Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies

EGF, but not TGF alpha, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF alpha, whereas the ubiquitination was more sustained by incubation with EGF than with TGF alpha. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF alpha, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF alpha dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.     

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

Background

Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.

Methodology

In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.

Results

Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.

Conclusions

Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.     

Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.     

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V,caspase cleavage and dUTP incorporation assays

H. P. Dong, A. Holth, M. G. Ruud, E. Emilsen, B. Risberg and B. Davidson Measurement of apoptosis in cytological specimens by flow cytometry: comparison of annexin V, caspase cleavage and dUTP incorporation assays Objective: To compare the performance of different assays for measuring apoptosis in cytological specimens. Methods: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase‐3 and ‐8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. Results: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V‐positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). Conclusions: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.     

Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.     

Habitual physical activity facilitates stress-induced HSP72 induction in brain,peripheral, and immune tissues

The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.     

TCTP Is an Androgen-Regulated Gene Implicated in Prostate Cancer

TCTP has been implicated in a plethora of important cellular processes related to cell growth, cell cycle progression, malignant transformation and inhibition of apoptosis. In addition to these intracellular functions, TCTP has extracellular functions and plays an important role in immune cells. TCTP expression was previously shown to be deregulated in prostate cancer, but its function in prostate cancer cells is largely unknown. Here we show that TCTP expression is regulated by androgens in LNCaP prostate cancer cells in vitro as well as human prostate cancer xenografts in vivo. Knockdown of TCTP reduced colony formation and increased apoptosis in LNCaP cells, implicating it as an important factor for prostate cancer cell growth. Global gene expression profiling in TCTP knockdown LNCaP cells showed that several interferon regulated genes are regulated by TCTP, suggesting that it may have a role in regulating immune function in prostate cancer. In addition, recombinant TCTP treatment increased colony formation in LNCaP cells suggesting that secreted TCTP may function as a proliferative factor in prostate cancer. These results suggest that TCTP may have a role in prostate cancer development.     

Reconstruction of environmental and climate changes at Braamhoek wetland,eastern escarpment South Africa,during the last 16,000 years with emphasis on the Pleistocene–Holocene transition

A paleo-environmental record covering the last 16 ka (16,000 cal yrs BP) from the eastern areas of the summer rainfall region in South Africa is presented. This area is until now sparsely investigated due to the lack of well preserved natural archives. For this study, we used a peat section from a wetland situated close to the Drakensberg escarpment, where the high annual rainfall amounts supported a continuous peat accumulation since c. 16 ka. One peat core was analysed in terms of fossil pollen composition, carbon and nitrogen content, isotope composition (δ¹³C, δ¹⁵N) and microscopic charcoal concentration. The greatest degree of temporal resolution was achieved from the late Pleistocene and early Holocene section, where proxy-records indicate relatively dry conditions between ca. 16–13.7 ka, 12.8–10.5 ka, 9.5–8.2 ka, and wet conditions between c. 13.7–12.8 ka and 10.5–9.5 ka. A weak moisture signal is also evident at c. 8.2–7.5 ka. The late Pleistocene to early Holocene period was relatively cool, while conditions became generally warmer after 11–8 ka. The interpretation of the mid- and late-Holocene sequence is limited due to a slow accumulation and low sample resolution, but the available data suggest relatively dry conditions until c. 1.5 ka, followed by more humid conditions until c. 0.5 ka. We suggest that the millennial scale variability within the record can be attributed to shifts in the circulation systems dominating the region, i.e. the latitudinal movements of the inter-tropical convergence zone (ITCZ) and the dynamics of the mid-latitude low pressure belts.     

DNA microarray study on gene expression profiles in co-cultured endothelial and smooth muscle cells in response to 4- and 24-h shear stress

Shear stress, a major hemodynamic force acting on the vessel wall, plays an important role in physiological processes such as cell growth, differentiation, remodelling, metabolism, morphology, and gene expression. We investigated the effect of shear stress on gene expression profiles in co-cultured vascular endothelial cells (ECs) and smooth muscle cells (SMCs). Human aortic ECs were cultured as a confluent monolayer on top of confluent human aortic SMCs, and the EC side of the co-culture was exposed to a laminar shear stress of 12 dyn/cm² for 4 or 24 h. After shearing, the ECs and SMCs were separated and RNA was extracted from the cells. The RNA samples were labelled and hybridized with cDNA array slides that contained 8694 genes. Statistical analysis showed that shear stress caused the differential expression (p ≤ 0.05) of a total of 1151 genes in ECs and SMCs. In the co-cultured ECs, shear stress caused the up-regulation of 403 genes and down-regulation of 470. In the co-cultured SMCs, shear stress caused the up-regulation of 152 genes and down-regulation of 126 genes. These results provide new information on the gene expression profile and its potential functional consequences in co-cultured ECs and SMCs exposed to a physiological level of laminar shear stress. Although the effects of shear stress on gene expression in monocultured and co-cultured EC are generally similar, the response of some genes to shear stress is opposite between these two types of culture (e.g., ICAM-1 is up-regulated in monoculture and down-regulated in co-culture), which strongly indicates that EC–SMC interactions affect EC responses to shear stress.