Chris H.M.M. De Bruyn

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps. The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD+, cyclic AMP, NADP+ and uridinediphosphoglucose (UDPG). In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.

Sequence-, time- and dose-dependent synergism of methotrexate and 6-mercaptopurine in malignant human T-lymphoblasts☆

Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.

Partial hypoxanthine-guanine phosphoribosyl transferase deficiency with full expression of the Lesch-Nyhan syndrome.

SummaryA patient with the full clinical expression of the classical Lesch-Nyhan syndrome is presented with a residual hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity of 5–10% in erythrocyte lysate and about 30% in fibroblast lysate. The activities of other erythrocyte enzymes of purine metabolism were typical for a classical Lesch-Nyhan patient. The effects of allopurinol therapy on the excretion of urinary purine metabolites were studied by a newly developed isotachophoretic technique.The unusually high residual activity of HGPRT in erythrodytes and fibroblasts of the patient enabled the enzymologic characterization of the mutant enzyme: in fibroblasts the affinities for the substrates hypoxanthine and guanine were normal. However, there was an increased apparent Km for phosphoribosylpyrophosphate (PRPP), a complete absence of product inhibition by IMP and GMP, and a decreased heat stability. Addition of PRPP did not stabilize the mutant enzyme. In addition to the altered properties of the fibroblast enzyme, the Km of the erythrocyte enzyme for hypoxanthine was also increased.Immunoprecipitation experiments revealed the presence of an approximately normal amount of material cross-reacting with anti-human HGPRT antiserum. However, it appeared that this cross-reacting material had a decreased stability. When intact erythrocytes were incubated with radiolabeled purine bases, no formation of IMP or GMP could be detected, despite the relatively high residual activity of HGPRT in the hemolysate. The results fit the following hypothesis: as a consequence of a structural mutation affecting the PRPP-site of the enzyme and a decreased heat stability, the activity of the mutant enzyme under in vivo conditions is virtually zero.In the erythrocytes of the patients mother a normal HGPRT-activity was found. However, the activity in her fibroblasts was lower than normal, while a decreased heat stability and an intermediate behavior towards IMP could be shown.Hair root analysis of several members of the patients family confirmed the heterozygosity of the mother, whereas no other heterozygotes could be detected. The family anamnesis did not show other cases of Lesch-Nyhan syndrome. These findings were taken as evidence that the patient described in this paper might represent a mutation orginating from the gametes in either of the maternal grandparents.

Hypoxanthine-guanine phosphoribosyl transferase deficiency.

SummaryIn man congenital lack of an enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan syndrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation between HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT deficiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of uric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency.There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant enzymes may exhibit different kinetic and electrophoretic properties, indicating that there might be a mutation on the structural gene coding for HG-PRT.Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine bases hypoxanthine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT. Uptake of purine bases into intact red blood cells occurs according to a two component mechanism, one component probably involving a phosphoribosyl transferase system.The inheritance of HG-PRT deficiency is X-linked recessive and it is transmitted by asymptomatic carrier females. Several methods have been introduced for carrier detection. As a consequence of X chromosome inactivation, in these females a mosaicism of HG-PRT positive and HG-PRT negative fibroblasts can be demonstrated after cloning or after selection of HG-PRT negative cells in a selective medium. A more rapid method involves direct measurements of HG-PRT activities in single hair roots from the scalp. Because hair roots develop more or less clonally, in heterozygote females HG-PRT positive and negative hair roots are encountered. HG-PRT deficiency can be detected antenatally by demonstrating the presence or absence of enzyme activity in ammiotic fluid derived fibroblasts qualitatively by autoradiography and quantitatively by ultramicrochemical measurements of enzyme activities in single or small numbers of cells.In studies with isolated cells the metabolic defect can be corrected in several ways. Metabolic cooperation between HG-PRT positive and HG-PRT negative cells leads to apparently normal phenotype of all cells, provided there is cell to cell contact. There is evidence that a missing enzyme product or a derivative might be transferred from the normal to the mutant cells. Apparent correction of the enzyme defect is also observed when HG-PRT deficient lymphocytes are stimulated with phytohaemagglutinin.The first data suggestive of genetic complementation between two human HG-PRT deficient cell strains by which hybrid cells can synthesize a functionally active HG-PRT, are consistent with the view that HG-PRT deficiency in man is due to a structural gene mutation. Recent results show that other interesting findings might come from experiments in which HG-PRT deficient cells are treated with exogenous genetic material (isolated DNA or metaphase chromosomes) to reactivate or induce HG-PRT activity.

High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides in biological fluids

A method is presented for the separation and quantitative determination of compounds normally related to purine and pyrimidine metabolism in biological material. The retention behaviour of nucleobases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides has been systematically investigated by reversed-phase high-performance liquid chromatography using a non-linear gradient. Ultimately a separation of the purine and pyrimidine compounds was achieved in a 35-min run with an average detection limit of 5-10 pmol per injection. Recoveries of standards added to urine, plasma or serum were 96 +/- 5%.

BIPHASIC EFFECT OF ADENOSINE ON CELL GROWTH AND CELL CYCLE OF HUMAN LYMPHOID CELL LINES: 219

Accumulation of adenosine is detected in plasma of ADA-deficient children(1). We incubated human lymphoid cell lines with adenosine in presence of the ADA inhibitor deoxycoformycin. The malignant T cell line Moltland the non-malignant B cell line RPMI 1788 were both more affected by adenosine than the malignant B cell line RAJI was. However, adenosine had a remarkable biphasic effect on cell growth of all three cell lines. After 48 hours incubation with 40μM adenosine growth of all three cell lines was severely inhibited (Molt 493%, RPMI 1788 87%, RAJI 63%). When 100μM adenosine was used the inhibitory effect was less severe (Moltμ58%, RPMI 1788 72%, RAJI 49%). This biphasic effect was also reflected in the cell cycle. After 24 hours incubation addition of 20μM adenosine to the cell cultures resulted in accumulation of cells in the G1 phase while addition of 50μM adenosine resulted in a decrease of cells in the G1 phase. Drawing conclusions about the inhibitory effects of adenosine should be done with caution.

A radiochemical microassay of S-adenosylhomocysteine hydrolase in lymphoid cells

Adenosine is known to affect the immune system through its effects on lymphoid cells. In the human lymphocyte adenosine together with homocysteine can be converted to S-adenosylhomocysteine (SAH) by SAH hydrolase (EC 3.3.1.1) [1]. Elevation of adenosine concentrations in cells of various origins leads to accumulation of SAH, which is a potent inhibitor of several transmethylation reactions [2-4]. Both for diagnostic and fundamental purposes we have measured the SAH-hydrolase activity in human T-lymphoid cells with a procedure that requires less material than the conventional methods. Here we present a simple radiochemical microassay for the SAH-hydrolase activity in about 12000 human peripheral T lymphocytes or human thymocytes.

INFLUENCE OF METHOTREXATE ON PURINE AND PYRIMIDINI POOLS AND ON CELL PHASE DISTRIBUTION OF CULTURED HUMAN LYMPHOBLASTS: 50

Human leukemic lymphoblastic cell lines (Molt-4, Raji) were treated for 24 hrs with 0.2 μM Methotrexate (MTX). After 12 hrs, intracellular dCTP and dTTP concentrations decreased to 20% of the values of untreated cells. We noted a significant increase of UMP, UTP and CTP concentrations under these conditions. At the same time dATP and dGTP levels dropped to 30% and 50%, respectively. Accumulation of cells in G1 phase and decrease of cells in G2+M phase (to almost zero) was observed, after treatment with 0.2 μMMTX. The effect of 0.02 μM MTX on intracellular purine and pyrimidine concentrations was less pronounced, than that of 0.2 μM MTX, except for dCTP. In contrast to a decrease of dCTP, observed in experiments with 0.2 μM MTX, an increase of dCTP was measured in experiments with 0.02 μM MTX. Cell cycle phase distribution studies with 0.02 μM MTX, revealed an accumulation of cells in early S phase and decrease of cells in G1 phase and G2+M phase.

INHIBITION OF 3H-THYMIDINE INCORPORATION BY ADENOSINE AND DEOXYADENOSINE IN HUMAN PERIPHERAL LYMPHOCYTES AND MALIGNANT LYMPHOID CELL LINES: 218

Children with ADA deficiency have an impairment of both cellular and humoral immunity. We performed a detailed study of the relative toxicities of the ADA substrates adenosine and deoxyadenosine with regard to 3H-thymidine incorporation in mitogen-stimulated human peripheral blood lymphocytes and malignant lymphoid cell lines. Lymphocytes, isolated by Percoll gradient centrifugation and counterflow elutriation, were stimulated with various mitogens (PHA, ConA, PWM, SpA, StA) known to activate different lymphocyte subpopulations. Both lymphocytes and lymphoid cell lines were incubated with various adenosine and deoxyadenosine concentrations in presence of the ADA inhibitor deoxycoformycin. Combinations of deoxycoformycin and adenosine or deoxyadenosine gave inhibition of 3H-thymidine incorporation in both lymphocytes and cell lines. Adenosine affected 3H-thymidine incorporation of mitogen-stimulated lymphocytes to a similar extent as deoxyadenosine. However, to the malignant T cell line Molt 4 deoxyadenosine was more toxic than adenosine. This was not observed with the cell lines REH (nonBnonT), KM-3 (nonBnonT) and RAJI (B).Lymphocytes stimulated with B- or T cell mitogens were affected in their thymidine incorporation to a comparable extent by adenosine and deoxyadenosine. The cell line Molt was more sensitive to deoxyadenosine than both stimulated lymphocytes and the cell lines REH, KM-3 and RAJI.

ADENOSINE RECEPTORS ON HUMAN T LYMPHOCYTES AND HUMAN THYMOCYTES: 55

Adenosine (ado) can effect the immune system via binding to cell-surface receptors on lymphoid cells, resulting in an increase in the intracellular level of cAMP. The basal amount of cAMP in T lymphocytes (range 1.6-9.0; mean 5.5 pmoles/107 cells) was significantly higher than that in thymocytes (range 0.2-1.7; mean 0.7 pmoles/107 cells). Incubation of thymocytes with 10 μM ado resulted in a rapid (1 min), transient increase in cAMP, whereas in T lymphocytes a prolonged (> 15 min) cAMP increase occurred. The effects of ado were most pronounced after incubation (15 min) with the non-metabolizable 2-chloro-ado and the phosphodiesterase inhibitor Ro20-1724. This resulted in a net increase in cAMP levels of 10-35 pmoles for thymocytes and 20-60 pmoles for T lymphocytes. Binding of ado to the ado receptor and the subsequent increase in the intracellular cAMP level may be a major event with respect to the effect of ado on T-lymphoid cells.