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Biomedical subjects

G Rijksen

Publications and source records attributed to G Rijksen.

At least 55 records · Page 3Linked to original sources
PubMed1993

Protein tyrosine kinase activity in laryngeal squamous cell carcinoma.

Oncogenes play an important role in the process of malignant transformation. Since many of the protein tyrosine kinases (PTK) are products of oncogenes, the aim of this study was to demonstrate whether an increased PTK activity could be found in head and neck tumors. By using a non-radioactive dot-blot assay, PTK activity was measured in tumor and normal tissues of 38 patients with laryngeal cancer. The control group consisted of 19 healthy persons. PTK activity in tumor cells was significantly higher (P < 0.001) than in normal cells of the tumor patients and normal controls. Additionally, the PTK activity in the normal mucosa of the tumor patients was significantly higher than in the normal mucosa of the control group.

Carcinoma, Squamous Cell↗
PubMed1993

Adsorption and uptake of the alkyllysophospholipid ET-18-OCH3 by HL-60 cells during induction of differentiation by dimethylsulfoxide.

The human leukemic cell line HL-60 is highly sensitive to the antineoplastic agent alkyllysophospholipid, 1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3). We investigated the adsorption and uptake of radiolabeled ET-18-OCH3 in undifferentiated HL-60 cells and during differentiation to granulocytes induced by dimethylsulfoxide. HL-60 cells become less sensitive to the cytotoxic action of ET-18-OCH3 during differentiation. The decrease in sensitivity is correlated with a decrease in both adsorption and uptake of [3H]ET-18-OCH3 during differentiation. Binding studies revealed that the binding of ET-18-OCH3 to both undifferentiated and differentiated HL-60 cells is non-saturable which renders the existence of a specific binding place highly unlikely.

Adsorption↗
PubMed1992

Characterization of protein tyrosine kinases from human breast cancer: involvement of the c-src oncogene product.

Tyrosine phosphorylation is an important regulatory mechanism in response to the action of growth factors and oncogenes. Since many oncogenes code for tyrosine kinases, increased or altered oncogene expression may be reflected in increased tyrosine kinase activity. In a recent study (Hennipman et al., Cancer Res., 49: 516-521, 1989), we found that the tyrosine kinase activity of the cytosolic and membrane fractions of malignant human breast tissue was significantly higher compared to the benign or the normal breast tissue. Moreover, the increase in the cytosolic fractions was found to be of prognostic value. In the present study we determined the protein tyrosine kinase (PTK) activity of another 72 breast cancer specimens, and it could be shown again that the PTK activity in all 72 of these tumors was elevated compared to normal controls. We characterized these cytosolic PTKs by anion exchange chromatography using fast protein liquid chromatography, and it could be shown that at least two different forms of PTK exist. Using antibodies against a number of known oncogene products, we could determine that at least 70% of the PTK activity in the cytosol originated from the presence of the c-src oncogene product. Both of the PTK activity peaks seen in the fast protein liquid chromatography patterns could be precipitated with the anti-Src antibody. Furthermore, using the MCF-7 breast cancer cell line, it could be shown that the antibody against c-src also precipitated a part of the cytosolic PTK activity. In normal human peripheral lymphocytes, no precipitation of the cytosolic and membrane PTK activity could be achieved using the anti-Src antibody. Inasmuch as the cytosolic PTK activity parallels the malignancy in breast tumors (Hennipman et al., Cancer Res., 49: 516-521, 1989), and the majority of this activity is precipitated by anti-Src antibodies, the c-src protooncogene may play a key role in the manifestation of breast cancer.

Blotting, Western↗
PubMed1992

Phosphorylation of pyruvate kinase type K is restricted to the dimeric form.

In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.

Astrocytoma↗
PubMed1992

Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase.

Neuron specific enolase (NSE) is widely used as a neuro-endocrine marker. However the presence of NSE in many non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), gamma-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and gamma-enolase activity with the doubling-time (Td) and NSE-ag content with gamma-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of gamma-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of gamma-enolase activity and both NSE-ag content and gamma-enolase activity increased linearly with Td. It is concluded that gamma-enolase seems to be associated with the regulation of growth rate and that a compound with the gamma-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.

Carcinoma, Small Cell↗
PubMed1992

Protein tyrosine kinases in human brain and gliomas.

Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low- (grade I and grade II) and high-grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low-grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two- to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high-grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high-grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane-bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, high-grade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p = 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low-grade astrocytomas.

Adult↗
PubMed1992

The cytotoxicity of alkyl-lysophospholipid on clonogenic leukemia cells and on normal bone marrow progenitor cells is highly, but differentially, increased by cryopreservation.

We studied the cytotoxic effect of alkyl-lysophospholipid (ALP) in combination with cryopreservation on human clonogenic leukemia cells from 10 patients with acute leukemia and on normal bone marrow progenitors (committed stem cells) from 10 donors, in order to assess the applicability of ALP as a purging agent in autologous bone marrow transplantation (ABMT). The tumoricidal effect of ALP was greatly increased by the cryopreservation procedure, both on leukemic progenitors and to a lesser extent on normal bone marrow progenitors. The cytotoxic effects of ALP and of cryopreservation were synergistic. As a consequence, the ALP dose for ex vivo purging has to be adjusted. Furthermore, the cryopreservation procedure itself is more cytotoxic for leukemic progenitors than for normal marrow progenitors, indicating that cryopreservation has a purging effect in AMBT.

Bone Marrow Purging↗
PubMed1991

Cellular expression of K-type pyruvate kinase in normal and neoplastic human tissues.

The cellular expression of K-type pyruvate kinase was studied immunohistochemically in several normal and neoplastic tissues of human origin. The authors used the monoclonal antibody, designated as ES1, which was raised against human K-type pyruvate kinase. In contrast to the normal counterparts, a strong immunoreactivity was found in a rhabdomyosarcoma (n = 1), in a carcinoma of the pancreas (n = 1), and in neurofibromas (n = 2). Furthermore, the staining in leiomyosarcomas (n = 2) was shown to be more intense when compared with both normal smooth muscle cells and leiomyomas (n = 2). These findings show that knowledge about the cellular expression of the K-type pyruvate kinase identifies cell types for which its expression serves as oncodevelopmental marker. In addition, these immunohistochemical studies give information whether shifts toward K-type containing isozymes of pyruvate kinase, which are determined by electrophoresis in whole cytosolic extracts of various tumors, are due to an altered gene expression or due to proliferation of cells which normally express already the K-type pyruvate kinase. The first possibility probably occurs in rhabdomyosarcomas. The latter possibility seems to be valid for astrocytomas because astrocytes express the K-type pyruvate kinase in normal brain.

Antibodies, Monoclonal↗
PubMed1991

Membrane transport of natural folates and antifolate compounds in murine L1210 leukemia cells: role of carrier- and receptor-mediated transport systems.

L1210-B73 cells, variants of L1210 cells grown in medium containing nanomolar concentrations of folates, express a membrane associated folate binding protein (mFBP) in addition to the classical reduced folate/methotrexate carrier (RF/MTX-carrier) present in L1210 cells grown in standard high folate medium (G. Jansen et al., Cancer Res., 49: 1959-1963, 1989). In this study we used L1210-B73 and L1210 cells as a model system to study the affinity of the RF/MTX-carrier and the mFBP for the natural folate compounds folic acid and 5-formyltetrahydrofolate (5-CHO-THF), as well as a number of antifolate compounds. Furthermore we studied the contribution of the RF/MTX-carrier and the mFBP in membrane transport of these (anti)folates, and finally we analyzed the role of the mFBP and RF/MTX-carrier in the cytotoxic effects of the antifolates. The antifolates used were either inhibitors of dihydrofolate reductase, including methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM), or two folate-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and 2-deamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583). The affinity of the RF/MTX-carrier for natural and antifolate compounds declined in the order 10-EdAM greater than or equal to ICI-198,583 greater than or equal to 5-CHO-THF greater than MTX much greater than CB3717 much greater than folic acid. The mFBP exhibited a high binding affinity for CB3717 and ICI-198,583 but a poor binding affinity for MTX and 10-EdAM. Binding affinities of the mFBP decreased in the order CB3717 greater than or equal to folic acid = ICI-198,583 greater than or equal to 5-CHO-THF much greater than MTX = 10-EdAM. Over 24 h, at 25 nM, [3H]folic acid uptake in L1210-B73 cells was found to proceed for more than 98% via the mFBP. Uptake of [3H]-5-CHO-THF, at 50 nM extracellular concentration, occurred via both the mFBP (81%) and the RF/MTX-carrier (19%). With respect to antifolates, the mFBP in L1210-B73 cells contributed for less than 30% in the uptake of [3H]MTX but was the predominant route (92%) in the uptake of [3H]ICI-198,583. Results from affinity and membrane transport observations were consistent with growth inhibition studies on L1210-B73 cells demonstrating that the mFBP played only a minor role in the cytotoxic effects of MTX or 10-EdAM. On the other hand, L1210-B73 cells were significantly more sensitive to CB3717 (220-fold) and ICI-198,583 (10-fold) than parental L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Alkyllysophospholipid ET-18-OCH3 acts as an activator of protein kinase C in HL-60 cells.

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH3 on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH3 at a concentration of 20 micrograms/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser25]-protein kinase C (19-31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH3 compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH3, dependent on the mode of addition of ET-18-OCH3 and phospholipids.

Cell Membrane↗
PubMed1991

Pyruvate kinase activity and isozyme composition in normal fibrous tissue and fibroblastic proliferations.

Pyruvate kinase (PK) was studied in 57 fibroblastic and fibrohistiocytic proliferations and normal fibrous tissue (n = 10). The specific activity was significantly increased in malignant tumors (1.67 +/- 0.25) compared with normal tissue (0.26 +/- 0.04; P less than 0.001) and benign proliferations (0.52 +/- 0.05; P less than 0.005). Although an overlap exists between aggressive fibromatosis and the benign group, high values of PK activity are indicative of Grade 2 and 3 malignancy. Significant shifts in isozyme pattern, favoring the expression of K-type subunits were found in tumors with a metastasizing potential and aggressive fibromatosis. These changes in the isozyme pattern of PK in aggressive fibromatosis may act as another argument to place them in the category of malignant fibroblastic tumors.

Adult↗
PubMed1991

Growth-inhibitory effects of 5,10-dideazatetrahydrofolic acid on variant murine L1210 and human CCRF-CEM leukemia cells with different membrane-transport characteristics for (anti)folate compounds.

5,10-Dideazatetrahydrofolic acid (DDATHF) is a potent inhibitor of glycinamide ribonucleotide transformylase, one of the folate-dependent key enzymes in de novo purine biosynthesis. The present report demonstrates that multiple membrane-transport routes may be involved in the cellular uptake of DDATHF. These routes include the classic reduced folate carrier and a membrane-associated folate-binding protein (mFBP). The role of an mFBP in the uptake of DDATHF was suggested from observations that (a) the mFBP showed a very high binding affinity for DDATHF, (b) murine and human leukemia cells expressing an mFBP were highly sensitive to growth inhibition by DDATHF, and (c) protection against this growth inhibition could be achieved using folic acid rather than reduced folate compounds.

Animals↗
PubMed1991

Partial characterization of protein tyrosine kinase activity in normal and leukemic human myeloid cells.

We have examined the expression of the protein tyrosine kinase (PTK) encoding oncogenes fes and abl in normal and malignant human myeloid cells in immunoblotting experiments. fes was markedly present in all cytosolic and most membrane fractions of normal and malignant cells. abl was only visible in normal cells, and occurred mostly in the cytosolic fractions. Molecular weights of identified proteins were different from the known products of fes and abl, possibly by alternative splicing at the mRNA level or by proteolysis. PTKs in myeloid cells were further purified by fast liquid protein chromatography (FPLC). PTK-activities of column fractions were assayed using a solid-phase non-radioactive dot-blot assay. Cytosolic and membrane fractions showed a FPLC pattern with a constant as well as a variable part in both normal and malignant cells, possibly indicative for PTKs with specialized functions in normal cell growth and transformation. Partial characterization of PTKs from different eluted peaks of AML-M4 blast cells demonstrated that PTKs from these peaks are kinetically distinct from each other.

Cell Membrane↗
PubMed1991

Phosphorylation of pyruvate kinase and glycolytic metabolism in three human glioma cell lines.

Three cell lines established from human gliomas were found to differ in the capacity to phosphorylate the glycolytic enzyme pyruvate kinase in vitro. Phosphorylation in the glioblastoma cell line U-138 was more pronounced than in the glioma cell line Hs 683 and in the glioblastoma cell line A-172. All 3 cell lines showed similar pyruvate kinase isozyme patterns and expressed about 90% K-type and 10% M-type subunits. So, differences in pyruvate kinase phosphorylation could not be explained by differences in the availability of the appropriate substrate, being pyruvate kinase type K. As in gliomas, phosphorylation could specifically and almost completely be inhibited by fructose-1,6-bisphosphate. In order to investigate a potential physiological significance of the phosphorylation of pyruvate kinase, we have characterized these cell lines for several glycolytic parameters. In U-138 cells, the production of lactate appeared to be 2 times higher as compared with A-172 and Hs 683 cells under normal growth conditions and even 4 times higher under low glucose culture regime. The efflux of lactate correlated with the pyruvate kinase phosphorylation pattern in the cell lines. In none of the cell lines could the lactate production be stimulated by glutamine as additional energy source under low glucose culture conditions. The higher glycolytic flux in U-138 cells was not accompanied by higher glycolytic enzyme activities. The isozyme patterns of hexokinase, pyruvate kinase, aldolase, enolase and lactate dehydrogenase in the cell lines were nearly identical and resembled the patterns previously described for solid gliomas. However, the isozyme composition of phosphofructokinase in the cell lines differed from the situation in gliomas. While in gliomas the expression of L-type phosphofructokinase is favored, in the glioma cell lines, we found an increase in the expression of C-type subunits.

Adenosine Triphosphate↗
PubMed1991

31P nuclear magnetic resonance and zero-point titration compared for measuring free magnesium concentration in erythrocytes.

Intracellular ionized magnesium concentrations ([Mg2+]i) were measured in erythrocytes by 31P nuclear magnetic resonance (NMR) and zero-point titration in 14 controls and seven patients with renal magnesium loss. The mean intracellular ionized magnesium concentration in controls measured by 31P NMR was 0.20 (SD 0.03) mmol/L cell water, compared with 0.55 (SD 0.12) mmol/L cell water by zero-point titration. Total erythrocyte magnesium content measured with the lysate method was 0.63 mmol/L cell water higher than estimated by 31P NMR, probably because not all magnesium complexes are fully visible to the NMR technique. We found a positive correlation between plasma ultrafiltrable magnesium and [Mg2+]i irrespective of the [Mg2+]i assay used. [Mg2+]i measured with 31P NMR correlated modestly but significantly with [Mg2+]i determined by zero-point titration (r = 0.58, P less than 0.02). Washing erythrocytes before the zero-point titration decreased the ATP content and the cell water fraction, which led to overestimation of [Mg2+]i by zero-point titration. Although absolute values for [Mg2+]i differ with the assay used, both methods determined significantly lower values for [Mg2+]i in patients with isolated renal magnesium loss.

Adenosine Triphosphate↗
PubMed1990

Multiple membrane transport systems for the uptake of folate-based thymidylate synthase inhibitors.

N10-Propargyl-5,8-dideazafolic acid (CB3717) and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI-198,583) are potent folate-based inhibitors of thymidylate synthase. We studied the membrane transport and the growth-inhibitory effects of the two thymidylate synthase inhibitors on human CCRF-CEM leukemia cells with different transport properties for folic acid, reduced folates, and methotrexate (MTX). Membrane transport of [3H]ICI-198,583 can proceed via the high affinity/low capacity reduced folate carrier as supported by findings that (a) uptake of [3H]ICI-198,583 was significantly impaired in CEM cells which have a transport defect for MTX, (b) variants of CEM cells which overproduce the reduced folate carrier system showed a concomitant increase in the uptake of [3H]ICI-198,583 as for [3H]MTX, (c) MTX inhibited transport of [3H]ICI-198,583, and (d) uptake of [3H]ICI-198,583 was inhibited after treatment of CEM cells with an N-hydroxysuccinimide ester of MTX, which is a potent inhibitor of MTX transport. However, a membrane-associated folate-binding protein (FBP) offers another route for entry of CB3717 and ICI-198,583. CEM-FBP cells that have an elevated amount of FBP and do not have a functional reduced folate carrier were 640- and 61-fold more sensitive to CB3717 and ICI-198,583, respectively, compared to control CEM cells expressing the reduced folate/MTX carrier. This high sensitivity was related to a high affinity of the FBP for CB3717 and ICI-198,583 (Kd 2-3 nM), which is only 3-fold lower than for folic acid (Kd 1 nM) but significantly higher than for MTX (Kd 100 nM). Furthermore, after incubation of CEM-FBP cells for 24 h at 10 nM [3H]ICI-198,583, the high affinity binding of the FBP for ICI-198,583 allowed a 600-fold concentrative uptake of [3H]ICI-198,583 and its conversion to polyglutamate forms. These results indicate that multiple folate transport systems may be involved in the uptake of folate-based thymidylate synthase inhibitors.

Antineoplastic Agents↗