ABSTRACT
BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.
Subject(s)
Antibodies, Monoclonal/chemistry , Biomarkers, Tumor/immunology , DNA-Binding Proteins/immunology , Endonucleases/immunology , Protein Array Analysis/methods , Antibodies, Monoclonal/immunology , Antibody Specificity , Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/chemistry , Carcinoma, Non-Small-Cell Lung/metabolism , Choline-Phosphate Cytidylyltransferase/immunology , Choline-Phosphate Cytidylyltransferase/metabolism , Cross Reactions , DNA-Binding Proteins/metabolism , Endonucleases/metabolism , HEK293 Cells , Humans , Immunohistochemistry/methods , Lung Neoplasms/chemistry , Lung Neoplasms/metabolismABSTRACT
To allow genome-scale identification of genes that regulate cellular signaling, we cloned >90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase activity-deficient mutants. To establish the utility of this resource, we tested the effect of expression of the kinases on three different cellular signaling models. In all screens, many kinases had a modest but significant effect, apparently due to crosstalk between signaling pathways. However, the strongest effects were found with known regulators and novel components, such as MAP3K10 and DYRK2, which we identified in a mammalian Hedgehog (Hh) signaling screen. DYRK2 directly phosphorylated and induced the proteasome-dependent degradation of the key Hh pathway-regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2 and the known Hh pathway component, GSK3beta. Our results establish kinome expression screening as a highly effective way to identify physiological signaling pathway components and genes involved in pathological signaling crosstalk.
Subject(s)
Hedgehog Proteins/metabolism , Protein Kinases/isolation & purification , Protein Kinases/metabolism , Signal Transduction , Animals , COS Cells , Chlorocebus aethiops , Fibroblasts/metabolism , Gene Expression , Gene Library , Kruppel-Like Transcription Factors/metabolism , MAP Kinase Kinase Kinases/metabolism , Mammals , Mice , NIH 3T3 Cells , Oncogene Proteins/metabolism , Protein Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Trans-Activators/metabolism , Vero Cells , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2 , Dyrk KinasesABSTRACT
Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.
Subject(s)
Actins/metabolism , Endopeptidases/metabolism , Luciferases/metabolism , Proteomics/methods , Apoptosis , Autophagy , Endopeptidases/analysis , Recombinant Fusion ProteinsABSTRACT
Glial-derived nexin (GDN) is a proteinase inhibitor secreted from glial cells and it can enhance neuronal function. However, its expression and function in neuronal differentiation are not, as yet, well-known. In the present study, we analyzed glial-derived nexin gene expression in dissociated neural stem/progenitor cells (NS/PCs) (D0) from the embryonic mouse cerebral cortex, expanded NS/PC cultures (D4 and D10 cultures) and cultured neurons (E15) using a semi-quantitative RT-PCR assay. Our data suggest that mouse GDN, homologue of human GDN, was significantly up-regulated in the expanded NS/PC cultures and cultured neurons. To analyze its function in neuronal differentiation, human GDN cDNA was cloned into bicistronic plasmids containing green fluorescent protein (GFP) and the resulting plasmids were transfected into rodent primary NS/PCs and non-neuronal human embryonic kidney (HEK) cells. Our data suggest that the ectopic expression of human GDN triggered the expression of the neuronal marker TuJ1 in both NS/PCs and HEK cells. We conclude that GDN is up-regulated during neuronal differentiation and plays a role in transforming non-neuronal HEK cells into neuron-like cells.
Subject(s)
Amyloid beta-Protein Precursor/physiology , Cell Differentiation/physiology , Gene Expression/physiology , Neurons/physiology , Receptors, Cell Surface/physiology , Animals , Blotting, Northern/methods , Bromodeoxyuridine/metabolism , Cells, Cultured , Cloning, Molecular/methods , Embryo, Mammalian , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry/methods , Indoles , Mice , Neurons/cytology , Protease Nexins , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Stem Cells/physiology , Transfection/methods , Tubulin/metabolismABSTRACT
Three cell groups, neural stem/progenitor cells (NS/PCs) dissociated from the embryonic day 11 (E11) rodent cerebral cortex, expanded NS/PC cultures, and cultured neurons from E15, were used to conduct a genomic study with differential display (DD). The mouse Af1q, homologue of human AF1q, was found to be significantly up-regulated during the neuronal production from NS/PCs. The ectopic expression of human AF1q triggered the expression of the neuronal marker TuJ1 in non-neuronal human embryonic kidney (HEK) cells.
