title: Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.
abstract: The laminin-binding alpha7beta1 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 alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.
authors.affiliation: Department of Stomatology, University of California at San Francisco, 94143-0512, USA.
authors.forename: I S
authors.initials: IS
authors.lastname: Vizirianakis
authors.forename: C C
authors.initials: CC
authors.lastname: Yao
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: B L
authors.initials: BL
authors.lastname: Ziober
authors.forename: A S
authors.initials: AS
authors.lastname: Tsiftsoglou
authors.forename: R H
authors.initials: RH
authors.lastname: Kramer
mesh_terms: D016475:3T3 Cells; D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D019556:COS Cells; D002448:Cell Adhesion; D002460:Cell Line; D002469:Cell Separation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D039001:Integrin alpha Chains; D007797:Laminin / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018076:DNA, Complementary; D039001:Integrin alpha Chains; D007797:Laminin; D012333:RNA, Messenger; C085725:integrin alpha7; C121346:laminin 1; D001710:Biotin