Overexpression of EGFR and c-erbB2 causes enhanced cell migration in human breast cancer cells and NIH3T3 fibroblasts.

Overexpression of EGFR and c-erbB2 frequently occurs in human breast cancers, correlating with poor prognosis. Here we show that overexpression of EGFR and c-erbB2 in cell lines increases cell migration, an important step in metastasis formation. The effect of EGFR on migration is dependent on the addition of EGF to the cells. In contrast, c-erbB2 seems to act independently of its ligand in these assays. Overexpression of this receptor is sufficient to induce cell migration. In addition, we investigated the involvement of a number of signal transduction pathways known to be activated by the EGFR. We found that inactivation of MAPKK results in a decreased migration, while inactivation of PI3K increases migration.

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.

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.

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.

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.

Changes in glycosylation of L1210 cells after exposure to various antimetabolites.

This study establishes that antimetabolites do have the potency to change cellular glycosylation, as was suggested in our previous review (Eur J Cancer 1990, 26, 516-523). Murine leukaemia L1210 cells were exposed to various antimetabolites under non-lethal conditions. The antimetabolites 5-fluorouracil (5FU), arabinofuranosylcytosine (AraC), methotrexate (MTX) and 6-mercaptopurine (6MP), but not 6-thioguanine, induced considerable changes in the metabolic incorporation of radioactively labelled monosaccharides. Each antimetabolite exhibited a different effect. Significant differences were found between the radioactivity incorporated from the monosaccharides glucosamine, fucose, mannose and galactose, relative to control values. Polyacrylamide gel electrophoresis indicated that changes were induced in the glycosylation of individual glycoproteins. 5FU, AraC, MTX and 6MP all influenced both pyrimidine- and purine-mediated sugar incorporation. This excludes, therefore, direct effects of the antimetabolites on their analogue nucleotide-sugars. The antimetabolite-induced changes in glycosylation did not directly correlate with the observed cell-cycle effects of the antimetabolites.