Invasion of tumorigenic HT1080 cells is impeded by blocking or downregulating the 37-kDa/67-kDa laminin receptor.

The 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) acting as a receptor for prions and viruses is overexpressed in various cancer cell lines, and their metastatic potential correlates with LRP/LR levels. We analyzed the tumorigenic fibrosarcoma cell line HT1080 regarding 37-kDa/67-kDa LRP/LR levels and its invasive potential. Compared to the less invasive embryonic fibroblast cell line NIH3T3, the tumorigenic HT1080 cells display approximately 1.6-fold higher cell-surface levels of LRP/LR. We show that anti-LRP/LR tools interfere with the invasive potential of HT1080 cells. Anti-LRP/LR single-chain variable fragment antibody (scFv) iS18 generated by chain shuffling from parental scFv S18 and its full-length version immunoglobulin G1-iS18 reduced the invasive potential of HT1080 cells significantly by 37% and 38%, respectively. HT1080 cells transfected with lentiviral plasmids expressing small interfering RNAs directed against LRP mRNA showed reduced LRP levels by approximately 44%, concomitant with a significant decrease in the invasive potential by approximately 37%. The polysulfated glycans HM2602 and pentosan polysulfate (SP-54), both capable of blocking LRP/LR, reduced the invasive potential by 20% and 35%, respectively. Adhesion of HT1080 cells to laminin-1 was significantly impeded by scFv iS18 and immunoglobulin G1-iS18 by 60% and 68%, respectively, and by SP-54 and HM2602 by 80%, suggesting that the reduced invasive capacity achieved by these tools is due to the perturbation of the LRP/LR-laminin interaction on the cell surface. Our in vitro data suggest that reagents directed against LRP/LR or LRP mRNA such as antibodies, polysulfated glycans, or small interfering RNAs, previously shown to encompass an anti-prion activity by blocking or downregulating the prion receptor LRP/LR, might also be potential cancer therapeutics blocking metastasis by interfering with the LRP/LR-laminin interaction in neoplastic tissues.

Targeted degradation of ABC transporters in health and disease.

ATP binding cassette (ABC) transporters comprise an extended protein family involved in the transport of a broad spectrum of solutes across membranes. They consist of a common architecture including two ATP-binding domains converting chemical energy into conformational changes and two transmembrane domains facilitating transport via alternating access. This review focuses on the biogenesis, and more precisely, on the degradation of mammalian ABC transporters in the endoplasmic reticulum (ER). We enlighten the ER-associated degradation pathway in the context of misfolded, misassembled or tightly regulated ABC transporters with a closer view on the cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP), which plays an essential role in the adaptive immunity. Three rather different scenarios affecting the stability and degradation of ABC transporters are discussed: (1) misfolded domains caused by a lack of proper intra- and intermolecular contacts within the ABC transporters, (2) deficient assembly with auxiliary factors, and (3) arrest and accumulation of an intermediate or 'dead-end' state in the transport cycle, which is prone to be recognized by the ER-associated degradation machinery.

Therapeutic approaches for prion disorders.

Prion diseases are lethal for both humans and animals, and affected individuals die after several months following a rapid disease progression. Although researchers have attempted for decades to develop effective therapeutics for the therapy of human prion disorders, until now no efficient drug has been available on the market for transmissible spongiform encephalopathy (TSE) treatment or cure. Approximately 200 patients worldwide have died or suffer from variant Creutzfeldt-Jakob disease (CJD). Incidences for sporadic and familial CJD are approximately 1.5-2 per million per year and one per 10 million per year, respectively, in Europe. This review summarizes classical and modern trials for the development of effective anti-TSE drugs, introduces potential effective delivery systems, such as lentiviral and adeno-associated virus systems for antiprion components, including antibodies and siRNAs, and presents vaccination trials. Most of the antiprion drugs target prion protein PrP(c) and/or PrP(Sc). Alternative targets are receptors and coreceptors for PrP, that is, the 37/67-kDa laminin receptor and heparan sulfate proteoglycanes. We review clinical trials for the treatment of TSEs and describe hindrances and chances for a breakthrough in therapy of prion disorders.

Circumventing tolerance to the prion protein (PrP): vaccination with PrP-displaying retrovirus particles induces humoral immune responses against the native form of cellular PrP.

Passive immunization with antibodies directed against the cellular form of the prion protein (PrPC) can protect against prion disease. However, active immunization with recombinant prion protein has so far failed to induce antibodies directed against native PrPC expressed on the cell surface. To develop an antiprion vaccine, a retroviral display system presenting either the full-length mouse PrP (PrP209) or the C-terminal 111 amino acids (PrP111) fused to the transmembrane domain of the platelet-derived growth factor receptor was established. Western blot analysis and immunogold electron microscopy of the retroviral display particles revealed successful incorporation of the fusion proteins into the particle membrane. Interestingly, retroviral particles displaying PrP111 (PrPD111 retroparticles) showed higher incorporation efficiencies than those displaying PrP209. Already 7 days after intravenous injection of PrPD111 retroparticles, PrPC-deficient mice (Prnp(o/o)) showed high immunoglobulin M (IgM) and IgG titers specifically binding the native PrPC molecule as expressed on the surface of T cells isolated from PrPC-overexpressing transgenic mice. More importantly, heterozygous Prnp(+/o) mice and also wild-type mice showed PrPC-specific IgM and IgG antibodies upon vaccination with PrPD111 retroparticles, albeit at considerably lower levels. Bacterially expressed recombinant PrP, in contrast, was unable to evoke IgG antibodies recognizing native PrPC in wild-type mice. Thus, our data show that PrP or parts thereof can be functionally displayed on retroviral particles and that immunization with PrP retroparticles may serve as a novel promising strategy for vaccination against transmissible spongiform encephalitis.