Expression of EGFR, HER2, HER3, and HER4 in metastatic squamous cell carcinomas of the oral cavity and base of tongue.

The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

Two-dimensional gel analysis of [35S]methionine labelled and phosphorylated proteins present in virions and light particles of herpes simplex virus type 1, and detection of potentially new structural proteins.

Cells infected with herpes simplex virus (HSV) synthesize both infectious viruses and non-infectious light particles (L-particles). The latter contain the envelope and tegument components of the virions, but lack virus capsid and DNA. Electrophoresis in SDS-polyacrylamide gels (SDS-PAGE) has been used extensively for analysis of structural proteins in virions and L-particles. Two-dimensional (2-D) gel electrophoresis, however has a markedly higher resolution, and in the present work we have used this technique to study both [35S]methionine labelled and phosphorylated structural proteins in virions and L-particles. Proteins were assigned to the tegument or the envelope by the analysis of L-particles. Localization of structural proteins was also determined by stepwise solubilization in the presence of the neutral detergent NP-40 and NaCl, and by isolation of capsids from nuclei of infected cells. Different steps in posttranslational modification can be detected by 2-D gel electrophoresis such that a single polypeptide may appear as several spots. This was most clearly observed for some of the HSV-encoded glycoproteins which were shown to exist in multiple forms in the virion. Some polypeptides apparently not identified previously were either capsid associated, or localized in the tegument or envelope. The degrees of phosphorylation in L-particles and virions are almost identical for some proteins, but markedly different for others. Thus, glycoprotein E of HSV-1 is for the first time shown to be phosphorylated, and most heavily so in virions. The IE VMW)110 protein represents a group of proteins which are more phosphorylated in L-particles than in virions. Attempts are made to correlate the proteins detected by 2-D analysis with those previously separated by SDS-PAGE.

Synthesis of a water-soluble serine-based neoglycolipid which can be covalently linked to solid phases.

N alpha-Fmoc-serine pentafluorophenyl ester was glycosylated with perbenzoylated lactosyl bromide. The resulting product was coupled to a resin functionalized with 6-aminohexanoic acid and then N alpha-acylated to give a serine-based analogue of lactosylceramide. The water-soluble neoglycolipid was covalently linked to microtiter plates via its carboxyl group and was recognized by a lactose-binding lectin in an ELISA.

Synthesis of 3'- and 4'-deoxyfluorolactose and its Me3SiCH2CH2 and ceramide derivatives.

(2-Trimethylsilyl)ethyl (Me3SiCH2CH2)3'- and 4'-deoxyfluorolactosides (1 and 3) were synthesized by glycosylation of Me3SiCH2CH2 2,3,6-tri-O-benzyl-beta-D-glucopyranoside with 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-beta-D-galactopyranosyl bromide and 2,3,6-tri-O-benzyl-4-deoxy-4-fluoro-beta-D-galactopyranosyl bromide. Anomeric deblocking of the fully acetyled Me3SiCH2CH2 glycosides (12 and 13) gave the corresponding hemiacetals 14 and 15. Removal of the acetyl groups gave 3'- and 4'-deoxyfluorolactose (2 and 4). The deoxyfluorolactosylceramides 5 and 6 were synthesized via boron trifluoride etherate- or silver triflate-activation of the trichloroacetimidates prepared from 14 and 15. Silver triflate-mediated glycosylations showed lower reaction rates, and fewer byproducts were formed.

Monoclonal antibody against a lactose epitope of glycosphingolipids binds to melanoma tumour cells.

Mice were immunized with a neoglycoprotein consisting of a chemically modified carbohydrate moiety (reductively aminated 3'-sialyllactose) linked to human serum albumin. By this procedure an antibody response to the normally non-immunogenic carbohydrate structure was obtained. Hybridomas were established, and monoclonal antibodies were selected in ELISA based on their binding to the saccharide hapten, or to a lactosylceramide-mimicking neoglycolipid, lactose-bis-sulfone. One of the selected antibodies, 2H4, was of particular interest, since it also bound to glycolipids present on melanoma cells. FACS analysis of a panel of 14 melanoma cell lines showed that the 2H4 antibody bound to the majority of these. In frozen, non-fixed sections or paraffin sections of biopsies the monoclonal antibody 2H4 stained melanoma cells, but not tumour infiltrating lymphocytes or normal skin. Detailed immunochemical analysis of 2H4, using thin layer chromatography revealed that it recognized an internal lactose epitope in several glycosphingolipids.

Synthesis of the monodeoxy derivatives of 2-(trimethylsilyl)ethyl beta-lactoside.

Monodeoxy derivatives of 2-(trimethylsilyl)ethyl (Me3SiEt) beta-lactoside were synthesized, by deoxygenation at the disaccharide level, for the 2'-, 3'-, 4'-, and 6'-monodeoxylactosides. The 2-, 3-, and 6-deoxy derivatives were synthesized by beta-D-galactosylation of suitably protected monodeoxygenated Me3SiEt glucosides. Silver silicate was shown to be an efficient glycosylation promoter in the preparation of the 2- and 3-deoxylactosides.