ABSTRACT
BACKGROUND: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah- 1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating beta-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. METHODS: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. RESULTS: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. CONCLUSION: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , beta Catenin , Carcinogenesis , Cell Proliferation , Clinical Coding , Clone Cells , Colon , Colorectal Neoplasms , Diagnosis , DNA, Complementary , Ecthyma, Contagious , Enzyme-Linked Immunosorbent Assay , Sensitivity and SpecificityABSTRACT
Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.
Subject(s)
Humans , Phosphatidylinositol 3-Kinase/metabolism , Drug Synergism , Enzyme Activation , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/enzymology , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/antagonists & inhibitors , Stem Cell Factor/pharmacology , Tacrolimus Binding Protein 1A/metabolismABSTRACT
BACKGROUND: The S100A2 gene, also known as S100L or CaN19, encodes a protein comprised of 99-amino acids, is a member of the calcium-binding proteins of EF-hand family. According to a recent study, this gene was over-expressed in several early and malignant carcinomas compared to normal tissues. To elucidate the role of S100A2 protein in the process during carcinogenesis, production of monoclonal antibody specific to the protein is essential. METHODS: First, cDNA sequence coding for ORF region of human S100A2 gene was amplified and cloned into an expression vector to produce GST fusion protein. Recombinant S100A2 protein and subsequently, monoclonal antibody to the protein were produced. The specificity of anti-S100A2 monoclonal antibody was confirmed by immunoblot analysis of cross reactivity to other recombinant proteins of S100A family (GST-S100A1, GST-S100A4 and GST-S100A6). To confirm the relation of S100A2 to cervical carcinogenesis, S100A2 protein in early cervical carcinoma tissue was immunostained using the monoclonal antibody. RESULTS: GST-S100A2 recombinant protein was purified by affinity chromatography and then fusion protein was cleaved and S100A2 protein was isolated. The monoclonal antibody (KK0723; Korean patent pending #2001-30294) to the protein was produced and the antibody did not react with other members of EF-hand family proteins such as S100A1, S100A4 and S100A6. CONCLUSION: These data suggest that anti-S100A2 monoclonal antibody produced in this study can be very useful for the early detection of cervical carcinoma and elucidation of mechanism during the early cervical carcinogenesis
Subject(s)
Animals , Humans , Calcium-Binding Proteins , Carcinogenesis , Chromatography, Affinity , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.
Subject(s)
Animals , Humans , Amino Acid Sequence , Antibodies, Monoclonal , Cell Division , Cell Line , Cell Line, Tumor , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Melanoma , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.
Subject(s)
Animals , Humans , Amino Acid Sequence , Antibodies, Monoclonal , Cell Division , Cell Line , Cell Line, Tumor , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Melanoma , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
No abstract available.
Subject(s)
Cell Proliferation , Interleukin-1 , Leukemia, Myelogenous, Chronic, BCR-ABL PositiveABSTRACT
No abstract available.