Transfection of MCF-7 Carcinoma Cells with Human Integrin α7 cDNA Promotes Adhesion to Laminin

Abstract

The laminin-binding α7β1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin α7 subunit contributes to a form of muscular dystrophy in integrin α7 null mice, whereas specific mutations in the α7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin α7 in human-related muscular disorders, we cloned α7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin α7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human α7X2B alternative splice form. Expression of human α7 was further confirmed by transfection of chimeric human/mouse α7 cDNA constructs. To demonstrate the functionality of expressed human α7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human α7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human α7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human α7 cDNA. These results show for the first time the exogenous expression of functional full-length human α7 cDNA, as well as the development of monoclonal antibodies against the human α7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving α7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.