ABSTRACT
A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.
Subject(s)
Antibodies , Antibodies, NeutralizingABSTRACT
Circulating tumor cells (CTCs) are shed from cancerous tumors, enter the circulatory system, and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer. Identification and characterization of CTCs provides a means to study, monitor, and potentially interfere with the metastatic process. Isolation of CTCs from blood is challenging because CTCs are rare and possess characteristics that reflect the heterogeneity of cancers. Various methods have been developed to enrich CTCs from many millions of normal blood cells. Microfluidics offers an opportunity to create a next generation of superior CTC enrichment devices. This review focuses on various microfluidic approaches that have been applied to date to capture CTCs from the blood of patients with cancer.
Subject(s)
Microfluidics , Neoplastic Cells, Circulating/pathology , Antigens, Surface/metabolism , Biomarkers, Tumor/metabolism , Cell Separation , Humans , Microfluidics/methods , Neoplasms/diagnosis , Neoplasms/pathology , Neoplastic Cells, Circulating/metabolismABSTRACT
The ubiquitous alpha(E)-catenin is an essential actin cytoskeletal linker. The transcription factor, serum response factor (SRF), induces transcription via binding to the serum response element (SRE) in gene promoters, and in many cases responds to actin dynamics. Here, we report that alpha(E)-catenin expression in HEK293 cells activates the SRE.L transcriptional reporter, a reporter containing the isolated SRF-binding site, and a stably integrated SRE.L reporter in fibroblasts. alpha-Catenin-induced reporter activity appears only partly dependent on RhoA GTPase and Rho kinase function. alpha-Catenin expression has no effect on RhoA activation or localization, and alpha-catenin-induced SRE.L reporter activation is insensitive to the actin-modulating agent latrunculin B. Ectopic alpha-catenin expression was not sufficient to induce actin filament assembly as measured by stress fiber formation. SRE.L reporter is activated by the C-terminal approximately 300 residue region of alpha(E)-catenin. These results suggest induction of SRF-mediated transcription by alpha(E)-catenin either downstream of RhoA or via a parallel pathway.
Subject(s)
Kidney/metabolism , Serum Response Factor/metabolism , Transcriptional Activation/physiology , alpha Catenin/metabolism , rho-Associated Kinases/metabolism , rhoA GTP-Binding Protein/metabolism , Cell Line , Humans , Protein Structure, Tertiary , Signal Transduction/physiologyABSTRACT
A key issue regarding the role of alpha6beta4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique beta4 intracellular domain in the absence of contributions from the alpha6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the beta4 transmembrane and intracellular domains. Expression of this chimeric receptor in beta4-null cancer cells enabled us to assess the signaling potential of the beta4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the beta4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact alpha6beta4. In contrast to alpha6beta4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the beta4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of alpha6beta4. These data also question the putative role of the beta4 intracellular domain as an "adaptor" for growth factor receptor signaling.
Subject(s)
Integrin beta4/chemistry , Cell Line, Tumor , Cell Movement , Cluster Analysis , Humans , Integrin beta4/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/metabolism , Receptor, trkB/chemistry , Recombinant Fusion Proteins/chemistry , Signal Transduction , Tyrosine/chemistry , src-Family Kinases/metabolismABSTRACT
Galpha12/13 or Galphaq signals induce activation of Rho GTPase, leading to serum response factor (SRF)-mediated gene transcription and actin cytoskeletal organization; however, less is known regarding how Rho pathway signals are down-regulated. Here we report that Galphaz signals inhibit serum response factor (SRF)-dependent transcription. Galphaz expression inhibits Galpha12/13-, Galphaq-, and Rho guanine nucleotide exchange factor (GEF)-induced serum response element (SRE) reporter activation in human embryonic kidney 293T and PC-12 cells. Expression of Galphaz mutants with defective fatty acylation has no inhibitory effect. Expression of Galphaz, but not Galphai, attenuates serum-induced SRE reporter activation, suggesting that Galphaz can down-regulate endogenous signals leading to SRF. Whereas Galphaz also blocks SRE reporter induction by the activated mutant RhoAL63, it does not affect Galpha12- or Rho GEF-induced RhoA activation or RhoAL63-GTP binding in vivo. Moreover, Galphaz does not inhibit SRE reporter induction by an activated form of Rho kinase. Because Galphaz inhibits RhoAL63/A188-induced reporter activation, phosphorylation of RhoA on serine 188 does not seem to be involved; furthermore, RhoA subcellular localization was not affected. Use of pharmacologic inhibitors implies that Galphaz-induced reduction of SRE reporter activation occurs via a mechanism other than adenylate cyclase modulation. These findings suggest that Galphaz signals may attenuate Rho-induced stimulation of SRF-mediated transcription.
Subject(s)
GTP-Binding Protein alpha Subunits/physiology , Rho Factor/antagonists & inhibitors , Serum Response Factor/antagonists & inhibitors , Transcription, Genetic/physiology , Amino Acid Substitution , Cell Line , Gene Expression Regulation/physiology , Genes, Reporter , Humans , Kidney , Mutagenesis, Site-Directed , Plasmids , Recombinant Proteins/metabolism , Signal Transduction , TransfectionABSTRACT
Alpha-catenin, an integral part of cadherin-catenin adhesion complexes, is a major binding partner of beta-catenin, a key component of the Wnt pathway, which activates T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription and is often upregulated in cancers. Recently, we identified an alpha-catenin-related protein, alpha-catulin, whose function is poorly understood, as part of a Rho GTPase signaling complex. Here, based on evidence suggesting that alpha-catulin may associate with a beta-catenin fraction, we investigated the role of alpha-catenin family members in beta-catenin-mediated signals. Expression of the full length or a 103-residue region of alpha-catenin strongly inhibits the induction of the TCF/LEF-responsive TOPFLASH reporter in HEK293T cells expressing activated beta-catenin or in cancer cells with constitutively upregulated Wnt signaling, whereas alpha-catulin expression had no effect. Interestingly, alpha-catulin expression attenuates the activation of the cyclin D1 promoter, a target of Wnt pathway signals. Alpha-catulin appears to inhibit Ras-mediated signals to the cyclin D1 promoter, rather than beta-catenin signals, and the synergy between Ras and beta-catenin required to fully activate this promoter. Data suggesting the involvement of Rho in this response are presented and discussed. These results suggest a novel function for alpha-catulin and imply that alpha-catenin and alpha-catulin have distinct activities that downregulate, respectively, beta-catenin and Ras signals converging on the cyclin D1 promoter.
Subject(s)
Cytoskeletal Proteins/metabolism , Trans-Activators/metabolism , Active Transport, Cell Nucleus/drug effects , Amino Acid Sequence , Cell Line , Cyclin D1/genetics , Cytoskeletal Proteins/genetics , Genes, Reporter , Humans , In Vitro Techniques , Lithium Chloride/pharmacology , Models, Biological , Molecular Sequence Data , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Signal Transduction , Trans-Activators/genetics , Transcription, Genetic , Vinculin/genetics , Vinculin/metabolism , alpha Catenin , beta Catenin , ras Proteins/genetics , ras Proteins/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/metabolismABSTRACT
The Rho GTPase signaling pathway is required for actin cytoskeletal organization and serum response factor-dependent gene transcription. Lbc is a Rho-specific guanine nucleotide exchange factor that contains a modulatory C-terminal region. To elucidate Lbc regulatory mechanism(s), a yeast two-hybrid screen for proteins that interact with the Lbc C-terminal region was carried out, resulting in multiple isolation of cDNAs encoding the same 734-amino acid Lbc interacting protein. The Lbc interacting protein has homology with the alpha-catenin cell adhesion component and is identical to the alpha-catenin-like alpha-catulin protein of unknown function. The human alpha-catulin gene (CTNNAL1) maps to 9q31-32. Here we identify the predicted endogenous alpha-catulin product, document alpha-catulin and Lbc co-expression in multiple human cell lines, and show alpha-catulin and Lbc subcellular co-fractionation and intracellular localization. The required regions for Lbc and alpha-catulin interaction were mapped, and complex formation between Lbc and alpha-catulin in mammalian cells was detected. Functionally, alpha-catulin co-expression leads to increased Lbc-induced serum response factor activation in vivo as measured by a transcriptional reporter assay. Furthermore, alpha-catulin co-expression enhances Lbc-induced GTP-Rho formation in vivo. These results support the concept that the recently identified alpha-catulin protein may modulate Rho pathway signaling in vivo by providing a scaffold for the Lbc Rho guanine nucleotide exchange factor.