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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.

Deoxyadenosine and its nucleotides have been implicated in the pathogenesis of the immunodeficient state associated with an inherited deficiency of adenosine deaminase (ADA) (1,2), In ADA deficient patients, T lymphocytes may selectively phosphorylate deoxyadenosine released by other tissues. Both dividing and resting T cells have abundant deoxyadenosine phosphorylating activity, mediated primarily by deoxycytidine kinase, but minimal deoxynucleotide dephosphorylating activity, mediated by cytoplasmic deoxynucleotidase (3,4). For this reason, normal and malignant T lymphocytes exposed to micromolar concentrations of deoxyadenosine, in the presence of an ADA inhibitor, progressively accumulate dATP until cell death ultimately ensues (5,6).

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).

Both 19S and 7S anti-gamma-globulins in rheumatoid arthritis sera are enriched in kappa light chain bearing antibody molecules when compared to total 19S and 7S globulins from the same individuals. In patients with subacute bacterial endocarditis 19S anti-gamma-globulins are also, to a degree, enriched in kappa light chains, whereas the 7S anti-gamma-globulins have kappa to lambda light chain ratios indistinguishable from total 7S globulin.

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.

Antipeptide antisera were raised to the second and third complementarity-determining regions of the light chain derived from a human monoclonal IgM (Sie) which had antigammaglobulin activity and belonged to the Wa cross-reactive idiotypic group of human rheumatoid factors, two of whose members (Sie, Wo1) had been previously sequenced in our laboratory (Andrews and Capra, Biochemistry 20, 5816-5822, 1981). These antisera were found to react with the light chain of another human monoclonal IgM (Go1) that shared the Wa idiotype while antipeptide antisera made to the third CDR of the Sie heavy chain failed to react. The amino acid sequence of the variable region of the Go1 light chain was found to be highly homologous to the light chain of Sie from which the synthetic peptides were derived, particularly in the framework regions and the second and third CDR. This study illustrates that antipeptide antisera are valuable and specific probes for determining the relationship between molecules which exhibit similar antigen binding or idiotypic specificities and, furthermore, such antisera are able to predict amino acid sequences with surprising precision.

An inherited deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) produces selective lymphopenia and immunodeficiency disease in humans. Previous experiments have suggested that lymphospecific toxicity in this condition might result from the selective accumulation of toxic deoxyadenosine nucleotides by lymphocytes with high deoxycytidine kinase, levels and low deoxynucleotide dephosphorylating activity. The present experiments were designed to determine if deoxyadenosine analogs which are not substrates for adenosine deaminase might similarly be toxic toward lymphocytes and lymphoid tumors. Two such compounds, 2-chlorodeoxyadenosine and 2-fluorodeoxyadenosine, at concentrations of 3 nM and 0.15 microM, respectively, inhibited by 50% the growth of human CCRF-CEM malignant lymphoblasts in vitro. Each was phosphorylated in intact cells by deoxycytidine kinase accumulated as the nucleoside triphosphate, and inhibited DNA synthesis more than RNA synthesis. Both deoxynucleosides had significant chemotherapeutic activity against lymphoid leukemia L1210 in mice.

In humans, a genetic deficiency of the enzyme adenosine deaminase (ADA) leads to the specific impairment of the development of the lymphoid system, with resulting immunodeficiency disease1. Recent studies have suggested that the remarkable lymphospecific toxicity seen in ADA deficiency may result from the selective accumulation of deoxyadenosine nucleotides in lymphoid tissues, particularly the thymus, which when compared to other tissues have high deoxyadenosine phosphorylating activity, and low deoxyribo-nucleotide dephosphorylating activity2–5. The dATP thereby produced inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase and perhaps via other as yet undescribed mechanisms.

The mechanism via which cytotoxic T lymphocytes (CTL)1-lyse their target cells remains a mystery. The recent development of monoclonal antibodies that react with function-associated molecules on the surfaces of human or murine T cells has provided useful reagents for the delineation of the mechanism of CTL function. One such function-associated molecule is the T3 molecular complex present on the surfaces of all mature human T cells (1). Monoclonal antibodies to the T3 complex have diverse effects on T cell function including induction of prolIferatlon of resting T cells (2,3), inhibition of CTL generation invitro (4), and inhibition of effector CTL function in lytic assays (3,5–7). In the present report, we will discuss these properties of the monoclonal antibodies placing emphasis on the inhibition of CTL- medfated lysis of target cells.

With a sensitive radioimmunoassay we have found elevated IgA rheumatoid factor (IgA-RF) levels in the sera of patients with rheumatoid arthritis, Sjogren's syndrome, and systemic lupus erythematosus. The IgA--RF showed a pattern of reaction with human IgG subclasses and animal gammaglobulins similar to that of IgM-RF from the same patients. Rheumatoid factors of both classes were shown to be present in saliva of patients with rheumatoid arthritis and Sjogren's syndrome.

Growth and differentiation of thymocytes and mature T lymphocytes is regulated by cellular interactions that are in part mediated by soluble factors. We identify IL-6, formerly called B cell stimulating factor (BSF-2). IFN-beta 2, or hybridoma-plasmacytoma growth factor (HPGF) as a novel T cell costimulant rIL-6 induced a six-to seven-fold increase in proliferation of human thymocytes stimulated with suboptimal doses of PHA. A similar effect with added IL-6 could be observed using peripheral blood T lymphocytes, but only if the cultures were first rigorously depleted of monocytes that release high levels of IL-6. Analysis of the mechanism of the IL-6 effect on thymocytes and T lymphocytes showed that IL-6 did not lead to an increase in IL-2-R expression. Concentrations of antibody to IL-2-R inhibiting IL-2 effects did not block the IL-6-induced proliferation, indicating that the IL-6 effect was relatively IL-2 independent. These results identify IL-6 as a novel costimulant of human thymocytes and mature T lymphocytes, and suggest that IL-6 is also an important regulatory of cellular immunity.

This paper describes a monoclonal antibody (B532) that detects a membrane antigen present on greater than or equal to 95% of the B cells from lines carrying the Epstein-Barr virus (EBV) genome. Evidence suggesting that B532 is EBV-related was originally obtained by using a cell-binding radioassay with different cell line substrates. Immunofluorescence and cell-sorter analysis confirmed that the antigen was present in high density on all EBV-infected lymphoblastoid B-cell lines, but not on EBV-negative B-, T-, myeloid, or null cell lines. Isolated normal peripheral blood B and T lymphocytes and monocytes failed to bind B532. The monoclonal antibody did not inhibit in vitro EVB infection nor did it block the killing of EBV-infected targets by cytotoxic T lymphocytes. The cell surface antigen recognized by B532 was shown by immunoprecipitation to have a molecular weight of approximately 45,000.

Primary human monocyte-derived macrophages (MDM) were shown to have diminished deoxynucleoside kinase activities compared to T lymphoblasts, and a reduced ability to phosphorylate dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity. These drugs, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyadenosine (ddA), which are potent anti-HIV agents in CD4 lymphocytes, did not inhibit HIV replication in MDM, even at concentrations of 100 microM. This drug concentration of AZT is approximately 100-fold higher than the levels attained in the serum of treated patients and the levels required to inhibit HIV replication in lymphocytes. These observations may explain the failure of AZT therapy to clear viremia, consistent with the presence of a drug-resistant reservoir of infected cells in vivo. New therapeutic approaches to inhibit the replication of HIV in MDM may be needed.

The role of immunoglobulin structural genes in the generation of autoantibodies in humans has not been elucidated. Human monoclonal IgM anti-IgG autoantibodies (rheumatoid factors, RFs) from unrelated people often share idiotypic antigens. Antibodies against synthetic peptides have localized two of the shared idiotypic determinants to the second and third complementarity-determining regions of the kappa light chain. The reported sequences of several human RF light chains are remarkably homologous in these regions. Animal studies have shown that some shared idiotypic antigens represent serological markers for immunoglobulin variable (V)-region genes. Therefore, we hypothesized that human RF light chains derived from a single germ-line gene, designated V kappa-(RF), or from a small family of very closely related genes. In the present experiments, we have isolated and sequenced two human V kappa germ-line genes that encode kappa light chains, which are identical or closely related to the light chains of human RF. The data indicate that the shared idiotypic antigens on RF are phenotypic markers for a kappa V-region gene that is highly conserved in the human population. The results also imply that the light chains of IgM anti-IgG autoantibodies can be encoded by germ-line genes without any somatic mutation.

Primary infection with EBV results in a life-long carrier state with a small number of circulating B lymphocytes harboring the virus and infective virus being excreted into pharyngeal secretions.

2'-deoxyNAD was examined as a substrate for both mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation reactions. 2'-deoxyNAD is a substrate for the diphtheria toxin-catalyzed mono(ADP-ribosyl)ation of elongation factor-2, inactivating its function to enhance protein synthesis. On the other hand, 2'-deoxyNAD is a poor substrate for poly(ADP-ribose) polymerase. 2'-deoxyNAD was not synthesized intracellularly from deoxyATP, even when deoxyATP content was markedly increased by incubation of cells with deoxyadenosine and an adenosine deaminase inhibitor. 2'-deoxyNAD, because of its specificity, could be a quite useful reagent for the investigation of cellular mono(ADP-ribosyl)ation reactions.

6-methylpurine 2′-deoxyriboside killed mouse macrophages infected with amastigotes of Leishmania donovani and Leishmania mexicana , but did not affect the growth of non-parasitized cells. Leishmania extracts cleaved the non-toxic 6-methylpurine 2′-deoxyriboside to 6-methylpurine, a potent adenine antimetabolite for mammalian cells. By eliminating macrophages latently infected with Leishmania donovani amastigotes, 6-methylpurine 2′-deoxyriboside could augment the effects of leishmanicidal agents in vivo .

Previously, we isolated a Vk gene (Humkv325) from a human placenta that encodes RF light chains bearing the PSL2 and PSL3 CRI markers. Here we report the isolation and characterization of a second human Vk gene (Humkv328) that can be used for RF synthesis. This Vk gene probably encodes at least two 6B6.6 CRI+ RF light chains (Les and Pom) from unrelated subjects, and thus may be related to the light chain-associated 6B6.6 CRI.

The synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (1b) from 1,3-di-O-acetyl-5-O-benzoyl-2-deoxy-2-fluoro-D-arabinofuranose (2a) and 2,6-dichloropurine in six steps using an enzymatic deamination as the last step is reported. The target compound was found to be stable to purine nucleoside phosphorylase cleavage and was cytotoxic in two cell lines, one a T-cell line. Incubation of L1210 cells with 1b results in an inhibition of DNA synthesis as judged by the reduced incorporation of labeled thymidine into DNA, while RNA and protein syntheses were unaffected.

Since B lymphocytes of patients with rheumatoid arthritis (RA) have a higher rate of Epstein-Barr virus (EBV)-induced transformation than normal B cells, we have investigated whether this phenomenon is related to defective cytotoxic T-cell control of emerging B-cell clones. We have studied the in vitro generation of cytotoxic T lymphocytes specific for antigens appearing on autologous B cells after EBV infection using lymphocytes from six patients with RA. Cytotoxic cells were generated by two cycles of stimulation with mitomycin C-treated autologous B cells, and their lytic activity was assessed against 51Cr-labeled targets. As previously reported for lymphocytes of normal individuals, RA cytolytic T lymphocytes were induced only by EBV-infected B cells, not by noninfected B cells, and expression of the cytotoxicity required EBV-transformed targets. After primary in vitro stimulation with autologous EBV-infected B cells, the RA cytotoxic cells expressed higher lytic activity than the normal counterparts, but the difference was not statistically significant (P > 0.1). However, after secondary in vitro stimulation both groups displayed similar cytotoxic activities. In the presence of complement, monoclonal antibodies OKT8 (characterizing cytotoxic/suppressor cells), SC1, and OKT3 (characterizing most T cells) reduced cytotoxicity by 80–90%; antibody OKT3 also reduced the lytic activity by 68% in the absence of complement. We conclude that the higher EBV-induced transformation rate seen with RA lymphocytes is not explained by a deficiency in this EBV-specific cytotoxic T cell.

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.

The chromatin-bound enzyme poly(ADP-ribose) polymerase (ADPRP) is strongly stimulated by DNA with single- or double-stranded breaks, and transfers the ADP-ribose moiety of NAD to nuclear proteins. The activation of ADPRP is important for DNA repair and replication, and also has been postulated to play a role in the pathogenesis of lymphocyte dysfunction associated with chronic inflammatory diseases, and inborn errors of nucleoside metabolism. We have detected high titers of IgG autoantibodies to the ADPRP protein in six patients with rheumatic complaints. No other autoantibodies were detected in any of the six sera. The specificity of the anti-enzyme antibodies was established by (a) immunoprecipitation of ADPRP activity, (b) immunoprecipitation and immunoblotting of both the native 116-kD enzyme and its proteolytic digestion products. ADPRP was purified from human thymus and calf thymus. The autoantibodies reacted equivalently with both enzymes. The anti-ADPRP antibodies had a distinctive immunofluorescent pattern with HEp-2 cells, reacting intensely with nucleoli and metaphase chromosomes, and diffusely with the nucleus. Autoantibodies to ADPRP have not been described previously. The presence of a specific immune response against an enzyme that has been associated with various immunodeficiency syndromes raises intriguing possibilities concerning the relationship between DNA damage, immunodeficiency, and autoimmunity.

The idiotype of the mouse myeloma protein with anti-α-1,3 dextran activity, J558, has been characterized by a solid-phase radioimmunoassay. The idiotype of the J558 protein depends on a specific light and heavy chain interaction and is altered in the presence of the hapten, nigerose. Cross-reacting idiotypes were found on another mouse myeloma protein with α1,3 dextran specificity, normal anti-dextran antibody, and certain reconstructed myeloma proteins composed of the J558 heavy chain and heterologous light chains.

Our experiments have led us to conclude that the rheumatoid arthritis shared epitope may act as a peptide that is important for positive and negative selection of T lymphocytes, that T lymphocytes are skewed by positive selection to recognize epitopes that are similar but not identical to self, and that peptide sequences that are similar to the RA-shared epitope are abundantly expressed by microorganisms that chronically infect most people. This combination of events could partly explain the association of the shared epitope with the severe forms of RA. The hypothesis cannot be tested directly, because we do not postulate that any unique population of autoreactive T cells is expanded in RA; however, the role of positive selection in molding the human T-cell repertoire to exogenous antigens can be tested by mapping T-cell antigenic determinants on the E. coli dnaJ protein or the gp110 protein of EBV in people with different HLA-DR types. Moreover, positive selection models imply that maternal antigens that cross the placenta can influence the T-cell repertoire. Thus, one might expect to find that the frequency of HLA-DR4 in the mothers of patients with RA who themselves lack the DR4 antigen, would be more frequent than predicted by chance alone. As the principles of positive selection are more precisely delineated in animal systems, it should become possible to ascertain more clearly how the shared epitope on HLA-DR molecules enhances the severity of autoimmune reactions; however, RA only occurs in humans; possibly because of the unique inability of human macrophages to replicate. Thus, only the direct analysis of patients can directly reveal the mechanisms of disease pathogenesis.

Several clinical features are consistent with nervous system involvement in the pathogenesis of rheumatoid arthritis. The neuropeptide substance P is one possible mediator of this interaction, since it can be released into joint tissues from primary sensory nerve fibers. The potential effects of the peptide on rheumatoid synoviocytes were examined. The results show that substance P stimulates prostaglandin E2 and collagenase release from synoviocytes. Furthermore, synoviocyte proliferation was increased in the presence of the neuropeptide. Similar effects were observed with a truncated form of substance P. Synoviocytes were sensitive to very small doses of the neuropeptide (10(-9) M), and its effects were inhibited by a specific antagonist. Thus, the specific stimulation of synoviocytes by the neuropeptide substance P represents a pathway by which the nervous system might be directly involved in the pathogenesis of rheumatoid arthritis.

Somatic cell genetics has been used to probe the mechanism of adenosine (Ado) toxicity to mammalian cells deficient in Ado deaminase and Ado kinase. Ado resistant clones of an Ado kinase deficient murine lymphoblastoid cell line (R1.1) were isolated and characterized. In deoxycoformycin supplemented medium, the mutant clones were 10-30 fold more resistant than parental cells to the anti-proliferative actions of Ado, 3-deaza-Ado, carbocyclic-Ado, adenine, 5'-methylthioadenosine, and several other Ado analogs. The mutants were normally sensitive to the toxic effects of deoxyadenosine and adenine arabinoside. Levels of S-adenosylmethionine (SAM) were 50 pmols/106 cells in the parental line, compared to 250-350 pmois/106 cells in the mutant. Methionine adenosyitransferase activity was 1.5-3.2 fold higher in the Ado-resistant cells than in parental lymphoblasts, and varied inversely with the medium methionine concentration. Ado induced the accumulation of equivalent amounts of S-adenosylhomocystelne (SAH) in both cell types. However, the SAH to SAM ratio in the Ado-resistant mutants never exceeded 0.1. These results show that (i) methionine adenosyltransferase is an inducible enzyme in mammalian cells, (ii) the toxic effects of Ado, adenine, and many Ado analogs, can be averted by increasing the velocity of SAM synthesis.

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.

• Concentrations of testosterone, androstenedione, dehydroepiandrosterone (DHA), DHA sulfate (DHAS), progesterone, 17α-hydroxyprogesterone (17-OHP), and hydrocortisone were determined in amniotic fluid obtained at amniocentesis or at elective cesarean section. Male fetuses had significantly higher concentrations of testosterone and androstenedione than female fetuses had between 15 and 21 weeks of gestation but not near term (36 to 40 weeks). In both sexes, progesterone and 17-OHP concentrations fell and DHA, DHAS, and hydrocortisone concentrations increased significantly with advancing gestational age. Amniotic fluid 17-OHP, testosterone, DHA, and androstenedione levels from female fetuses with congenital adrenal hyperplasia (CAH) were more elevated in the second trimester than in the third. Three female fetuses at risk for CAH, but not affected, had normal steroid concentrations. Steroid concentrations from two fetuses with Klinefelter's syndrome were not abnormal. ( Am J Dis Child 1982;136:218-222)

Mice homozygous for the mutation wasted (wst/wst) have been postulated to be a model for the form of human severe combined immunodeficiency disease (SCID) that is secondary to a genetic deficiency of adenosine deaminase (ADA). To test this hypothesis more critically, we transplanted raarrrow from wst/wst and littermate control mice into lethally irradiated normal recipients. If there was an inherent ADA deficiency in the hematopoietic stem cells of the wst/wst mutant, the defect would be expected to be passed on to the irradiated marrow chimeras, resulting in decreased Vmax values for ADA, and/or changes in the Km for adenosine. However, the Vmax and Km values for ADA and adenosine, repsectively, in recipient's hematologic and non-hematologic tissues did not differ significantly from control values. In addition, we also found no significant differences in ADA activities between lymphoid and liver tissue from untreated 24-to-28-day-old wst/wst and littermate control mice. These results indicate that the wasted mouse is not a model for ADA deficiency and SCID.

Poly(ADP-ribose) polymerase (ADPRP) is highly activated by DNA with strand breaks and transfer poly (ADP-ribose)chains outo nuclear proteins. Poly (ADP-ribosyl)action rapidly consumes cellular NAD and leads cells to die. This reaction has been implied to be involved in the deoxyadenosine toxicity toward resting human lymphocytes, and could be an important mechanism in the toxicity of all DNA-damaging agents. To analyze the characteristics of ADPRP, we have utilized human autoantibodies to ADPRP which we have identified in the sera of rheumatic patients. cDNA clones encoding ADPRP were isolated from human libraries and used in these studies. The results of these studies indicate that ADPRP is an abundant nuclear enzyme. Eukaryotic cells synthesize ADPRP throughout S-phase, and keep the cellular ADPRP level constantly in proportion to the amount of DNA. Interestingly more than 99% of ADPRP is present in cells as inactive form. These data may suggest the importance of ADPRP as a structural protein in chromatin conformation. In addition, these results are consistent with the hypothesis of “suicide mechanism” of ADPRP. Eukaryotic cells have approximately 100-fold more enzyme than that is required for the regular cell growth, and keep it for an emergency event. When cellular DNA is severely damaged, ADPRP is activated and eliminates the cell. This mechanism could be applicable to the toxicity of any DNA damaging agents, including many purine and pyrimidine analogs.

BIOCHEMICAL BASIS FOR DEOXYADENOSINE AND 2-CHLORODEOXYADENOSINE TOXICITY TO BESTING HDMAN LYMPHOCYTES: 187

Exogenous addition of methylthioadenosine (MeSAdo) inhibits proliferation of cells which are deficient in MeSAdo phosphorylase. A series of evidences has shown that the target of toxic mechanism of MeSAdo is not totally dependent on polyamine synthesis. Eukaryotic cells contain unique modified amino acid in elongation factor 2 (EF-2), designated as diphthamide. This residue is the specific target of mono(ADP-ribosyl)ation catalyzed by diphtheria toxin (DT) or Pseudomonous toxin. The structure of diphthamide is 2- [ 3-carboxyamido-3-(trimethylammonio)propyl]histidine, and the first reaction to modify histidine involves the transfer of an aminocarboxypropyl group from S-adenosylmethionine. MeSAdo should be the nucleoside product of this reaction. By these reasons, we have analyzed the effect of MeSAdo on the biosynthesis of diphthamide. A mutant cell line H3 which is deficient in MeSAdo phosphorylase and resistant to MeSAdo, has been isolated from murine lymphoma cell line R1.1. for the study. As measured by susceptibility to DT induced ADP-ribosylation, MeSAdo inhibits the formation of diphthamide in a dose dependent manner. In addition, MeSAdo substantially protected H3 cells from the lethal effect of DT. These results suggest that the modulation of diphthamide synthesis can be, at least a part of, the mechanism of MeSAdo toxicity toward eukaryotic cells.

The DHL-9 wild type cell line is a human histiocytic lymphoma naturally devoided of deoxyadenosine deaminase activity. From this cell line a mutant, DHL-9, dAR-2, resistant to high concentrations of deoxyadenosine has been selected. To be cytotoxic deoxyadenosine has to be converted to the corresponding deoxyribonucleoside triphosphate, which is an inhibitor of ribonucleotide reductase. In this study we tested if the high resistance of DHL-9,dAR-2 to deoxyadenosine was due to either a higher level of ribonucleotide reductase or to an alteration of the structure of the nucleotide binding subunit, M1 of ribonucleotide reductase. The mutant was found to have a two to five times higher ribonucleotide reductase activity than the wild type in crude cell extracts. The mutant activity appeared to be as sensitive to dATP inhibition as the wild type enzyme. By SDS-gel electrophoresis followed by immunoblotting it was found that the M1-subunit level was 2-3-fold higher in the dAR-2 extracts than in the wild type. This increased protein M1-level may well explain the 10-20-fold higher resistance to deoxyadenosine of the dAR-2 cell line, but the reason for increased M1-production remains to be clarified.

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.

dAdo is known to be toxic to both proliferating and resting lymphocytes that lack adenosine deaminase (ADA) activity. We now show that human monocytes are also highly sensitive in vitro to nM concentrations of dAdo plus the ADA inhibitor deoxycoformycin, and to the ADA-res istant analog CdA. The dose- and time-dependent toxicity of dAdo or CdA to monocytes is blocked by deoxycytidine, implicating deoxycytidine kinase in the formation of toxic dAdo or CdA nucleotides. Monocytes exposed to dAdo plus deoxycoformycin, or to CdA accumulate massive DNA damage detectable within 1 hour. The accumulation of DNA strand breaks in lymphocytes stimulates the lethal consumption of NAD and ATP for poly(ADP-ribose) synthesis. However, monocytes lack the poly(ADP-ribose) polymerase enzyme and therefore show no significant NAD or ATP depletion until cell viability declines (12 hr). The DNA damage in monocytes exposed to CdA is associated with a decrease in protein synthesis in vitro, and with inhibition of IL-6 secretion. The selective toxicity of CdA to monocytes was confirmed by in vivo studies. Thus, the blood monocyte counts, but not the neutrophil counts, fell to 0 in one week in nearly all patients receiving CdA infusion chemotherapy for cutaneous lymphoma. These results show that dAdo and CdA cause DNA strand break formation and inhibit protein synthesis in human monocytes in vitro, and cause profound monocytopenia in vivo. These compounds may have potential use in the therapy of immune disorders associated with monocyte/macrophage activation.

Unlike other nucleoside anti-metabolites, 2-chlorodeoxyadenosine is selectively toxic at nanomolar concentrations to human lymphocytes and monocytes. In susceptible cells, the drug causes a dose- and time-dependent accumulation of DNA strand breaks, with resultant activation of poly(ADP-ribose) polymerase. Furthermore, the actions of 2-chlorodeoxyadenosine are entirely independent of replicative DNA synthesis. For this reason, we reasoned that 2-chlorodeoxyadenosine would represent a useful agent for the treatment of slowly replicating lymphoid malignancies, and for the therapy of chronic autoimmune diseases. In the present study, 2-chlorodeoxyadenosine was administered to 18 patients wich advanced chronic lymphocytic leukemia, 4 of whom had concurrent autoimmune hemolytic anemia. An overall response rate of 56% was achieved. Only minor and reversible bone marrow suppression occurred during treatment, indicating a high degree of lymphocyte selectivity. Moreover, 3 of the 4 patients with autoimmune hemolytic anemia had complete resolution of hemolysis. Three patients with hairy cell leukemia also received 2-chlorodeoxyadenosine therapy. Two of the patients achieved clinical remission after one course of the drug. These results demonstrate that 2-chlorodeoxyadenosine is a safe and potent anti-lymphocyte and immunosuppressive agent. Further trials of the drug in autoimmune and lymphoproliferative diseases are warranted.

The accumulation of deoxyadenosine (dAdo) and its metabolites may produce immunodeficiency in children who lack adenosine deaminase. The metabolism of dAdo in dividing human lymphoblasts, and the mechanism of dAdo toxicity, have aroused considerable controversy. To investigate this problem, we have selected stable mutant human lymphoblastoid cell lines resistant to the anti-proliferative effects of dAdo and have compared their biochemical phenotypes. The dAdo-resistant mutants differed from wild type cells in one of three ways: (1) an increase in the activity of ribonucleotide reductase, (2) an increase in cytoplasmic nucleotidase activity, (3) a decrease in deoxycytidine kinase activity. All three genetic changes caused a secondary rise in de novo deoxycytidine formation and excretion, and a reciprocal inability to phosphorylate dAdo and to form dATP. Thus, human lymphocytes can avert dAdo toxicity by increasing deoxycytidylate synthesis and degradation, as well as by decreasing deoxycytidine kinase levels. The net result in each case is an impaired functional capacity to phosphorylate deoxyadenosine.