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Biomedical subjects

G Rijksen

Publications and source records attributed to G Rijksen.

At least 73 records · Page 4Linked to original sources
PubMed1990

Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates.

This study reports the isolation and characterization of a variant of the human CCRF-CEM leukemia cell line that overproduces the carrier protein responsible for the uptake of reduced folates and the folate analogue methotrexate. The variant was obtained by adapting CCRF-CEM cells for prolonged times to stepwise decreasing concentrations of 5-formyltetrahydrofolate as the sole folate source in the cell culture medium. From cells that were grown on less than 1 nM 5-formyl-tetrahydrofolate, a variant (CEM-7A) was isolated exhibiting a 95-fold increased Vmax for [3H]methotrexate influx compared to parental CCRF-CEM cells. The values for influx Km, efflux t0.5, and Ki for inhibition by other folate (analogue) compounds were unchanged. Affinity labeling of the carrier with an N-hydroxysuccinimide ester of [3H]methotrexate demonstrate an approximately 30-fold increased incorporation of [3H] methotrexate in CEM-7A cells. This suggests that the up-regulation of [3H]methotrexate influx is not only due to an increased amount of carrier protein, but also to an increased rate of carrier translocation or an improved cooperativity between carrier protein molecules. Incubation for 1 h at 37 degrees C of CEM-7A cells with a concentration of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate in the physiological range (25 nM) resulted in a 7-fold decline in [3H]methotrexate influx. This down-regulation during incubations with 5-formyltetrahydrofolate or 5-methyltetrahydrofolate could be prevented by either the addition of 10-25 nM of the lipophilic antifolate trimetrexate or by preincubating CEM-7A cells with 25 nM methotrexate. The down-regulatory effect was specifically induced by reduced folates since incubation of CEM-7A cells with 25 nM of either methotrexate, 10-ethyl-10-deazaaminopterin, aminopterin, or folic acid, or a mixture of purines and thymidine, had no effect on [3H]methotrexate influx. Similarly, these down-regulatory effects on [3H]methotrexate transport by 5-formyltetrahydrofolate, and its reversal by trimetrexate or methotrexate, were also observed, though to a lower extent, for parental CCRF-CEM cells grown in folate-depleted medium rather than in standard medium containing high folate concentrations. These results indicate that mediation of reduced folate/methotrexate transport can occur at reduced folate concentrations in the physiological range, and suggest that the intracellular folate content may be a critical determinant in the regulation of methotrexate transport.

Biological Transport↗
PubMed1990

Phosphorylation of pyruvate kinase type K in human gliomas by a cyclic adenosine 5'-monophosphate-independent protein kinase.

In recent years, we reported the isozyme shift of pyruvate kinase from the M- toward the K-type in human neuroectodermal tumors. To investigate whether this shift enables phosphorylation of pyruvate kinase in these tumors, we studied 29 different specimens of human brain tumors for endogenous pyruvate kinase phosphorylation. While in normal human brain no phosphorylation of pyruvate kinase was detected, in all brain tumors pyruvate kinase became phosphorylated. There was no correlation between the extent of the pyruvate kinase phosphorylation and the histological classification and grading or the pyruvate kinase isozyme composition of the tumors. Only pyruvate kinase type K, and not type M, served as a substrate in the phosphorylation reaction. The phosphorylation of pyruvate kinase could be completely inhibited by addition of fructose 1,6-bisphosphate, a positive effector of pyruvate kinase type K; alanine, however, a negative effector, and phospho-enol-pyruvate, a substrate in the pyruvate kinase reaction, had no effect. While pyruvate kinase type L in liver is phosphorylated by a cyclic AMP-dependent protein kinase, the incorporation of phosphate into pyruvate kinase in human brain tumors appeared to be cyclic AMP independent and occurred exclusively on serine residues.

Biopsy↗
PubMed1990

Selective killing of malignant cells from leukemic patients by alkyl-lysophospholipid.

We studied the effects of the alkyl-lysophospholipid, 1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ALP), on human leukemia cells from 56 patients with various leukemias and on normal bone marrow progenitors in order to assess the application of ALP as an in vitro marrow-purging agent. The tumoricidal activity was analyzed by the elimination of clonogenic leukemia cells (leukemic colony-forming cells), by the inhibition of the proliferative capacity [( 3H]thymidine incorporation) of leukemia cells, and by the elimination of viable leukemia cells measured with flow cytometry. The tumoricidal activity of ALP was dose and incubation time related, as, although to a lesser extent, held true for normal marrow progenitors. For some leukemias the ALP dose necessary for the elimination of 100% of the leukemic colony-forming cells is probably too toxic for normal marrow cells. The results of this study strongly support the possibility that ALP is a promising purging agent in the majority of patients with leukemias.

Adolescent↗
PubMed1990

Diagnosis of pyruvate kinase deficiency in a transfusion-dependent patient with severe hemolytic anemia.

In a 2-yr old girl a hemolytic anemia was present since birth requiring multiple blood transfusions. Pyruvate kinase deficiency was suspected on the basis of a marginal enzyme activity, but could not be established due to the presence of massive numbers of donor cells in her peripheral blood. However, by density fractionation we succeeded in the isolation of a small fraction of the patient's own cells, in which a severe pyruvate kinase deficiency could be detected. In contrast hexokinase and glucose-6-phosphate dehydrogenase activities were extremely high, which is indicative that a very immature cell population is present in this fraction. In immunofluorescence studies a clear crossreaction was apparent with anti M2-type pyruvate kinase antibodies, whereas only a faint reaction with anti L-type could be detected. Despite the presence of a slight amount of L-type immunoreactive material, the residual activity in the patient's cell fraction could only be attributed to M2-type pyruvate kinase as was shown by cellulose acetate electrophoresis.

Anemia, Hemolytic↗
PubMed1990

Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation.

Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to -M shift of pyruvate kinase.

Animals↗
PubMed1990

Activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase in smooth muscle proliferation.

The specific activity of hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase and glucose-6-phosphate dehydrogenase was measured in 41 smooth muscle cell tumors: 20 leiomyomas and 21 cases of leiomyosarcoma. Statistical analysis revealed no significant differences in specific activity between normal smooth muscle tissue and the benign and malignant tumors originating from it. Quantification of the isozyme composition of pyruvate kinase showed a significant shift in isozyme pattern towards K-type subunits in leiomyosarcomas as compared to leiomyomas.

Fructose-Bisphosphate Aldolase↗
PubMed1990

Activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase in lipoblastic and neurogenic proliferations.

The alterations in specific activity and/or isozyme pattern of hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase and glucose-6-phosphate dehydrogenase were studied in the tissue specimens of 26 patients with lipoblastic tumors and 28 patients with tumors of neurogenic origin. Although the biochemical data demonstrated that the activities of most enzymes studied were elevated in the specimens of the malignant tumors, only the differences in activity of hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase measured between benign and malignant neurogenic tumors were significant. In malignant tumors, especially those of neurogenic origin, the isozyme pattern of pyruvate kinase showed a shift towards K-type subunits.

Adolescent↗
PubMed1989

Interaction of N-hydroxy(sulfo) succinimide active esters with the reduced folate/methotrexate transport system from human leukemic CCRF-CEM cells.

The membrane impermeant protein cross-linker 3,3'-dithiobissulfosuccinimidyl propionate (DTSSP) is a well-known inhibitor of human erythrocyte band 3-mediated inorganic anion transport. We observed that DTSSP is also a potent inhibitor of reduced folate/methotrexate transport in human CCRF-CEM leukemia cells. An interaction of DTSSP with the reduced folate/MTX is substantiated by findings that: (a) like MTX transport itself, the concentration of DTSSP required for half-maximal inhibition of [3H]methotrexate transport varied substantially with the anionic composition of the external medium. In a saline buffer and an anion-deficient buffer the I50 values were 7 and 1 microM, respectively; (b) saturation of the carrier with 1-5 microM methotrexate completely protected the transport system from interaction by DTSSP; (c) methotrexate transport activity in DTSSP-treated cells could be restored after cleavage of the disulfide bond in DTSSP under mild reducing conditions; and (d) pretreatment of cells with DTSSP reduced the incorporation of [3H]methotrexate after labeling with an N-hydroxysuccinimide ester of [3H]methotrexate (NHS-MTX), another potent inhibitor of methotrexate transport. Comparison of DTSSP- and NHS-MTX-induced inhibition of methotrexate transport showed that DTSSP inhibition, in contrast to NHS-MTX inhibition, was (a) less potent, (b) dependent on buffer conditions, (c) reversible by reducing agents, and (d) required only a very low molar ratio of methotrexate over DTSSP to afford maximal protection.

Biological Transport↗
PubMed1989

Identification of a membrane-associated folate-binding protein in human leukemic CCRF-CEM cells with transport-related methotrexate resistance.

CEM/MTX cells, a subline of CCRF-CEM cells resistant to methotrexate (MTX) by virtue of impaired transport by the reduced folate/methotrexate transport system, were grown in media containing folate levels in the physiological range rather than in standard media supplemented with high folate concentrations. Over a 7-month period folic acid concentrations were gradually lowered from 2 microM to 2 nM without subsequent changes in the transport-defective phenotype. In these cells we observed the up regulation of a membrane-associated folate-binding protein with high affinities for folic acid and reduced folates, but poor affinities for the folate antagonists MTX and 10-ethyl-10-deazaaminopterin. The binding capacity for [3H]folic acid was 12.5 pmol/10(7) cells, but could be further increased to 30 pmol/10(7) cells following cell transfer to completely folate-deficient medium for 3 days, except that in the latter situation cell growth stopped. The affinities of the folate-binding protein for 5-methyltetrahydrofolate, folinic acid, and MTX were 0.33, 0.11, and 0.009, respectively, relative to that of folic acid. Growth of CEM/MTX cells was promoted by nanomolar concentrations of either folic acid (median effective concentration, 0.35 nM) or folinic acid (median effective concentration, 0.75 nM), suggesting that the folate-binding protein is not only involved in folate binding, but also in the uptake of folates. The insensitivity of CEM/MTX cells to MTX was correlated with the poor affinity of the folate-binding protein for MTX, compared to folic acid. MTX was only growth inhibitory when added at concentrations at least 30-fold exceeding those of folic acid in the culture medium. On the other hand, CEM/MTX cells grown at 2 microM or 2 nM folic acid were equally sensitive to the lipophilic antifolate trimetrexate. Despite the low affinity for MTX, the folate-binding protein could be specifically labeled by an N-hydroxysuccinimide ester of [3H]MTX and appeared to have a molecular weight of 44,000 as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that an alternative folate uptake system, a folate-binding protein, car play an important role in transport-related methotrexate resistance. Moreover, since all these effects were observed for CEM/MTX cells grown at folate levels in the physiological range, it is conceivable that this mechanism of methotrexate resistance can also be of significance in leukemic cells in vivo.

Biological Transport↗
PubMed1989

Phosphofructokinase in alveolar type II cells isolated from fetal and adult rat lung.

The glycolytic enzyme 6-phosphofructokinase (EC 2.7.1.11) was studied in adult and fetal type II pneumocytes which had been isolated from rat lung at different days of development. In addition, the activities of the enzymes hexokinase (EC 2.7.1.1), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were assayed. The specific activities of the latter enzymes decrease during perinatal development and reach about adult values shortly after birth. In contrast, 6-phosphofructokinase activity increases slightly until 2 days before birth, and drops sharply afterwards. The 6-phosphofructokinase subunit composition was determined in fetal and adult type II cells. The ratio of the three subunits of 6-phosphofructokinase in type II cells isolated on fetal days 19 and 21 (term is at day 22) and in adult type II cells was identical: the three subunits were present in a ratio of 68: 14: 18 for types L, M and C, respectively. In addition, we investigated some regulatory properties of 6-phosphofructokinase from alveolar type II cells. 6-Phosphofructokinase from alveolar type II cells is strongly inhibited by increasing MgATP concentrations. This inhibition is reflected by an increase in the S0.5 for fructose 6-phosphate. Fructose 2,6-bisphosphate stimulates alveolar type II 6-phosphofructokinase. Half-maximal stimulation occurs at 1.6 and 2.0 microM fructose 2,6-bisphosphate for fetal and adult type II cells, respectively. The level of the most potent positive effector of 6-phosphofructokinase, fructose 2,6-bisphosphate, was also determined. The level of the hexose bisphosphate decreases during prenatal development; however, the level in the adult type II cells is considerably lower. The concentration of fructose 2,6-bisphosphate appears to be sufficient to fully activate 6-phosphofructokinase both in fetal and adult type II cells.

Adenosine Triphosphate↗
PubMed1989

Expression of a folate binding protein in L1210 cells grown in low folate medium.

We have isolated variants of L1210 cells (L1210B) expressing, in addition to the "classical" high affinity/low capacity system for reduced folate uptake, high levels of a membrane-associated folate binding protein. This folate binding protein was expressed in L1210 cells grown at low physiological folate levels (less than 0.5 nM), but down-regulated after transfer in standard high folate (2 microM) medium. The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5-11 pmol/10(6) cells) but affinities were different. The affinities relative to folic acid were 0.5 for 5-methyltetrahydrofolate, 0.25 for 5-formyltetrahydrofolate, 0.08 for 10-ethyl-10-deazaaminopterin, and 0.05 for methotrexate, respectively. L1210B cells exposed to low extracellular concentrations of [3H]folic acid (25 nM) accumulated 15 pmol [3H]folic acid/10(7) cells over a 5-h period. [3H]Folic acid accumulation by wild-type L1210 cells could not be demonstrated under these conditions. The folate binding protein in L1210B cells could be specifically and covalently labeled at 4 degrees C with a N-hydroxysuccinimide ester of [3H]-methotrexate or [3H]folic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent-solubilized membrane proteins showed a major labeled band with Mr 42,000-44,000.

Animals↗
PubMed1989

Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy.

The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.

2,3-Diphosphoglycerate↗
PubMed1989

Characterization of pyruvate kinase from human rhabdomyosarcoma in relation to immunohistochemical and morphological criteria.

We have compared the pyruvate kinase (PK) isoenzyme pattern of three rhabdomyosarcomas with foetal skeletal muscle tissue of 19 and 23 weeks of gestation, together with adult muscle in relation to immunohistochemical and morphological criteria. In foetal tissue of 19 weeks of gestation a focal immunopositivity for desmin and myoglobin was observed, whereas in tissue of 23 weeks an overall positivity for these proteins was present. Two of the three neoplasms were poorly differentiated and of the alveolar subtype. They were desmin immunoreactive. Some large spindle-shaped cells expressed myoglobin. The third one was more differentiated in microscopic characteristics and all cells showed immunoreactivity for desmin and myoglobin. In the foetal tissues five forms of pyruvate kinase isoenzymes were present with K2M2 as the predominant form. In adult muscle tissue only M4 was present. The tumors were characterized by a profound shift to the K-type, whereas the M4-type was not expressed at all. A difference in isoenzyme composition of pyruvate kinase was found between the morphologically less differentiated tumors and the more differentiated tumor; in the latter more M-subunits were expressed.

Adolescent↗
PubMed1989

Tyrosine kinase activity in breast cancer, benign breast disease, and normal breast tissue.

Tyrosine specific protein kinase activity was determined in 70 specimens of the human mammary gland. These included 28 cancers of the breast, 21 benign breast diseases, and 21 normal breast tissues. We measured tyrosine kinase activity in the cytosol fraction and in the membrane fraction of the homogenates. In addition cytosolic aldolase activity was measured. Tyrosine kinase activity was determined using poly(glutamic acid:tyrosine = 4:1) as an artificial substrate. Cancers of the breast exhibited considerable higher tyrosine kinase activities in both cytosol and membrane fractions, compared to benign breast tumors (P less than or equal to 0.001). Benign tumors demonstrated increased activities in cytosol in comparison to normal breast tissues (P less than 0.001). Furthermore, there appears to be a strong association of an enhanced expression of activity of tyrosine kinase in cytosol of primary carcinomas and early systemic relapse. In combination with aldolase activity a nearly complete discrimination is achieved between malignant specimens on one hand and benign and normal tissues on the other.

Adult↗
PubMed1989

Erythrocyte Na+/K+ ATPase activity measured with 23Na NMR.

A 23Na NMR assay for measurement of erythrocyte Na+/K+ ATPase activity is presented. Using the nonpermeant shift reagent dysprosium tripolyphosphate the signals of intra- and extracellular sodium are separated, enabling measurement of sodium fluxes nondestructively, without the need to physically separate the cells from their environment. By increasing membrane permeability with nystatin we have shown that the assay allows the detection of differences in membrane permeability. With low doses of nystatin the ouabain-sensitive sodium flux increased more than twofold. With high doses of nystatin the Na+/K+ pump could not prevent an almost total equilibration of intra- and extracellular sodium. All sodium that entered the cells remained NMR visible, proving that sodium influx can be measured quantitatively. 31P NMR spectra taken before and after the assay revealed a slight acidification of the cells and no significant change in ATP concentration. No evidence of Dy3+ entering the cell was observed.

Cell Membrane Permeability↗
PubMed1989

A nonradioactive dot-blot assay for protein tyrosine kinase activity.

A new procedure for the assay of protein tyrosine kinase, based on the detection of phosphorylated tyrosyl residues by using monoclonal antibodies to phosphotyrosine, is described. After incubation of a protein tyrosine kinase sample with the substrates poly-(GluNa,Tyr)4:1 and unlabeled ATP an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining procedure. The signal is quantified by densitometry. The assay is linear with time and is quantitative in a wide range of sample protein concentrations. Its sensitivity allows the kinetic characterization of protein tyrosine kinases at low substrate concentrations, whereas on the other hand the avoidance of radioactivity enables the use of high ATP concentrations as well. Protein tyrosine kinase activities of human breast carcinomas and normal breast tissues measured with this method correlated well with the conventional assay, in which the incorporation of [32P]phosphate is measured by TCA precipitation and liquid scintillation counting. Compared to the latter, the new assay is at least as sensitive and accurate and harbors the advantage of the avoidance of radioactivity, thus enabling one to perform a large number of protein tyrosine kinase assays simultaneously.

Adenosine Triphosphate↗
PubMed1989

Phosphorylation of 6-phosphofructokinase in rat lung.

1. 6-Phosphofructokinase of both fetal and adult rat lung consists of L, M and C subunits in a ratio of 65:25:10. 2. 6-Phosphofructokinase was purified to homogeneity from adult rat lung and subjected to phosphorylation in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. 3. This resulted in phosphorylation of the L and M subunit of 6-phosphofructokinase. 4. The C subunit was not phosphorylated. 5. However, if the phosphorylation of 6-phosphofructokinase was studied in the cytosol fraction of either fetal or adult lung using endogenous protein kinase(s), only the L subunit was phosphorylated. 6. This phosphorylation was dependent on cyclic AMP. 7. No influence of calcium, calmodulin or phosphatidylserine/diolein on the phosphorylation was observed. 8. It is concluded that although both L and M subunits of rat lung 6-phosphofructokinase are potential substrates for cyclic AMP-dependent protein kinase, their phosphorylation in situ is differentially regulated.

Aging↗