Development of monoclonal antibodies to integrin receptors.

Integrins are heterodimeric cell surface receptors composed of an alpha and a beta subunit. They are involved in homotopic and heterotopic cell adhesion and also function as receptors for extracellular matrix molecules such as collagen, fibronectin and laminin. The family to which an integrin belongs is defined by the presence of a particular beta subunit paired with a unique alpha subunit. In this chapter we describe methods to produce monoclonal antibodies to the family of integrin subunits characterized by beta1 and provide detailed instructions for the development of a monoclonal antibody to the alpha6 integrin receptor expressed by human prostate carcinoma cells (PC3 cells). Data are presented that correlate the functional capabilities of an antibody with its biochemical characterization.

Active zones on motor nerve terminals contain alpha 3beta 1 integrin.

Active zones are the sites along nerve terminals where synaptic vesicles dock and undergo calcium-dependent exocytosis during synaptic transmission. Here we show, by immunofluorescent staining with antibodies generated against Xenopus laevis integrins, that alpha3beta1 integrin is concentrated at the active zones of Xenopus motor nerve terminals. Because integrins can link extracellular matrix molecules to cytoskeletal elements and participate in the formation of signaling complexes, the localization of integrin at active zones suggests that it may play a role in the adhesion of the nerve terminals to the synaptic basal lamina, in the formation and maintenance of active zones, and in some of the events associated with calcium-dependent exocytosis of neurotransmitter. Our findings also indicate that the integrin composition of the terminal Schwann cells differs from that of the motor nerve terminals, and this may account at least in part for differences in their adhesiveness to the synaptic basal lamina.

Inhibition of pp125FAK in cultured fibroblasts results in apoptosis.

The tyrosine kinase called pp125FAK is believed to play an important role in integrin-mediated signal transduction. pp125FAK is associated both functionally and spatially with integrins, which are the cell surface receptors for extracellular matrix components. Although the precise function of pp125FAK is not known, two possibilities have been proposed: pp125FAK may regulate the assembly of focal adhesions in spreading or migrating cells, or pp125FAK may participate in a signal transduction cascade to inform the nucleus that the cell is anchored. To test these models in living cells, a peptide representing the focal adhesion kinase (FAK)-binding site of the beta 1 tail was coupled to carrier protein and injected into cultured cells to competitively inhibit the binding of pp125FAK to endogenous integrin, thus inhibiting activation of pp125FAK on a cell-by-cell basis. In addition, an antibody directed against an epitope adjacent to the focal adhesion targeting sequence on pp125FAK was microinjected, as an alternative means of inhibiting pp125FAK activation. It was observed that when rounded cells were injected with either the integrin peptide or the anti-FAK antibody, the cells rapidly began to apoptose, within 4 h after injection. These results indicate that pp125FAK may play a critical role in suppressing apoptosis in fibroblasts.

Selective assembly of laminin variants by human carcinoma cells.

BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble basement membranes, the pattern of production of various laminin subunits remains to be explored. EXPERIMENTAL DESIGN: The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies. For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains. RESULTS: All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was demonstrated by immunofluorescence staining. The cells contained both B1 and S chain laminin in the cell layer, but preferentially secreted the B1 chain into the culture supernatant as shown by Western blotting. The 300 to 400 kDa M chain immunoreactive band was found in laminin secreted into the culture medium of HU-1 cells. Immunoprecipitation of biosynthetically labeled proteins showed that the M chain was synthesized as a complex with B chains. Little or no A chain laminin was detected in the culture medium supernatant. HU-1 cells also synthesized the newly described laminin variant, epiligrin which was secreted into the medium. Thus, the HU-1 cells secreted two laminin variants: M-B1-B12 laminin and epiligrin into the culture medium. Immunostaining of HU-1 nude mice tumors showed that tumor basement membranes contained M, B1, and B2 laminin and epiligrin immunoreactivity but apparently no S chain. CONCLUSIONS: All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion and deposition of laminin variants into tumor basement membranes. These data indicate that the tumor cells vary among themselves with regards to laminin production and that some of them, like HU-1 may produce essentially all laminin chains simultaneously.