The aim of this study was to evaluate in vitro and in vivo imaging of HER-2-over-expressing tumours using near-infrared optical imaging. A fluorochrome probe was designed by coupling Cy5.5 to ...anti-HER-2 antibodies. Cells over-expressing (SK-BR-3 cells) or normally expressing (PE/CA-PJ34 cells) the HER-2 protein were incubated with the probe. After removing unbound probe molecules, fluorescence intensities were determined (a.u.: arbitrary units). Cells were additionally investigated using FACS and laser scanning microscopy. The probe was also injected intravenously into tumours bearing SK-BR-3 ( n=3) or PE/CA-PJ34 ( n=3). Whole-body fluorescence images were generated and analysed. The incubation of SK-BR-3 cells with the probe led to higher fluorescence intensities 2,133 (+/-143) a.u. compared to controls 975 (+/-95) a.u.. The results from FACS and immunocytochemical analysis were in agreement with these findings. A distinct dependency between the fluorescence intensity and the cell number used in the incubations was detected. In vivo, the relative fluorescence intensities in SK-BR-3 tumours were higher than in PE/CA-PJ34 tumours at 16-24 h after probe application. HER-2-over-expressing tumours were depictable in their original size. Labelling of HER-2 with Cy5.5 is suitable for in vitro and in vivo detection of HER-2-over-expressing tumour cells.
Genetic and genomic studies have enhanced our understanding of complex neurodegenerative diseases that exert a devastating impact on individuals and society. One such disease, age-related macular ...degeneration (AMD), is a major cause of progressive and debilitating visual impairment. Since the pioneering discovery in 2005 of
complement factor H
(
CFH
) as a major AMD susceptibility gene, extensive investigations have confirmed 19 additional genetic risk loci, and more are anticipated. In addition to common variants identified by now-conventional genome-wide association studies, targeted genomic sequencing and exome-chip analyses are uncovering rare variant alleles of high impact. Here, we provide a critical review of the ongoing genetic studies and of common and rare risk variants at a total of 20 susceptibility loci, which together explain 40-60% of the disease heritability but provide limited power for diagnostic testing of disease risk. Identification of these susceptibility loci has begun to untangle the complex biological pathways underlying AMD pathophysiology, pointing to new testable paradigms for treatment.
The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small‐molecule drugs targeting this network are increasingly available. However, often only ...specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model‐based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR‐dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF‐ as well as KRAS‐mutated tumor cells, which we confirmed using a xenograft model.
Analysis of the signaling response of colon cancer cells to systematic perturbations reveals an EGF receptor‐mediated cross‐talk between the MAPK and AKT pathways. Accordingly, the predicted combinatorial treatment is shown to inhibit tumor growth in vivo.
Synopsis
Analysis of the signaling response of colon cancer cells to systematic perturbations reveals an EGF receptor‐mediated cross‐talk between the MAPK and AKT pathways. Accordingly, the predicted combinatorial treatment is shown to inhibit tumor growth in vivo.
A modular response analysis model trained on perturbation data in a panel of colon cancer cells revealed a negative feedback from MAPK signaling to the EGF receptor, which leads to AKT cross‐activation after MEK inhibition.
The model predicted that successful inhibition of growth‐factor signaling in colon cancer requires combined inhibition of MEK and EGF receptor to prevent AKT activation and tumor cell survival, which was confirmed by growth assays.
A xenograft tumor model of KRAS‐mutant colon cancer showed that EGFR receptor inhibition alone has no effect, but in combination with a MEK inhibitor successfully reduces tumor growth.
Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known ...about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of ‘DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)’. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted ‘skin aging–associated secreted proteins (SAASP)’. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of ‘metabolism’ and ‘adherens junction interactions’ was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.
A frequent deletion of complement factor H (CFH)-related genes CFHR3 and CFHR1 (ΔCFHR3/CFHR1) is considered to have a protective effect against age-related macular degeneration (AMD), although the ...underlying mechanism remains elusive. The deletion seems to be linked to one of the two protective CFH haplotypes which are both tagged by the protective allele of single nucleotide polymorphism rs2274700 (CFH:A473A). In a German cohort of 530 AMD patients, we now show that protection against AMD conferred by ΔCFHR3/CFHR1 is independent of the effects of rs2274700 and rs1061170 (CFH:Y402H). This suggests a functional role of CFHR1 and/or CFHR3 in disease pathogenesis. We therefore characterized the CFHR3 function and identified CFHR3 as a novel human complement regulator that inhibits C3 convertase activity. CFHR3 displays anti-inflammatory effects by blocking C5a generation and C5a-mediated chemoattraction of neutrophils. In addition, CFHR3 and CFHR1 compete with factor H for binding to the central complement component C3. Thus, deficiency of CFHR3 and CFHR1 results in a loss of complement control but enhances local regulation by factor H. Our findings allude to a critical balance between the complement regulators CFHR3, CFHR1 and factor H and further emphasize the central role of complement regulation in AMD pathology.
Age-related macular degeneration (AMD) is a prevalent cause of blindness in Western societies. Variants in the genes encoding complement factor H (CFH), complement component 3 (C3) and age-related ...maculopathy susceptibility 2 (ARMS2) have repeatedly been shown to confer significant risks for AMD; however, their role in disease progression and thus their potential relevance for interventional therapeutic approaches remains unknown.
Here, we analyzed association between variants in CFH, C3 and ARMS2 and disease progression of geographic atrophy (GA) due to AMD. A quantitative phenotype of disease progression was computed based on longitudinal observations by fundus autofluorescence imaging. In a subset of 99 cases with pure bilateral GA, variants in CFH (Y402H), C3 (R102G), and ARMS2 (A69S) are associated with disease (P = 1.6x10(-9), 3.2x10(-3), and P = 2.6x10(-12), respectively) when compared to 612 unrelated healthy control individuals. In cases, median progression rate of GA over a mean follow-up period of 3.0 years was 1.61 mm(2)/year with high concordance between fellow eyes. No association between the progression rate and any of the genetic risk variants at the three loci was observed (P>0.13).
This study confirms that variants at CFH, C3, and ARMS2 confer significant risks for GA due to AMD. In contrast, our data indicate no association of these variants with disease progression which may have important implications for future treatment strategies. Other, as yet unknown susceptibilities may influence disease progression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein levels within signal transduction pathways vary strongly from cell to cell. Here, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity ...in protein levels. We systematically perturbed the protein levels of Erk, the terminal kinase in the MAPK signalling pathway in a panel of human cell lines. We found that the steady‐state phosphorylation of Erk is very robust against perturbations of Erk protein level. Although a multitude of mechanisms exist that may provide robustness against fluctuating protein levels, we found that one single feedback from Erk to Raf‐1 accounts for the observed robustness. Surprisingly, robustness is provided through a fast post‐translational mechanism although variation of Erk levels occurs on a timescale of days.
Synopsis
Protein levels within signal transduction pathways vary strongly from cell to cell. For example, it has been reported that concentrations of the last kinase within the MAPK signalling module, Erk, varies about four‐fold between clonal cells under the same conditions. In the present study, we analysed how signalling pathways can still process information quantitatively despite strong heterogeneity in protein levels. Mathematical analysis of isolated phosphorylation–dephosphorylation cycles predicts that phosphorylation of a signalling molecule is proportional to the protein concentration. We systematically perturbed the protein levels of Erk in human cell lines by siRNA. We found that the steady‐state phosphorylation of Erk is very robust against perturbations of Erk protein level, suggesting that there are mechanisms that provide robustness to the pathway against protein fluctuations. Using mathematical modelling, we identified three potential mechanisms that may provide robustness against fluctuating protein levels:
Kinetic effects (saturation of the activating kinase Mek),
Transcriptional negative feedbacks,
Negative feedbacks on the post‐translational level.
By experimental analysis of the systems, which included analysis of Erk phosphorylation under Mek overexpression, measuring transcript levels of negative feedback regulators, and application of generic inhibitors of transcription and translation, we could exclude kinetic effects and transcriptional negative feedback as mechanisms of robustness.
By analysing a panel of cell lines, we found that cells are robust as long as the signal passes through Raf‐1. In contrast, cells where the pathway is activated by a mutation in B‐Raf lose robustness. Detailed molecular analysis of the system shows that a single post‐translational feedback to Raf mediates robustness. Thus, robustness is provided through a fast post‐translational mechanism although variation of Erk levels occurs on a timescale of days.
MAPK signalling is robust against variation in protein level.
Robustness is mediated by a negative feedback to Raf.
Loss of negative feedback due to mutation in B‐Raf opens the door for targeted intervention.
The adult mammalian CNS has a limited capacity for nerve regeneration and structural plasticity. The presence of glia-derived inhibitory factors myelin-associated glycoprotein (MAG) and Nogo-A have ...been suggested to provide a nonpermissive environment for elongating nerve fibers. In particular, Nogo-A, an integral membrane protein predominantly expressed by oligodendrocytes, has been demonstrated to impair neurite growth in vitro and in vivo. Structure function analysis revealed that Nogo-A protein contains at least two active domains, NiG and Nogo-66, with diverse effects on neurite outgrowth and cell spreading. We now provide evidence that these inhibitory domains mediate their effects via an antagonistic regulation of the small GTPases RhoA and Rac1, resulting in activation of RhoA and suppression of Rac1. By inactivating RhoA with C3 transferase or the downstream effector Rho-kinase ROCK with, the inhibitory effects of both Nogo-A fragments and MAG on neurite outgrowth and oligodendrocyte-mediated growth cone collapse were abolished. Furthermore, we show that the recently cloned receptor for Nogo-66 and MAG, NgR, is not necessary for either NiG- or MAG-induced RhoA activation.
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