Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages.

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Morphology and incidence of the "posttherapeutic lymphoid cell" in the bone marrow of children with acute lymphoblastic leukemia.

In the posttherapeutic bone marrow of a group of 30 children with acute lymphoblastic leukemia (ALL), small numbers of a particular lymphoid cell with a comparatively large size and large dark nucleus were found. This cell was called the "posttherapeutic lymphoid cell." This type of cell is easily distinguishable in the May-Grünwald-Giemsa-stained smears as well as in semi- and ultrathin Epon sections. Immunoelectron-microscopically it proved to be positive for common ALL. It is hypothesized that the cell might be characteristic for ALL. However, it appeared that this cell could equally be found in non-Hodgkin's malignant lymphoma after a treatment comparable to that in ALL. Furthermore, the cell could be detected in the posttherapeutic bone marrow of children with nonlymphoid malignancies as well as the marrow of very young children (under 2 years of age) with nonmalignant diseases. The results showed that the cell in question is not associated with a particular disease but, rather, represents a special type of lymphoid cell in the regenerating or actively proliferating bone marrow.

6-Mercaptopurine: total body autoradiograms and plasma concentration-time curves of 6MP and metabolites from marmoset monkeys.

To study the body distribution of 6-mercaptopurine (6MP), [8-14C]6MP was given by infusion to 2 marmoset monkeys at a dose rate of 5 mg/kg body weight/h for 1 and 4 h, respectively. Both experimental animals were sacrificed 2 h after the end of the drug infusion and instantly frozen at -70 degrees C. Whole-body sagittal sections were made later. Blood samples were obtained regularly during the experiments to quantitate 6MP, 6MP riboside (6MPR), 6-thioxanthine, and 6-thiouric acid in plasma. The autoradiograms revealed extensive distribution of the 14C label. High levels of radioactivity were seen in liver, bile, and intestinal contents. Labeling of the central nervous system and bone marrow was obvious. The plasma concentration-time curves of 6MP and 6MPR attained steady-state concentrations of 30-40 microM and 6-12 microM, respectively. After stopping the infusion of the drug, the concentrations of 6MP and 6MPR became equal. 6MPR contributes to the biological effect of 6MP, as degradation of 6MPR results in 6MP. In studies on the pharmacokinetics and dynamics one should try to include all relevant metabolites of 6MP, both in plasma and in the cells.

6-mercaptopurine: high-dose 24-h infusions in goats.

In vitro investigations have indicated the need for both prolonged exposure to 6-mercaptopurine (6MP) and the use of high concentrations to achieve maximal cell kill. After the customary oral administration the bioavailability of 6MP appeared to be low, and i.v. bolus injections resulted in short-lived high concentrations of 6MP, so prolonged infusions seemed rational. To test the feasibility of this approach 24-h infusions were given to goats. We used our improved HPLC method to quantitate 6MP and 6MP riboside (6MPR) in plasma, CSF, and urine. The concentrations of 6MPR were in excess of those of 6MP. Since 6MPR can easily be converted to 6MP, 6MPR acts as a depot for 6MP. Penetration of both 6MP and 6MPR into CSF was excellent. Of the total dose administered, 38% to 68% could be accounted for in the urine, with about equal amounts of 6MP and 6MPR. At doses of 20 and 10 mg kg-1 h-1 total concentrations of 6MP and 6MPR in excess of 100 microM were reached during 24-h infusions. However, all three experimental animals died due to toxicity. A dose of 2 mg kg-1 h-1 was tolerated; the total steady state concentration of 6MP and 6MPR in two experiments was about 10 microM. We conclude that the prolonged infusion of 6MP is feasible, and in view of the excellent penetration of 6MP and 6MPR into CSF, studies using prolonged infusions of thiopurines are warranted in man.

6-Thioguanine: high-dose 2-H infusions in goats.

6-Thioguanine (6TG) is poorly absorbed after oral administration. Bolus injections of 6TG result in high peak concentrations with relatively short-lived plasma concentrations. In vitro studies have shown the importance of prolonged exposure to 6TG. Therefore we administered 6TG by infusion at a dose rate of 2 mg/h over 2 h. In three goats we determined the plasma concentration-time curves of 6TG and its riboside (6TGR). A steady state was reached for 6TG and was almost reached for 6TGR within the 2 h of infusion. In one experiment we obtained several samples of CSF and observed good penetration of 6TG and 6TGR into CSF. Urinary excretion of 6TG and 6TGR was also quantitated. The amount of drug and metabolite excreted later than 4 h after the end of the infusion was negligible. By infusing 6TG, the problems of both erratic absorption after oral administration and acute renal toxicity after bolus injection, can be averted. In our opinion prolonged infusions of 6TG may be of advantage in humans suffering from actively proliferating malignant diseases, and thus should be studied.

Increase of HLA-"DRw6" in patients with juvenile chronic arthritis.

In 43 patients with pauciarticular and 26 patients with polyarticular juvenile chronic arthritis (JCA), an increased frequency was found for HLA-B27 (36%) in male patients with onset of the disease over 9 years of age. The antigen "DRw6" was observed in high frequency (average 57%) in all subtypes of the disease. Indications for increased frequencies were obtained for DR3 in male JCA (40%) and DR5 (42%) in female JCA patients.

Purine metabolism in childhood acute lymphoblastic leukemia: biochemical markers for diagnosis and chemotherapy.

Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), 5'nucleotidase (5'NT), ecto-5'NT, hypoxanthine-guanine phosphoribosyltransferase(HGPRT), adenine phosphoribosyltransferase(APRT), adenosine kinase(AK), AMP deaminase (AMPD) and adenylate kinase(AdKin) activities were assayed in leukemic cells from bone marrow and/or peripheral blood of 43 newly diagnosed children with acute lymphoblastic leukemia(ALL). These enzyme activities have been investigated in relation to some immunological markers. ADA activity was higher in E-rosette positive leukemia(E+ ALL), while HGPRT, APRT, PNP, 5'NT, ecto-5'NT and AdKin activities were found to be lower in E+ ALL as compared to E- ALL. In common ALL (cALL) antigen positive leukemia, mean ADA activity was significantly lower as compared to cALL- leukemia, whereas PNP, 5'NT, ecto-5'NT and AdKin activities were significantly higher. cALL cells with cytoplasmic immunoglobulin M(IgM) heavy chains were found to have mean 5'NT activities twice as high as cALL cells lacking cytoplasmic IgM heavy chains. In two patients who had surface immunoglobulins on their cell membranes, low 5'NT activities were found. When measuring enzyme activities after 2-4 days of prednisone monotherapy, only mean ADA and HGPRT activities decreased in non-B, non-T ALL. These decreases were not significant in T-ALL patients. Mean enzyme activities in the leukemic cells of five patients with relapse were comparable to those in newly diagnosed patients, except for 5'NT, which was found to be within the activity range of control peripheral blood lymphocytes. It is concluded that ADA and AdKin activities are suitable as markers for E+ ALL and cALL+ leukemias respectively. 5'NT might help to distinguish between cALL cells having and lacking pre-B characteristics. Since 5'NT activity may also be decreased in B-ALL, it is not suitable as a T-ALL marker. Enzymes of purine metabolism in leukemic relapse need further investigation.

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%.

Sideroblastic anaemia. A review of seven paediatric cases.

Sideroblastic Anaemias are characterised by a) chronic hypochromic anemia, b) ringed sideroblasts in the bone marrow, c) an increase in total body iron, d) ineffective erythropoiesis and e) often abnormal concentrations of F.E.P. A classification of Sideroblastic Anaemia is given and the pathophysiology of Sideroblastic Anaemia is discussed. A series of seven paediatric cases with Sideroblastic Anaemia is presented and the results of studies of the iron, vitamin B6 and porphyrin metabolism are discussed. In two cases arguments for an ALA-synthetase deficiency are given. All five males were diagnosed as hereditary X-linked Sideroblastic Anaemia, one female as I.R.S.A. and the other female, who showed the features of the X-linked type, as congenital Sideroblastic Anaemia.

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.

Pyrimidine metabolism in erythrocytes of the newborn.

Activities of orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase were found to be significantly higher in erythrocytes from newborn infants than in erythrocytes from adults, and approximated those observed in patients with deficiency of hypoxanthine-guanine phosphoribosyltransferase. Enzyme activities were increased to a varying extent in patients with reticulocytosis. The results are discussed in relation to red cell age and stabilization of the enzymes by phosphoribosylpyrophosphate. Pyrimidine-5'-nucleotidase was assayed by a new radiochemical method involving thin-layer chromatography for separation of product from substrate. Enzyme activity was higher with orotidine monophosphate than with uridine monophosphate. The activity of this enzyme was similar in erythrocyte of newborns and adults.