Biomedical subjects

G Rijksen

Publications and source records attributed to G Rijksen.

At least 37 records · Page 2Linked to original sources

PubMed1997

Grb2 overexpression in nuclei and cytoplasm of human breast cells: a histochemical and biochemical study of normal and neoplastic mammary tissue specimens.

In 20-30 per cent of human breast cancers, the receptor tyrosine kinases epidermal growth factor receptor (EGFR) and c-erbB2 are overexpressed. This overexpression leads to increased mitogenic signalling and is correlated with poor prognosis. Overexpression of associated adaptor proteins, like Grb2, can also induce upregulation of signalling pathways. In this study, the expression of the Grb2 adaptor protein was determined in both normal human breast tissue and mammary cancers, using immunoblotting experiments and immunostaining on paraffin-embedded tissue sections. Both biochemical and immunohistochemical techniques revealed overexpression of Grb2 in all breast cancer specimens. In addition, although Grb2 protein is described as localized in the cytoplasm, it can also be detected in the nucleus, both in normal and in tumour breast tissue. In tumour breast tissue, 58 per cent of Grb2 protein is found in the nucleus, while 37 per cent is detected in the cytoplasm. In normal breast tissue, 22 per cent of Grb2 is found in the nucleus and 70 per cent in the cytoplasm. These findings indicate that in human breast cancer, Grb2 is overexpressed and appears to be predominantly localized in the nucleus.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Anti-adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: correlation with B cell differentiation stage.

We show that major histocompatibility complex (MHC) class II molecules on B cells transit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-gamma-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphoproliferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.

Animals↗

PubMed1996

Determination of specific protein kinase activities using phosphorus-33.

The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of 33P, compared with the traditionally applied 32P, allows the simultaneous examination of the amount of enzyme with 125I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.

Carcinoma↗

PubMed1996

c-Src protein expression is increased in human breast cancer. An immunohistochemical and biochemical analysis.

In human breast cancer, c-Src activity is elevated compared to normal breast tissue. It is not yet known whether this increase in c-Src activity is accompanied by an increase in c-Src protein expression. In this study, c-Src activity and protein expression were determined in a series of human breast cancers and in normal breast tissue, using immune complex kinase assays and immunoblotting. As the heterogeneity of breast cancer is not taken into account in these biochemical experiments, immunohistochemistry was also used to distinguish between normal and malignant cells. In human breast cancers, the c-Src activity is increased 4- to 30-fold, compared with normal breast tissue. This enhanced activity is accompanied by an increase in c-Src protein expression, as shown by both immunoblotting and immunohistochemistry. Immunohistochemistry indicates that the majority of c-Src appears to be concentrated around the nucleus in malignant cells, whereas in normal cells, it is distributed more evenly in the cytoplasm. These data confirm that c-Src activity is increased in human breast cancer. In addition, this study provides strong immunohistochemical evidence that the c-Src protein is also overexpressed, enabling a distinction to be made between normal and malignant cells.

Biomarkers, Tumor↗

PubMed1996

An enzyme-linked immunosorbent assay for the determination of src-family tyrosine kinase activity in breast cancer.

An enzyme-linked immunosorbent assay is described for the determination of protein tyrosine kinase activity originating from the presence of src-like tyrosine kinases in biological samples. In this assay a peptide derived from p34cdc2, cdc2(6-20)NH2, is coupled to the wells of a maleic anhydride-activated microtiter plate. This particular peptide has been described as an efficient and specific substrate for protein tyrosine kinases belonging to the src family kinases (Cheng et al., J.Biol.Chem. 267 (1992) 9248-9256). After incubation of the coated substrate with sample and ATP, the amount of phosphorylated tyrosyl residues is determined with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay appears to be very sensitive and is linear with sample protein concentration and phosphorylation time. Intra-assay variation is < 5%, whereas day-to-day variation is < 10%. The results of the assay have been compared with an ELISA in which the broad-specificity tyrosine kinase substrate poly(GluNa,Tyr)4:1 was coated. The results of both assays in 27 cytosolic breast cancer samples correlated very well (r = 0.94), in accordance with the predominant expression of src kinase activity in breast cancers (Ottenhoff-Kalff et al., Cancer Res. 52 (1992), 4773-4778). The present assay provides an easy, reproducible, and quick alternative for the usual radioactive methods used for the determination of src-kinase activities including immunecomplex kinase assay and TCA-precipitation assays. It allows the determination of src-like activities in human tumors for routine diagnostic purposes.

Animals↗

PubMed1996

Inhibitors of protein tyrosine phosphorylation reduce the proliferation of two human glioma cell lines.

Epidermal growth factor (EGF) and platelet-derived growth (PDGF) are suggested to be involved in the proliferation of human gliomas. We examined the effects of these growth factors on two human malignant glioma cell lines. Treatment of the A172 glioblastoma and the Hs683 glioma cell line with EGF and PDGF resulted in the tyrosine autophosphorylation, and hence activation, of the respective growth factor receptors. In addition, both cell lines responded to EGF and PDGF with increased deoxyribonucleic acid (DNA) synthesis. Because the intrinsic protein tyrosine kinase activity of this class of growth factor receptors is indispensable for their functioning, we tested the effects of specific protein tyrosine kinase inhibitors on growth factor-induced DNA synthesis and glioma cell proliferation. Genistein inhibited both EGF- and PDGF-stimulated autophosphorylation of the receptors and induction of DNA synthesis. However, genistein seemed to be cytotoxic to the cells. The tyrphostins RG 50875 and RG 13022 dose-dependently inhibited DNA synthesis induced by EGF, PDGF, and serum. RG 13022 completely blocked the EGF- and PDGF-induced DNA synthesis at a concentration of 50 mumol/L. The tyrphostins showed no selectivity in blocking either EGF or PDGF signaling. With concentrations up to mumol/L, no cytotoxic side effects of the tyrphostins were observed. Both tyrphostins also inhibit serum-driven cell growth in a dose-dependent manner. These results support the hypothesis that activated protein tyrosine kinase receptors are involved in the proliferation of A172 and Hs683 glioma cells. Selective inhibitors of protein tyrosine kinases, therefore, might have the potential to contribute to the treatment of growth factor-dependent gliomas.

Antineoplastic Agents↗

PubMed1996

Increased immunoreactivity and protein tyrosine kinase activity of the protooncogene pp60c-src in preneoplastic lesions in rat pancreas.

This study investigates the possibility that the c-Src protein tyrosine kinase is involved in experimental exocrine pancreatic carcinogenesis. Expression and activity of the protooncogene pp60c-src (c-Src) are investigated in acinar pancreatic (pre-) neoplastic lesions induced in rats by azaserine and compared with normal rat pancreas. Low or absent c-Src immunoreactivity and c-Src tyrosine kinase activity were found in the pancreas of untreated control rats. Compared with these controls, c-Src protein immunoreactivity was increased in "normal" acinar cells and even more in putative preneoplastic atypical acinar cell nodules (AACN) after azaserine treatment. In contrast, more advanced (secondary transformed) acinar cell lesions demonstrated no c-Src immunoreactivity. Rats treated with azaserine showed a 7-fold-higher c-Src tyrosine kinase activity in their pancreas. The level of c-Src tyrosine kinase activity correlated positively with the number of lesions in the pancreas, inasmuch as promotion of azaserine-initiated carcinogenesis by cerulein resulted in a more than 10-fold increase in the number of AACN, which was accompanied by a 6-fold increase in c-Src activity when compared with azaserine treatment alone. c-Src tyrosine kinase activity was responsible, on average, for 40% of the total tyrosine kinase activity in the pancreatic homogenates and was predominantly found in the cytoskeletal subcellular fraction. Furthermore, the transformation from normal to preneoplastic pancreatic tissue in azaserine-treated rats was accompanied by a change in the localization of the c-Src protein. With the use of immunohistochemistry and confocal laser scanning microscopy, the protein was detected in the cytoplasm in morphologically normal pancreatic acini, whereas in AACN it was detected both in the cytoplasm and in the nuclei. It is concluded that the protooncogene c-Src might be involved early in experimental pancreatic carcinogenesis: c-Src probably plays a minor role in pancreatic acinar cells after transformation to malignancy.

Animals↗

PubMed1995

Activation and translocation of c-Src to the cytoskeleton by both platelet-derived growth factor and epidermal growth factor.

We have examined the subcellular distribution and catalytic activity of c-Src tyrosine kinase after stimulation of A172 glioblastoma cells with peptide growth factors. Treatment of resting cells with platelet-derived growth factor resulted in an increase (3.5-fold) in the amount of c-Src protein associated with the cytoskeleton. In addition, an increase in specific c-Src kinase activity was observed in the cytoskeleton as well as in the cytosol and the membrane fraction. Similar effects on both c-Src redistribution and activity were seen after stimulation with epidermal growth factor. These data show that, like other signal transducing components, c-Src also becomes activated and associated to the cytoskeleton in response to growth factor stimulation.

Cell Compartmentation↗

PubMed1995

Protein tyrosine phosphatase activity as a diagnostic parameter in breast cancer.

Cellular phosphotyrosine levels are regulated by the balance between protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). It is supposed that this balance is disturbed in tumour cells, making the increased or altered activity of PTKs and PTPs likely hallmarks of tumour tissues. Indeed it could be shown that the PTK activity was increased in breast cancer in correlation with prognosis (Hennipman et al., Cancer Res. 49, 516-522, 1989). In the present report we measured the PTP activities in breast cancer and normal breast tissues. An increase of approximately three- to four-fold was measured in the cytosolic tumour fractions compared to normal, whereas the solubilized membrane fraction PTP activity showed an increase in tumours of approximately 1.5-fold. Remarkably, the membrane PTP activity correlated with the presence of tumour positive axillary lymph nodes (p = 0.004), whereas the cytosolic PTP activity correlated with the mitotic index, a higher PTP activity occurring when the mitotic index was higher than 10 (p = 0.0004). These results indicate the membrane PTP activity may be considered as an index of metastatic potential, whereas cytosolic PTP activity may be a measure of the growth capacity of the tumour. The increase of PTP activity in breast cancers was confirmed by enzyme-histochemical studies. In frozen sections of tumours a strong to moderate activity was found in both tumour cells and interstitial cells. In the interstitium membrane activity was most pronounced, whereas in the tumour cells diffuse staining of the cytoplasm together with a clear membrane staining was demonstrated. Immunoblotting with anti-phosphotyrosine antibodies also reveals differences between the tumours and normal tissues, confirming the disturbance of the balance between protein tyrosyl phosphorylation and dephosphorylation in the tumour cells.

Breast↗

PubMed1995

Influence of serum levels on leukemic cell destruction by the ether lipid ET-18-OCH3.

The effect of serum on the antineoplastic action of the alkyl-lysophospholipid 1-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3) was studied in two human leukemia cell lines, HL60 and K562, and in leukemic cells of patients. Decreasing amounts of serum in the culture medium enhanced the cytotoxic action of ET-18-OCH3 dramatically in both cell lines and in the leukemic cells, as measured by cell survival and proliferation. Uptake of ET-18-OCH3 was likewise increased at reduced serum levels. Similar effects were obtained when fetal calf serum (FCS) in the culture medium was substituted by bovine serum albumin (BSA, fatty acid free). Selectivity of the alkyl-lysophospholipid at reduced serum or BSA level was demonstrated by clonogenic assays of normal marrow progenitor cells. Our study provides an optimalization of the purging conditions in autologous bone marrow transplantation, by using a low concentration of BSA during ET-18-OCH3 treatment (20 micrograms/ml for 4 h) in serum-free culture medium.

Antineoplastic Agents↗

PubMed1995

Clinical relevance of protein-tyrosine (de-)phosphorylation in head and neck cancer.

Previous studies have shown that protein tyrosine (de)phosphorylation plays an important role in head and neck cancer. Protein-tyrosine kinases (PTK) and protein-tyrosine phosphatases (PTPase) activities in the cytosol of tumor tissue were significantly increased compared to normal tissue of cancer patients as well as controls. Additionally, the enzyme activities in normal tissue of tumor patients were significantly higher than enzyme activities in normal tissue of the control group. In this paper, we have correlated the cytosolic and membranous PTK and PTPase activity of tumor and nontumor tissue with several clinical and histological parameters known to influence the clinical outcome. Furthermore, we have analyzed the value of the enzyme activities as an independent predictor of clinical behavior and occurrence of second primary tumors. We confirmed our earlier observations that cytosolic and membranous PTK activities and cytosolic PTPase activities in tumor tissues are increased compared to activities in nontumor tissues and controls. Moreover, we also confirmed the findings of increased enzyme activities in nontumor tissues compared to findings in control tissues. This finding in histologically proven healthy mucosa is highly interesting because it indicates that these biochemical changes are obviously not (yet) translated into morphological changes. Significant differences were found in membranous PTK activity when the patients were grouped by sex, tumor localization, lymph node metastasis, and previous radiotherapy. During the follow-up period, no relation could be found between enzyme activities in tumor and/or nontumor tissues and disease-free interval or occurrence of second primary tumors.

Adult↗

PubMed1995

Aberrant tyrosine phosphorylation in head and neck tumours and potentially premalignant tissues.

Protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPase) activity in tumor tissue, obtained from 107 patients with squamous cell carcinoma of the upper aerodigestive tract, was determined. In 79 patients samples were also taken from a histologically proven non-tumourous part of the mucosa. In this extensive study we confirmed our previous findings of increased enzyme activities (cytosol PTK, membranes PTK, cytosol PTPase) in non-tumour tissues compared with control tissues. These biochemical changes suggesting a premalignant mucosa could not be correlated to a higher probability of secondary tumours.

Carcinoma, Squamous Cell↗

PubMed1994

Functional interaction between the epidermal growth factor receptor and c-Src kinase activity.

To study the relationship between the tyrosine kinase c-Src and the epidermal growth factor receptor (EGF-R), we used the breast cancer cell line ZR75-1, which was transfected with the EGF-R. The EGF-R transfected cell line expressed 60 times more EGF-R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF-R over-expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c-Src. However, the cell line over-expressing the EGF-R showed a twofold enhancement of c-Src kinase activity after EGF stimulation. The activation of c-Src kinase by EGF was confirmed in other EGF-R expressing cell types.

Breast Neoplasms↗

PubMed1994

Protein kinase C is not involved in the cytotoxic action of 1-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine in HL-60 and K562 cells.

We studied the effects of the alkyllysophospholipid 1-octadecyl-2-O-methyl-sn-glycerol-3-phosphocholine (ET-18-OCH3) on membrane associated protein kinase C (PK-C) activity in ET-18-OCH3 sensitive HL-60 cells and in two resistant cell types, Me2SO differentiated HL-60 cells and K562 cells. HL-60 cells expressed a lower PK-C activity level compared with both resistant cell types. However, membrane-bound PK-C activity in the sensitive HL-60 cells was approximately 3-fold increased in the presence of ET-18-OCH3, whereas in differentiated HL-60 cells and K562 cells PK-C was not influenced by ET-18-OCH3. The increase in PK-C activity in HL-60 cells was not due to translocation of cytosolic PK-C or synthesis de novo. The effect of ET-18-OCH3 on kinetic parameters of PK-C in all three cell types was investigated in order to elucidate the nature of the ET-18-OCH3 effects on PK-C activity in both sensitive and resistant cell types. A functional relationship between PK-C level and effect of ET-18-OCH3 on PK-C activity in the different cell types could not be found. Moreover, cells depleted of PK-C activity showed similar sensitivity or resistance to ET-18-OCH3 as cells expressing PK-C activity. These results suggest that a role of PK-C in the cytotoxic action of ET-18-OCH3 is very unlikely.

Alkaloids↗

PubMed1994

Protein tyrosine (de-)phosphorylation in head and neck squamous cell carcinoma.

Protein phosphorylation plays an important role in signal transduction of both normal and neoplastic cells. Since increased protein tyrosine phosphorylation may be associated with malignant transformation, we studied the activities of protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTPase) in patients with various head and neck tumors. Furthermore, we determined the patterns of tyrosine phosphorylated protein (P-tyr) in tissues by western blotting. Enzyme activities were studied in tumor and histologically normal, non-tumorous tissues of 54 patients and in 11 controls. P-tyr patterns were determined in 3 patients and 2 controls. PTK and PTPase activities were greater in tumor tissues than in normal tissue of the cancer patients as well as controls. P-tyr levels in tumors were also higher than in normal tissues. Additionally, PTK activity in normal tissue of tumor patients was significantly higher than in normal tissue of the control group. The same trend was observed for the PTPase activity and P-tyr levels.

Adult↗

PubMed1993

The role of protein tyrosine phosphatases in density-dependent growth control of normal rat kidney cells.

In normal rat kidney cells protein tyrosine phosphatases (PTPases) play a role in attaining density-dependent growth arrest after stimulation with mitogens. The PTPase inhibitor sodium orthovanadate prevents density-dependent growth inhibition of EGF-treated cells and mimicks in that respect the action of TGF beta and retinoic acid. However, enhanced PTPase activity is not obligatory for maintaining cells in a density-arrested state. In contrast to TGF beta and retinoic acid, vanadate is unable to restimulate density-inhibited cells, indicating that different mechanisms are operating. Yet, vanadate is strongly potentiating the effect of low concentrations of TGF beta but not of retinoic acid, implicating that tyrosine phosphorylation is linked to TGF beta action and that PTPase may represent a negative control element in the TGF beta signaling pathway.

Animals↗

PubMed1993

Orthovanadate both mimics and antagonizes the transforming growth factor beta action on normal rat kidney cells.

Normal rat kidney [NRK] cells grown in the presence of epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) have a normal phenotype and undergo density-dependent growth inhibition, whereas in the presence of multiple growth factors, density arrest is lost and the cells become phenotypically transformed. We studied the influence of the protein tyrosine phosphatase (PTPase) inhibitor sodium orthovanadate on the mitogenic stimulation of NRK cells by growth factors and on transformation-linked properties as loss of density-dependent growth inhibition and anchorage-independent growth. The fraction of cells in serum-deprived monolayer cultures that is induced to proliferate upon mitogenic stimulation by EGF or PDGF is only slightly enhanced upon addition of low concentrations (25-50 microM) of vanadate. Addition of vanadate per se induces proliferation of only a very limited amount of cells, but results in a shift of the dose-response curves for other growth factors to lower concentrations. Vanadate added in combination with EGF or PDGF is able to mimic the effect of transforming growth factor beta (TGF beta) in inducing phenotypic transformation. In monolayer cultures density-dependent growth inhibition is lost and anchorage-independent proliferation is observed on dishes coated with poly(2-hydroxy-ethyl methacrylate) (polyHEMA). The extent of these changes is similar to that induced by TGF beta. However, the morphology of the obtained colonies in polyHEMA-coated dishes is quite different. Cells transformed by TGF beta in the presence of EGF form rather amorphous colonies, whereas in the presence of orthovanadate colonies are formed that tend to fall apart in loose cells. The effect of vanadate on cell transformation is dependent on the growth factor conditions in a bimodal way. When a suboptimal dose of growth factor(s) is used, 25 microM vanadate is very effective in preventing density-induced growth inhibition and stimulating anchorage-independent proliferation. However, the same concentration of vanadate is inhibitory when cells are maximally stimulated and antagonizes the transforming effect of TGF beta added in combination with other growth factors. It is hypothesized that vanadate acts on a set of different protein tyrosine phosphatases. Some of these are positive and others negative regulators of growth.

Animals↗

PubMed1993

Standardization of an enzyme-linked immunosorbent assay for the determination of protein tyrosine kinase activity.

A procedure for an enzyme-linked immunosorbent assay for the determination of protein tyrosine kinase (PTK) activity from cytosolic and solubilized membrane fractions from breast cancers, is described. The general PTK substrate poly(GluNa, Tyr) 4:1 is coated to the wells of a microtiter plate. After incubation with PTK sample and ATP the amount of phosphorylated tyrosyl residues is quantitated with phosphotyrosine specific antibodies and a secondary peroxidase-labeled antibody. The assay is optimized with respect to coating and phosphorylation conditions. The signal is linear with phosphorylation time and with sample protein concentrations in a sufficiently wide range. The assay is standardized by using both internal and external standards. A lyophilized rat spleen extract is used as an external standard. Its PTK activity, determined by established quantitative methods, can be used to calculate the activity of the breast cancer samples. To eliminate day-to-day variations an internal standard, consisting of BSA-coupled phosphotyrosine, is coated to some wells of the microtiter plate. Interassay variation can be minimized by determination of the ratio of optical densities from internal and external controls. Its variation appeared to be less than 18%. Intraassay variation appears to be < 6%. PTK activities measured with this assay correlated well with those of a nonradioactive dot-blot assay and with conventional radioactive assays in which [32P]ATP is used as the substrate. Compared to these assays it appeared to be more sensitive and far more easy to perform.

Animals↗