Mutation analysis of "Endoglin" and "Activin receptor-like kinase" genes in German patients with hereditary hemorrhagic telangiectasia and the value of rapid genotyping using an allele-specific PCR-technique

<p>Abstract</p> <p>Background</p> <p>Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant disorder which is clinically characterised by recurrent epistaxis, mucocutaneous telangiectasia and visceral arteriovenous malformations. Genetic linkage studies identified two genes primarily related to HHT: endoglin (<it>ENG</it>) on chromosome 9q33-34 and activin receptor-like kinase1 (<it>ACVRL1</it>) on chromosome 12q13. We have screened a total of 41 unselected German patients with the suspected diagnosis of HHT. Mutation analysis for the <it>ENG </it>and <it>ACVRL1 </it>genes in all patients was performed by PCR amplification. Sequences were then compared to the HHT database <url>http://www.hhtmutation.org</url> sequences of the <it>ENG </it>mRNA (accession no. BC014271.2) and the <it>ACVRL1 </it>mRNA (accession no. NM000020.1).</p> <p>Results</p> <p>We identified 15 different mutations in 18 cases by direct sequencing. Among these mutations, one novel <it>ENG </it>mutation could be detected which has not yet been described in the literature before. The genotype-phenotype correlation was consistent with a higher frequency of pulmonary arteriovenous malformations in patients with <it>ENG </it>mutations than in patients with <it>ACVRL1 </it>mutations in our collective.</p> <p>Conclusion</p> <p>For rapid genotyping of mutations and SNPs (single nucleotide polymorphisms) in <it>ENG </it>and <it>ACVRL1</it>, allele-specific PCR methods with sequence-specific primers (PCR-SSP) were established and their value analysed.</p

Anomalous Features of EMT during Keratinocyte Transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike “conventional EMT”, these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated

Upregulation of calcium-sensing receptor and mitogen-activated protein kinase signalling in the regulation of growth and differentiation in colon carcinoma

In the present study, we demonstrate that Ca2+-induced growth inhibition and induction of differentiation in a line of human colon carcinoma cells (CBS) is dependent on mitogen-activated protein (MAP) kinase signaling and is associated with upregulation of extracellular calcium-sensing receptor (CaSR) expression. When CBS cells were grown in Ca2+-free medium and then switched to medium supplemented with 1.4 mM Ca2+, proliferation was reduced and morphologic features of differentiation were expressed. E-cadherin, which was minimally expressed in nonsupplemented medium, was rapidly induced in response to Ca2+ stimulation. Sustained activation of the extracellular signal-regulated kinase (ERK) occured in Ca2+-supplemented medium. When an inhibitor of ERK activation (10 μM U0126) was included in the Ca2+-supplemented culture medium, ERK-activation did not occur. Concomitantly, E-cadherin was not induced, cell proliferation remained high and differentiation was not observed. The same level of Ca2+ supplementation that induced MAP kinase activation also stimulated CaSR upregulation in CBS cells. A clonal isolate of the CBS line that did not upregulate CaSR expression in response to extracellular Ca2+ was isolated from the parent cells. This isolate failed to produce E-cadherin or undergo growth inhibition/induction of differentiation when exposed to Ca2+ in the culture medium. However, ERK-activation occurred as efficiently in this isolate as in parent CBS cells or in a cloned isolate that underwent growth reduction and differentiation in response to Ca2+ stimulation. Together, these data indicate that CaSR upregulation and MAP kinase signalling are both intermediates in the control of colon carcinoma cell growth and differentiation. They appear to function, at least in part, independently of one another

Visual Coding in Locust Photoreceptors

Information capture by photoreceptors ultimately limits the quality of visual processing in the brain. Using conventional sharp microelectrodes, we studied how locust photoreceptors encode random (white-noise, WN) and naturalistic (1/f stimuli, NS) light patterns in vivo and how this coding changes with mean illumination and ambient temperature. We also examined the role of their plasma membrane in shaping voltage responses. We found that brightening or warming increase and accelerate voltage responses, but reduce noise, enabling photoreceptors to encode more information. For WN stimuli, this was accompanied by broadening of the linear frequency range. On the contrary, with NS the signaling took place within a constant bandwidth, possibly revealing a ‘preference’ for inputs with 1/f statistics. The faster signaling was caused by acceleration of the elementary phototransduction current - leading to bumps - and their distribution. The membrane linearly translated phototransduction currents into voltage responses without limiting the throughput of these messages. As the bumps reflected fast changes in membrane resistance, the data suggest that their shape is predominantly driven by fast changes in the light-gated conductance. On the other hand, the slower bump latency distribution is likely to represent slower enzymatic intracellular reactions. Furthermore, the Q10s of bump duration and latency distribution depended on light intensity. Altogether, this study suggests that biochemical constraints imposed upon signaling change continuously as locust photoreceptors adapt to environmental light and temperature conditions

Multifaceted roles of GSK-3 and Wnt/β-catenin in hematopoiesis and leukemogenesis: opportunities for therapeutic intervention

Glycogen synthase kinase-3 (GSK-3) is well documented to participate in a complex array of critical cellular processes. It was initially identified in rat skeletal muscle as a serine/threonine kinase that phosphorylated and inactivated glycogen synthase. This versatile protein is involved in numerous signaling pathways that influence metabolism, embryogenesis, differentiation, migration, cell cycle progression and survival. Recently, GSK-3 has been implicated in leukemia stem cell pathophysiology and may be an appropriate target for its eradication. In this review, we will discuss the roles that GSK-3 plays in hematopoiesis and leukemogenesis as how this pivotal kinase can interact with multiple signaling pathways such as: Wnt/β-catenin, phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR), Ras/Raf/MEK/extracellular signal-regulated kinase (ERK), Notch and others. Moreover, we will discuss how targeting GSK-3 and these other pathways can improve leukemia therapy and may overcome therapeutic resistance. In summary, GSK-3 is a crucial regulatory kinase interacting with multiple pathways to control various physiological processes, as well as leukemia stem cells, leukemia progression and therapeutic resistance. GSK-3 and Wnt are clearly intriguing therapeutic targets

Breast cancer growth and metastasis: interplay between cancer stem cells, embryonic signaling pathways and epithelial-to-mesenchymal transition

Induction of epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSCs) can occur as the result of embryonic pathway signaling. Activation of Hedgehog (Hh), Wnt, Notch, or transforming growth factor-β leads to the upregulation of a group of transcriptional factors that drive EMT. This process leads to the transformation of adhesive, non-mobile, epithelial-like tumor cells into cells with a mobile, invasive phenotype. CSCs and the EMT process are currently being investigated for the role they play in driving metastatic tumor formation in breast cancer. Both are very closely associated with embryonic signaling pathways that stimulate self-renewal properties of CSCs and EMT-inducing transcription factors. Understanding these mechanisms and embryonic signaling pathways may lead to new opportunities for developing therapeutic agents to help prevent metastasis in breast cancer. In this review, we examine embryonic signaling pathways, CSCs, and factors affecting EMT

Patient-derived xenograft (PDX) models in basic and translational breast cancer research

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research

MicroRNAs Profiling in Murine Models of Acute and Chronic Asthma: A Relationship with mRNAs Targets

BACKGROUND: miRNAs are now recognized as key regulator elements in gene expression. Although they have been associated with a number of human diseases, their implication in acute and chronic asthma and their association with lung remodelling have never been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: In order to establish a miRNAs expression profile in lung tissue, mice were sensitized and challenged with ovalbumin mimicking acute, intermediate and chronic human asthma. Levels of lung miRNAs were profiled by microarray and in silico analyses were performed to identify potential mRNA targets and to point out signalling pathways and biological processes regulated by miRNA-dependent mechanisms. Fifty-eight, 66 and 75 miRNAs were found to be significantly modulated at short-, intermediate- and long-term challenge, respectively. Inverse correlation with the expression of potential mRNA targets identified mmu-miR-146b, -223, -29b, -29c, -483, -574-5p, -672 and -690 as the best candidates for an active implication in asthma pathogenesis. A functional validation assay was performed by cotransfecting in human lung fibroblasts (WI26) synthetic miRNAs and engineered expression constructs containing the coding sequence of luciferase upstream of the 3'UTR of various potential mRNA targets. The bioinformatics analysis identified miRNA-linked regulation of several signalling pathways, as matrix metalloproteinases, inflammatory response and TGF-β signalling, and biological processes, including apoptosis and inflammation. CONCLUSIONS/SIGNIFICANCE: This study highlights that specific miRNAs are likely to be involved in asthma disease and could represent a valuable resource both for biological makers identification and for unveiling mechanisms underlying the pathogenesis of asthma