ABSTRACT
The scaffold protein IQ motif containing GTPase activating protein 1 (IQGAP1) is an adherens junction component in the epithelial tissue that binds many signaling and structural molecules to regulate biological processes. It is known that IQGAP1 is overexpressed in some tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from three-dimensional (3D)-cultured DLD-1 cells to elucidate a characteristic feature of a tumor. In cancer research, 3D-cultured cancer cells are used as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. Our results showed that mAb 7E11 recognized increasing antigen in the lysate of 3D-cultured cells comparing with two-dimensional-cultured cells, and its antigen is the human IQGAP1. Furthermore, we indicated that mAb 7E11 was used in immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful in the analysis of human cancer.
Subject(s)
Antibodies, Monoclonal/pharmacology , Neoplasms/immunology , ras GTPase-Activating Proteins/immunology , Animals , Antibodies, Monoclonal/immunology , Cell Culture Techniques, Three Dimensional , Humans , Immunization , Neoplasms/therapy , Rats , Signal Transduction/genetics , ras GTPase-Activating Proteins/antagonists & inhibitorsABSTRACT
Cytokeratin (CK) 18 is an intermediate filament protein that plays a major functional role in the integrity and mechanical stability of cells. Since both CK8 and CK18 are major components of simple epithelia, in the context of tumors, they are expressed in most carcinomas, and have been studied as diagnostic and prognostic markers in tumor pathology. CK18 is also cleaved by some caspases during apoptosis. Three-dimensional (3D)-cultured cancer cells are useful for cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from 3D-cultured DLD-1 cells to elucidate a characteristic feature of a tumor, and our results showed that mAb 2H7 recognized human CK18. Furthermore, we indicated that mAb 2H7 was useful for immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful as a diagnostic tool for evaluating malignancy.
