines from decarboxylated S-adenosylmethlonine. The nucleoside is rapidly cleaved to adenine and methylthioribose-1-P by MTA phosphoryiase (MTAse). We have assigned the gene MTAP to chromosome 9pter->9q12 by enzymatic and electrophoretic analysis of somatic cell hybrids.All normal tissues and non-malginant cell lines contain MTAse. However, several human leukemic cell lines are deficient in the enzyme, and 5 patients with acute lymphoblastic leukemia (ALL) have been shown thus far to lack MTAse in their malignant cells. Karyotypic abnormalities involving fragile site 9p21 occur in ALL with lymphomatous clinical features. One of 5 such patients studied prospectively lacked MTAse in her leukemic cells but not in normal blood cells at remission. No inactive enzyme protein has been detected by immunoadsorption among 7 leukemic lines tested. The MTAse deficient cell lines excrete MTA up to 0.32 nmol/hr/mg protein. In mice, the growth of MTAse deficient mutant lymphoma cells (but not MTAse positive wild type cells) causes plasma MTA to rise from undetectable levels to > 800 nM pre-terminally. Assay of plasma or urine MTA may thus prove useful to screen leukemic patients for MTAse deficient malignant cell clones.