Naoyuki Kamatani

Lymphocyte dysfunction caused by deficiencies in purine metabolism

Two inborn errors of purine metabolism have been associated with autosomally inherited human immunodeficiency diseases. A lack of adenosine deaminase (ADA) produces severe lymphopenia and a combined immunodeficiency syndrome. A deficiency of purine nucleoside phosphorylase (PNP) is associated with a selective cellular immune depth. This article discusses the probable biochemical basis for lymphocyte-specific toxicity in these disorders.

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.

Differential Cyst(e)ine Requirements in Human T and B Lymphoblastoid Cell Lines

In an effort to find exploitable metabolic differences between human T and B lymphoblasts, we have compared the ability of lymphocytes of varying phenotype to grow in cystine-deficient medium. Only 6 of 12 human lymphoblastoid cell lines tested were able to utilize homocysteine thiolactone or cystathionine in place of cystine for growth. This difference in growth requirements was unrelated to the rate of cell division, the presence of Epstein-Barr viral genetic material, or whether or not the cell lines derived from benign or malignant tissues. Rather, all B lymphoblastoid cell lines grew in homocysteine thiolactone- or cystathionine-containing medium, while the T and non-T, non-B lymphoblastoid cell lines did not. Normal human peripheral blood T and B lymphoblasts did not respond to mitogens in the homocysteine thiolactone or cystathionine medium, but developed the ability to utilize these cysteine precursors after stimulation with concanavalin A, protein A, or Epstein-Barr virus. The differences in cysteine requirements among T and B cell lines may reflect a fundamental difference in de novo protein-synthesizing capacity of the two cell types.

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).

Biochemical Basis for Lymphocyte Dysfunction in Adenosine Deaminase and Purine Nucleoside Phosphorylase Deficiencies

Deoxypurine metabolism in human thymocytes and mature T lymphocytes differs remarkably from other cell types (1–3). When the normal pathways of deoxyadenosine and deoxyguanosine degradation are lacking in adenosine deaminase (ADA) and purine nucleoside Phosphorylase (PNP) deficient patients, human T cells exposed to even micromolar concentrations of the respective nucleosides progressively accumulate dATP or dGTP intracellularly. The dATP and dGTP increase to toxic levels primarily in this cell type because(i) in T cells deoxynucleoside phosphorylating activity substantially exceeds deoxynucleotide dephosphorylating activity, and (ii) nucleotides, as opposed to nucleosides, do not readily traverse cell membranes, and hence are trapped intracellularly.

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).

The importance of methylthioadenosine phosphorylase deficiency in human malignancy

Most investigators now accept that somatic mutations can lead to malignant transformation. Nonetheless, the exact means by which the metabolic products of altered genes actually influence carcinogenesis remains unresolved. We wish to describe how the loss of a purine metabolic enzyme, methylthioadenosine phosphorylase, may contribute to the growth of malignant cells by altering the regulation of putrescine synthesis.