Human and mouse IFN-beta gene therapy exhibits different anti-tumor mechanisms in mouse models.

Previously, we suggested that local human interferon-beta (IFN-beta) gene therapy with replication-defective adenoviral vectors can be an effective cancer treatment. Clinical trials to treat cancers with adenovirus expressing the human IFN-beta gene (IFNB1) has been planned. As a continued effort to explore the mechanisms of action of human IFN-beta gene therapy that can occur in the clinical setting, we tested mouse IFN-beta gene therapy in human xenograft tumors in both ex vivo and in vivo models. Delivery of the mouse IFN-beta gene (Ifnb) caused tumor inhibition; this effect was dependent on the indirect anti-tumor activities of IFN-beta, notably a stimulation of natural killer cells. IFN-beta does not show cross-species activity in its anti-proliferative effect and mouse IFN-beta does not cause as significant an anti-proliferative effect on mouse tumor cells as human IFN-beta causes on human tumor cells. Therefore, we believe that mouse models using either human IFN-beta or mouse IFN-beta gene transfer do not capture all aspects of the action of adenovirus-mediated human IFN-beta gene therapy that may be present in the clinical setting. Due to its multiple mechanisms of action, human IFN-beta gene therapy may be effective in treating human cancers that are either sensitive or resistant to the direct anti-proliferative effect of IFN-beta.

ECM signalling: orchestrating cell behaviour and misbehaviour.

The extracellular matrix (ECM) provides signalling cues that regulate cell behaviour and orchestrate functions of cells in tissue formation and homeostasis. The composition of the ECM, its three-dimensional organization and proteolytic remodelling are major determinants of the microenvironmental signalling context that controls cell shape, motility, growth, survival and differentiation. In recent years, the importance of ECM signalling has been underscored by the evidence that misregulation of cell-ECM interactions can contribute to many diseases, including developmental, immune, haemostasis, degenerative and malignant disorders. This review discusses recent insights into the regulatory functions of the ECM and their role in disease, focusing on cancer as a paradigm.

Perturbation of beta1-integrin function alters the development of murine mammary gland.

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the beta1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous beta1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the beta1-cytoplasmic domain and thus perturb beta1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and beta-casein gene transcripts were diminished, and the basement membrane component, laminin and the beta4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, beta1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso-apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.

Regulation of integrin function by the urokinase receptor.

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

Disruption of integrin function and induction of tyrosine phosphorylation by the autonomously expressed beta 1 integrin cytoplasmic domain.

The cytoplasmic domains of integrin beta subunits are essential for the function of integrins in cell adhesion and signaling. A chimera combining the transmembrane and cytoplasmic domains of the beta 1 integrin subunit with an irrelevant extracellular domain derived from L3T4 (murine CD4) was tested for its ability to interfere with integrin function. Expression of this construct in cultured human embryonic kidney cells under the control of the inducible metallothionein promoter resulted in cell rounding and detachment, and blocked cell adhesion mediated by the beta 1 and alpha v beta 5 integrins. Expression of the beta 1 chimera at basal levels interfered with the tyrosine phosphorylation of a 125-kDa protein induced by antibody-induced clustering of integrins. Induced expression of the chimera resulted in sustained tyrosine phosphorylation of this protein, which could be enhanced by clustering of the chimera but was insensitive to clustering of integrins. These results demonstrate that the autonomously expressed beta 1 integrin cytoplasmic domain can act as a trans-dominant inhibitor of integrin function, presumably via competitive interactions with cytoplasmic components that are required for integrin-mediated cell adhesion and tyrosine phosphorylation.

A novel phosphoglucomutase-related protein is concentrated in adherens junctions of muscle and nonmuscle cells.

Using five monoclonal antibodies raised against a human uterine smooth muscle extract, we have identified a novel antigen which runs as a closely spaced doublet in SDS-gels. The proteins (60/63 kDa) co-purify, are present in a 1:1 ratio as judged by Coomassie Blue staining, and are immunologically closely related, if not identical. No N-terminal sequence could be obtained from a mixture of the 60/63 kDa proteins, but the sequence of four polypeptides liberated by V8 protease or cyanogen bromide cleavage showed that the proteins are closely related to the glycolytic enzyme phosphoglucomutase type 1. Affinity-purified polyclonal antibodies and three different monoclonal antibodies to the 60/63 kDa proteins cross-reacted with rabbit skeletal muscle phosphoglucomutase type 1, whilst two additional monoclonal antibodies were specific for the 60/63 kDa proteins. Peptide maps of the 60/63 kDa proteins and phosphoglucomutase 1 are markedly different, and the purified proteins have no detectable phosphoglucomutase activity. Staining of cultured smooth muscle cells and fibroblasts with antibodies to 60/63 kDa proteins showed that the antigen is concentrated in focal contacts at the ends of actin bundles and is also associated with actin filaments. About 60% of the cellular 60/63 kDa proteins were found in the detergent-insoluble fraction, suggesting a physical association with the cytoskeleton. The highest levels of protein immunoreactivity were found in muscles. The antigen is concentrated in muscle adherens junctions, including smooth muscle dense plaques, cardiomyocyte intercalated disks, and striated muscle myotendinous junctions. Among epithelial cells, the 63 kDa isoform of the protein was found only in cultured keratinocytes where immunofluorescent staining was localized in cell-to-cell adherens junctions. Expression of the 60/63 kDa proteins in vascular smooth muscle cells is developmentally regulated and correlates with the differentiated contractile phenotype of these cells.

[Effect of a phorbol ester (PMA) and forskolin of the G-/F-actin equilibrium in human platelets]. / Der Einfluss von Phorbolester (PMA) und Forskolin auf des G-/F-Actin-Gleichgewicht humaner Thrombozyten.

Phorbolester (PMA) and forskolin (FSK) cause a dramatic reorganization of microfilaments in cultured cells. We have incubated human blood platelets with PMA and FSK and we investigated the G-/F-actin equilibrium by the DNase I inhibition assay. PMA incubation (0.8 microM, 5 min, 37 degrees C) leads to an increase of filamentous actin (14.4 +/- 4.0%) compared to control platelets. The effect is rapid, dose-dependent and specific, since the biologically inactive derivative phorbol 12,13-didecanoate has no effect. FSK incubation (4 microM, 5 min, 37 degrees C) causes a decrease of F-actin (12.8 +/- 9.0%), the effect is rapid and dose-dependent too. Since 8-bromoadenosine 3':5' cyclic monophosphate also decreases F-actin in human blood platelets, the FSK effect seems to be mediated by cyclic AMP due to affecting the adenylate cyclase.