P.M.P. van Bergen en Henegouwen

Crosstalk Between Epidermal Growth Factor Receptor- and Insulin-Like Growth Factor-1 Receptor Signaling: Implications for Cancer Therapy

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) can contribute to tumor development and -progression through their effects on cell proliferation, inhibition of apoptosis, angiogenesis, anchorage-independent growth and tumor-associated inflammation. EGFR-targeting monoclonal antibodies and small molecule tyrosine kinase inhibitors are currently in clinical use for the treatment of several types of cancer. However, primary and acquired resistance to these agents often occurs and thereby limits the clinical efficacy of mono-specific targeted therapy. Results from both in vitro and in vivo studies indicate that cross-talk between EGFR and IGF-1R can lead to acquired resistance against EGFR-targeted drugs. This review describes the interface between the EGFR and IGF-1R signaling networks and the implications of the extensive cross-talk between these two receptor systems for cancer therapy. EGFR and IGF-1R interact on multiple levels, either through a direct association between the two receptors, by mediating the availability of each others ligands, or indirectly, via common interaction partners such as G protein coupled receptors (GPCR) or downstream signaling molecules. This multi-layered cross-talk and its involvement in the induction of resistance to targeted therapies provide a clear rationale for dual targeting of EGFR and IGF-1R. We discuss several (potential) strategies to simultaneously inhibit EGFR and IGF-1R signaling as promising novel therapeutic approaches.

Ultrastructural immunocytochemical localization of B-50/GAP43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones

SummaryAccumulating evidence indicates that the neuron-specific B-50/GAP43, a substrate for protein kinase C, plays a role in neuronal differentiation and neuritogenesis during nervous tissue development and axonal regeneration. An ultrastructural immunocytochemical study on the localization of B-50 in presynaptic terminals (synaptosomes) and neuronal growth cones was carried out by means of cryoultramicrotomy with affinity-purified B-50 antibodies. Detection was accomplished with colloidal gold, conjugated either to protein-A or goat anti-rabbit immunoglobulins. In synaptosomes, isolated from the frontal cortex of 6-week-old rats, and in neuronal growth cones, isolated from forebrains of 5-day-old rats, the majority of B-50 is detected at the surrounding neuronal plasma membrane. In both neuronal growth cones and synaptosomes, a relatively small fraction of B-50 in the cytoplasm was not evidently associated with internal membranes. Our results indicate that B-50 is mainly located at the cytoplasmic face of the synaptosomal and neuronal growth cone plasma membrane. The similar B-50 localization in neuronal growth cones and synaptosomes suggests that, both in extending axons and mature synaptic terminals, B-50 may exert identical functions as a protein kinase C substrate at the plasma membrane.

Controlled growth of colloidal gold particles and implications for labelling efficiency

SummaryA new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm. The initial gold particle population, with an average diameter of 5.6±0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous. An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles. The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of β-galactosidase on ultrathin cryosections of Escherichia coli cells. We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method. In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading. Therefore these gold probes are suitable for qualitative double labelling experiments. The quantitative aspect of immunolabelling is discussed.

Ultrastructural double localization of B-50/GAP43 and synaptophysin (p38) in the neonatal and adult rat hippocampus.

SummaryB-50/GAP43, a neuron-specific phosphoprotein, is highly expressed in developing nervous tissue. Monospecific polyclonal affinity-purified B-50 antibodies were used to document the ultrastructural distribution of B-50 in the hippocampus of 90-day-old (P90) and 1-day-old (P1) rats. Double-labelling immunoprocedures were performed to compare the localization of B-50 and synaptophysin (p38), a protein specific for synaptic vesicles. By immunofluorescence light microscopy B-50 and p38 were similarly distributed in the CA1 neuropil of P90 rats. In contrast, in P1 rats B-50 was more widely distributed than p38. By electron microscopy of P90 rat hippocampus, B-50 was located at the plasma membranes of axon shafts and of p38-immunoreactive axon terminals. Some B-50 was found in the cytosol of axon terminals. B-50 was absent at the plasma membranes of apical dendrites and of pyramidal cells. In the P1 rat hippocampus, B-50 was detected at the plasma membrane of growth cones, axon terminals and axon shafts, but not in their cytosol. The plasma membranes of pyramidal cell bodies and their processes extending into the stratum radiatum were without B-50. B-50-immunoreactive organelles of the lysosomal family were found in the cytosol of pyramidal cells of the hippocampus of P1 and P90 rats. This ultrastructural study shows that during development of the stratum radiatum in the hippocampal field CA1, the localization of B-50 persists at the plasma membrane of axons and axon terminals in P1 and P90 rats. This localization of B-50 is consistent with the suggestion that B-50 acts as a regulator of neurotransmitter release and intracellular messengers.

Studies on a possible relationship between alterations in the cytoskeleton and induction of heat shock protein synthesis in mammalian cells

Heat shock-induced alterations in protein synthesis and the cytoskeleton of two mammalian cell types have been investigated. A hyperthermic treatment of 30 min at 43 degrees C causes an accumulation of heat shock proteins (HSPs). The apparent molecular weights of HSPs of Reuber H35 hepatoma cells and of N2A neuroblastoma cells are 28 000, 65 000, 68 000, 70 000, 84 000, 100 000 D and 68 000, 70 000, 84 000 and 100 000 D respectively. Hyperthermia induces the disruption of microfilaments in hepatoma cells. Microtubules and intermediate filaments (vimentin and cytokeratin) remain intact. In neuroblastoma cells microfilaments remain intact whereas microtubules become disorganized after heat shock. As a result vimentin is found as a perinuclear aggregate. These cells were still able to synthesize heat shock proteins after pretreatment with cytoskeleton disrupting drugs such as dihydroxycytochalasin B and colchicine. Therefore it is concluded that the alterations in the cytoskeleton observed after the heat treatment are unlikely to be the cause of heat shock protein synthesis. Our results suggest that these heat shock-induced alterations in the cytoskeleton can be considered as a part of the heat shock response.

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti-epidermal growth factor receptor nanobody

Intraoperative near‐infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real‐time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)‐targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti‐EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12‐800CW and R2‐800CW). Orthotopic tongue tumors were induced in nude mice using the OSC‐19‐luc2‐cGFP cell line. Tumor‐bearing mice were injected with 25 µg 7D12‐800CW, R2–800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12‐800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12‐800CW. A significantly higher tumor‐to‐background ratio (TBR) was observed in mice injected with 7D12–800CW compared to mice injected with R2‐800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR‐specific nanobody. After injection of 75 µg 7D12‐800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR‐targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Immunolocalization of B-50 (GAP-43 in the mouse olfactory bulb: predominant presence in preterminal axons

SummaryBecause the growth-associated protein B-50 (GAP-43) has been implicated in neurite outgrowth as well as in synaptic plasticity, we studied its light and electron microscopical distribution in the mouse olfactory bulb, an area of the nervous system which exhibits a high degree of synaptic plasticity. Immunofluorescent staining with monospecific affinity-purified anti-B-50 antibodies revealed that B-50 is most abundantly expressed in the olfactory nerve fibre layer and the granule cell layer neuropil, while little staining was observed in the external plexiform layer and in cell bodies. B-50 is absent from dendrites and myelinated axons as indicated by double labelling with monoclonal antibodies against microtubule-associated protein 2 and the large neurofilament protein, respectively. Using post-embedding immunogold labelling on ultrathin Lowicryl sections, B-50 was found to be highly concentrated in presumed growth cones in the olfactory nerve fibre layer and in thin unmyelinated axons and presynaptic terminals in the granule cell layer neuropil. Near background immunolabelling was seen in perikarya, dendrites and myelinated axons. In view of the implication of B-50 in plasticity-related phenomena, its abundance in the thin unmyelinated preterminal axons suggests that these are potential sites of extrasynaptic plasticity.

Localization and quantitation of hsp84 in mammalian cells

In order to investigate the function of heat shock protein 84 (hsp84) we have isolated the protein from mouse neuroblastoma cells and raised a polyclonal antiserum which was affinity-purified. The specificity of the antibody was established by immunoprecipitation and immunoblotting. Immunofluorescence studies revealed both a cytoplasmic and a nuclear localization of hsp84 in five different mammalian cell lines (mouse neuroblastoma cells and fibroblasts, rat hepatoma cells, and HeLa cells). In none of the five cell lines were found significant differences in the total cellular levels of hsp84 before and immediately after a heat shock (4 h, 42 degrees C) by immunoblot quantification. Furthermore after heat shock the fluorescence of anti-hsp84-labeled nuclei was increased relative to that of the surrounding cytoplasm. The increased fluorescence disappeared upon reincubation at 37 degrees C. The heat-induced increase in contrast between cytoplasmic and nuclear fluorescence could be explained by a combination of three factors: (a) decrease in nuclear projection area, (b) increase in cytoplasmic projection area, and (c) translocation of hsp84. The contribution of these factors to the increase after heat treatment was different for the cell lines.

Regulation of epidermal growth factor receptor expression in normal and transformed keratinocytes

SummaryTransformed keratinocytes (SCC-4, SCC-15, SCC-12F2, SVK14) or normal keratinocytes which differ in their differentiation programme were used to study the regulation of EGF-receptor expression. The capacity of the cells to differentiate was modulated by changing the extracellular calcium concentration. We were able to demonstrate that EGF-receptor expression in normal and transformed keratinocytes depends upon the cell type and one or more levels of regulatory control. At the DNA level, EGF-receptor gene amplification occurred in poorly differentiating cells. At the mRNA level, cells showing EGF-receptor gene amplification expressed elevated mRNA and protein levels when cultured under low Ca2+ conditions. Cells not exhibiting EGF-receptor gene amplification showed equal mRNA expression, regardless the Ca2+ concentration in the culture medium. At the protein level, EGF-receptor protein was decreased in cells exhibiting EGF-receptor gene amplification when extracellular Ca2+ was increased (to 1.6 mM) to stimulate differentiation, the decrease in protein being comparable to mRNA expression. Cells not exhibiting EGF-receptor gene amplification showed equal protein expression, regardless of the Ca2+ concentration in the culture medium. Under the same conditions, SV40 transformed keratinocytes showed equal mRNA but elevated protein expression in cells grown under low Ca2+ conditions. At the membrane level, normal keratinocytes and SCC-12F2 cells showed elevated numbers of cell surface exposed EGF-receptors in cells grown under low Ca2+ conditions, but equal mRNA and protein expression. These and previous findings demonstrate that EGF-receptor expression is regulated at the levels of DNA, mRNA and protein as well as by the plasma membrane composition, depending upon the cell type.

Analysis of EGF Receptor Oligomerization by Homo-FRET

Growth factor receptors are present in the plasma membrane of resting cells as monomers or (pre)dimers. Ligand binding results in higher-order oligomerization of ligand-receptor complexes. To study the regulation of receptor clustering, several experimental techniques have been developed in the last decades. However, many involve invasive approaches that are likely to disturb the integrity of the membrane, thereby affecting receptor interactions. In this chapter, we describe the use of a noninvasive approach to study receptor dimerization and oligomerization. This method is based upon the Förster energy transfer between identical adjacent fluorescent proteins (homo-FRET) and is determined by analyzing the change in fluorescence anisotropy. Homo-FRET takes place within a distance of 10nm, making this an excellent approach for studying receptor-receptor interactions in intact cells. After excitation of monomeric GFP (mGFP) with polarized light, limiting anisotropy values (r(inf)) of the emitted light are determined, where proteins with known cluster sizes are used as references. Dimerization and oligomerization of the epidermal growth factor receptor (EGFR) in response to ligand binding is determined by using receptors that have been fused with mGFP at their C-terminus. In this chapter, we describe the involved technology and discuss the feasibility of homo-FRET experiments for the determination of cluster sizes of growth factor receptors like EGFR.