Involvement of platelet-derived growth factor ligands and receptors in tumorigenesis.

Platelet-derived growth factor (PDGF) isoforms and their receptors have important roles during embryogenesis, particularly in the development of various mesenchymal cell types in different organs. In the adult, PDGF stimulates wound healing and regulates tissue homeostasis. However, overactivity of PDGF signalling is associated with malignancies and other diseases characterized by excessive cell proliferation, such as fibrotic conditions and atherosclerosis. In certain tumours, genetic or epigenetic alterations of the genes for PDGF ligands and receptors drive tumour cell proliferation and survival. Examples include the rare skin tumour dermatofibrosarcoma protuberance, which is driven by autocrine PDGF stimulation due to translocation of a PDGF gene, and certain gastrointestinal stromal tumours and leukaemias, which are driven by constitute activation of PDGF receptors due to point mutations and formation of fusion proteins of the receptors, respectively. Moreover, PDGF stimulates cells in tumour stroma and promotes angiogenesis as well as the development of cancer-associated fibroblasts, both of which promote tumour progression. Inhibitors of PDGF signalling may thus be of clinical usefulness in the treatment of certain tumours.

Unmanned aerial vehicles (drones) in out-of-hospital-cardiac-arrest.

BACKGROUND: The use of an automated external defibrillator (AED) prior to EMS arrival can increase 30-day survival in out-of-hospital cardiac arrest (OHCA) significantly. Drones or unmanned aerial vehicles (UAV) can fly with high velocity and potentially transport devices such as AEDs to the site of OHCAs. The aim of this explorative study was to investigate the feasibility of a drone system in decreasing response time and delivering an AED. METHODS: Data of Global Positioning System (GPS) coordinates from historical OHCA in Stockholm County was used in a model using a Geographic Information System (GIS) to find suitable placements and visualize response times for the use of an AED equipped drone. Two different geographical models, urban and rural, were calculated using a multi-criteria evaluation (MCE) model. Test-flights with an AED were performed on these locations in rural areas. RESULTS: In total, based on 3,165 retrospective OHCAs in Stockholm County between 2006-2013, twenty locations were identified for the potential placement of a drone. In a GIS-simulated model of urban OHCA, the drone arrived before EMS in 32 % of cases, and the mean amount of time saved was 1.5 min. In rural OHCA the drone arrived before EMS in 93 % of cases with a mean amount of time saved of 19 min. In these rural locations during (n = 13) test flights, latch-release of the AED from low altitude (3-4 m) or landing the drone on flat ground were the safest ways to deliver an AED to the bystander and were superior to parachute release. DISCUSSION: The difference in response time for EMS between urban and rural areas is substantial, as is the possible amount of time saved using this UAV-system. However, yet another technical device needs to fit into the chain of survival. We know nothing of how productive or even counterproductive this system might be in clinical reality. CONCLUSIONS: To use drones in rural areas to deliver an AED in OHCA may be safe and feasible. Suitable placement of drone systems can be designed by using GIS models. The use of an AED equipped drone may have the potential to reduce time to defibrillation in OHCA.

Effects of an EGFR-binding affibody molecule on intracellular signaling pathways.

Effects on intracellular signaling were studied in cells treated with the affibody molecule (ZEGFR:955)2 that targets the epithelial growth factor receptor (EGFR). EGFR is overexpressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Page. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or any other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (ZEGFR:955)2 is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues.

Hsp90 is expressed and represents a therapeutic target in human oesophageal cancer using the inhibitor 17-allylamino-17-demethoxygeldanamycin.

Heat shock protein 90 (Hsp90) has been demonstrated to protect oncogenic variants of signalling molecules from degradation and may consequently serve as a therapeutic target for the treatment of oesophageal cancer for which adequate therapy is often lacking. We studied the expression of Hsp90 in tumour tissues of human oesophageal cancer and the impact of Hsp90 inhibition on oesophageal cancer cell lines using the drug 17-allylamino-17-demethoxygeldanamycin (17-AAG). Quantitative immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues from patients with oesophageal cancer. In squamous cell carcinoma, a marked upregulation of Hsp90 could be noted in dysplastic epithelium and invasive cancer compared with normal epithelium. In adenocarcinoma, Hsp90 was expressed in neoplastic epithelium and also in normal non-neoplastic glands weakly. The inhibition of Hsp90 using 17-AAG led to a significant decrease in cell proliferation and viability in human oesophageal cancer cell lines. Using a clonogenic cell survival assay, Hsp90 inhibition significantly sensitised the cells for gamma-photon irradiation. Heat shock protein 90 was found to be critical for proper signalling induced by both epidermal growth factor and insulin-like growth factor-1, in which the inhibition of signalling by 17-AAG correlated with the observed reduction in cell proliferation and viability. These results showed that Hsp90 was selectively expressed in oesophageal cancer tissue compared with the corresponding normal tissue, and the inhibition of Hsp90 resulted in decreased proliferation and viability as well as radiosensitisation of oesophageal cancer cells. Heat shock protein 90 represents a potential therapeutic target in the treatment of patients with oesophageal cancer, alone or in combination with radiotherapy.

The stem cell factor receptor/c-Kit as a drug target in cancer.

Tyrosine phosphorylation has a key role in intracellular signaling. Inappropriate proliferation and survival cues in tumor cells often occur as a consequence of unregulated tyrosine kinase activity. Much of the current development of anti-cancer therapies tries to target causative proteins in a specific manner to minimize side-effects. One attractive group of target proteins is the kinases. c-Kit is a receptor tyrosine kinase that normally controls the function of primitive hematopoietic cells, melanocytes and germ cells. It has become clear that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loops or mutational activation. This makes c-Kit an excellent target for cancer therapies in these tumors. In this review we will highlight the current knowledge on the signal transduction molecules and pathways activated by c-Kit under normal conditions and in cancer cells, and the role of aberrant c-Kit signaling in cancer progression. Recent advances in the development of specific inhibitors interfering with these signal transduction pathways will be discussed.

DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation.

BACKGROUND: Phosphoinositide (PI) 3-kinase and its second messenger products, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), play important roles in signalling processes crucial for cell movement, differentiation and survival. Previously, we isolated a 32kDa PtdIns(3,4,5)P(3)-binding protein from porcine leukocytes. This protein contains an amino-terminal Src homology 2 (SH2) domain and a carboxy-terminal pleckstrin homology (PH) domain, and is identical to the recently described DAPP1 (also known as PHISH or Bam32) protein. Here, we characterised the subcellular distribution of DAPP1 in response to cell stimulation. RESULTS: When expressed transiently in porcine aortic endothelial (PAE) cells, DAPP1 translocated from the cytosol to the plasma membrane in response to platelet-derived growth factor (PDGF). This translocation was dependent on both PI 3-kinase activity and an intact DAPP1 PH domain. Following recruitment to the plasma membrane, DAPP1 entered the cell in vesicles. Similar responses were seen in DT40 chicken B cells following antibody treatment, and Rat-1 fibroblasts following epidermal growth factor (EGF) or PDGF treatment. Colocalisation studies in PAE cells suggested entry of DAPP1 by endocytosis in a population of early endosomes containing internalised PDGF-beta receptors. DAPP1 also underwent PI 3-kinase-dependent phosphorylation on Tyr139 in response to PDGF stimulation, and this event was involved in the vesicular response. CONCLUSIONS: This is the first report of plasma-membrane recruitment and endocytosis of a PI 3-kinase effector protein in response to cell stimulation. The results suggest a novel role for DAPP1 in endosomal trafficking or sorting.

Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction.

In this report we show that Tyr568 and Tyr570 are phosphorylated in vivo in the Kit/stem cell factor receptor (Kit/SCFR) following ligand-stimulation. By mutation of Tyr568 and Tyr570 to phenylalanine residues and expression of the mutated receptors in porcine aortic endothelial (PAE) cells, we could demonstrate a loss of activation of members of the Src family of tyrosine kinases when Tyr568 was mutated, while mutation of Tyr570 only led to a minor decrease in activation of Src family members. Mutation of both tyrosine residues led to a complete loss of Src family kinase activation. Phosphorylation of the adapter protein Shc by growth factor receptors provides association sites for Grb2-Sos, thereby activating the Ras/MAP kinase pathway. A much lowered degree of Shc phosphorylation, Ras and Erk2 activation and c-fos induction was seen in the Y568F mutant, while in the Y570F mutant these responses were less affected. In contrast, the mitogenic response was only slightly reduced. In a mutant receptor with both Tyr568 and Tyr570 mutated to phenylalanine residues, no phosphorylation of Shc and no activation of Ras and Erk2 was seen in response to stem cell factor stimulation, very weak induction of c-fos was seen and the mitogenic response was severely depressed. These data show that Ras/MAP kinase activation and c-fos induction by Kit/SCFR are mediated by members of the Src family kinases. However, the mitogenic response is only to a minor extent dependent on Src kinase activity.

Identification of Tyr-703 and Tyr-936 as the primary association sites for Grb2 and Grb7 in the c-Kit/stem cell factor receptor.

In this paper we demonstrate the presence of two novel in vivo autophosphorylation sites in the c-Kit/stem cell factor receptor (c-Kit/SCFR): Tyr-703 in the kinase insert and Tyr-936 in the C-terminal tail. We furthermore demonstrate that the adapter protein Grb2 is a specific binding partner for both phosphorylated Tyr-703 and phosphorylated Tyr-936, whereas the adapter protein Grb7 binds selectively to phosphorylated Tyr-936. It is shown that the association occurs through the Src homology 2 (SH2) domains of Grb2 and Grb7. Binding of Grb2 to Tyr-703 in the c-Kit/SCFR provides a link to the Ras/mitogen-activated protein kinase pathway.

On metabolic effects of diuretics and beta-blockers. Results from a cross-sectional and longitudinal population study of women.

A population sample of women in Göteborg, Sweden, was studied in 1968-69 and restudied in 1974-75. A number of metabolic variables could be studied cross-sectionally (in 1974-75) and longitudinally (intraindividual changes between 1968-69 and 1974-75). When studied cross-sectionally, increased serum urate concentrations were observed in women on diuretics and increased serum triglycerides in women on beta-blockers, compared to other women, while no obvious differences were found in blood glucose and serum cholesterol concentrations or in packed red cell volume (PCV). When studied longitudinally, a slight decrease in serum triglyceride concentration was found in women who started to take diuretics and an increase in women who started to beta-blockers compared to other women. Serum urate concentration tended to increase in women who started to take diuretics and PCV tended to decrease in women who started to take beta-blockers. Blood glucose was unimpaired. In conclusion, the differences observed were rather small and seemed to be rather unimportant compared to the threat presented by the arterial hypertension itself.

Renal stone disease--experience from a population study of women in Gothenburg, Sweden.

An interview concerning previous history of renal stone disease was included when a population study of 1 462 women aged 38--60 years was carried out in 1968-69. About 5% reported a history of renal stone disease. When the women were re-examined six years later, an annual incidence of 3.7 per 1 000 was found. Women with history of new attacks of renal stone disease usually had the same serum calcium and serum uric acid values as other women except for a few with markedly increased serum uric acid values.