RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression. It is mediated through small interfering RNAs (siRNAs), which trigger specific mRNA degradation. In mammalian systems, however, the application of siRNAs is severely limited by the instability and poor delivery of unmodified siRNA molecules into the cells in vivo. In this study, we show that the noncovalent complexation of synthetic siRNAs with low molecular weight polyethylenimine (PEI) efficiently stabilizes siRNAs and delivers siRNAs into cells where they display full bioactivity at completely nontoxic concentrations. More importantly, in a subcutaneous mouse tumor model, the systemic (intraperitoneal, i.p.) administration of complexed, but not of naked siRNAs, leads to the delivery of the intact siRNAs into the tumors. The i.p. injection of PEI-complexed, but not of naked siRNAs targeting the c-erbB2/neu (HER-2) receptor results in a marked reduction of tumor growth through siRNA-mediated HER-2 downregulation. Hence, we establish a novel and simple system for the systemic in vivo application of siRNAs through PEI complexation as a powerful tool for future therapeutic use.

The growth and metastatic spread of cancer is directly related to tumor angiogenesis, and the driving factors need to be understood to exploit this process therapeutically. However, tumor cells and their normal stroma express a multitude of candidate angiogenic factors, and very few specific inhibitors have been generated to assess which of these gene products are only innocent bystanders and which contribute significantly to tumor angiogenesis and metastasis. Here we investigated whether the expression in tumors of a secreted fibroblast growth factor (FGF)-binding protein (FGF-BP) that mobilizes and activates locally stored FGFs (ref. 11) can serve as an angiogenic switch molecule. Developmental expression of the retinoid-regulated FGF-BP gene is prominent in the skin and intestine during the perinatal phase and is down-modulated in the adult. The gene is, however, upregulated in carcinogen-induced skin tumors, in squamous cell carcinoma (SCC) and in some colon cancer cell lines and tumor samples. To assess the significance of FGF-BP expression in tumors, we depleted human SCC (ME-180) and colon carcinoma (LS174T) cell lines of their endogenous FGF-BP by targeting with specific ribozymes. We found that the reduction of FGF-BP reduced the release of biologically active basic FGF (bFGF) from cells in culture. Furthermore, the growth and angiogenesis of xenograft tumors in mice was decreased in parallel with the reduction of FGF-BP. This suggests that human tumors can utilize FGF-BP as an angiogenic switch molecule.

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Protein-loaded (bovine serum albumin (BSA) or luciferase) poly(vinyl alcohol) (PVA) nanofibers were obtained by electrospinning. Poly(p-xylylene) (PPX, also coined as parylene) coated PVA/BSA nanofibers were prepared by chemical vapor deposition (CVD). The release of BSA from PVA nanofibers under physiological conditions was monitored by absorption spectroscopy. Burst release of BSA was noted with uncoated PVA nanofibers. In contrast, PPX-coated nanofibers exhibited a significantly retarded release of BSA depending on the coating thickness of PPX (ranging from 40 to 300 nm). Luciferase was used here as model enzyme, which after electrospinning retained its enzyme activity. This preservation of enzyme activity and the continuous release of the intact enzyme from the immersed fibers meets a fundamental prerequisite for the application of enzymes or other sensitive agents released from electrospun nanofibers under physiological conditions.

Retroviral elements are found in abundance throughout the human genome but only rarely have alterations of endogenous genes by retroviral insertions been described. Herein we report that a human endogenous retrovirus (HERV) type C is inserted in the human growth factor gene pleiotrophin (PTN) between the 5 untranslated and the coding region. This insert in the human genome expands the region relative to the murine gene. Studies with promoterreporter constructs show that the HERV insert in the human PTN gene generates an additional promoter with trophoblastspecific activity. Due to this promoter function, fusion transcripts between HERV and the open reading frame of PTN (HERV-PTN) were detected in all normal human trophoblast cell cultures as early as 9 weeks after gestation (n ‫؍‬ 7) and in all term placenta tissues (n ‫؍‬ 5) but not in other normal adult tissues. Furthermore, only trophoblast-derived choriocarcinoma cell lines expressed HERV-PTN mRNA whereas tumor cell lines derived from the embryoblast (teratocarcinoma) or from other lineages failed to do so. We investigated the significance of HERV-PTN mRNA in a choriocarcinoma model by targeting this transcript with ribozymes and found that the depletion of HERV-PTN mRNA prevents human choriocarcinoma growth, invasion, and angiogenesis in mice. This suggests that the tissue-specific expression of PTN due to the HERV insertion in the human genome supports the highly aggressive growth of human choriocarcinoma and possibly of the human trophoblast.Pleiotrophin (PTN) is a secreted heparin-binding polypeptide growth factor (1) with mitogenic (1-3) and transforming effects (3) on fibroblasts and growth factor activity on epithelial (2, 4, 5) and endothelial (2, 5, 6) cells. Furthermore, PTN induces the release of proteolytic enzymes from endothelial cells (7) and stimulates neurite outgrowth (8) and tube formation by endothelial cells in vitro (5) as well as angiogenesis in the rabbit corneal pocket assay (6). PTN gene expression is regulated in a time-and tissue-specific manner during rodent development and PTN mRNA is found at high levels in the central nervous system during the perinatal period, is downregulated thereafter, and is present at low levels in a few adult tissues (1, 9-11). On the other hand, the PTN gene is upregulated in several human tumor tissues and tumor cell lines (2) but little is known about the regulatory elements in this gene (12).To understand the mechanisms that regulate expression of the human PTN gene, we studied 5Ј ends of PTN mRNA isolated from various human tissues that express the PTN gene. To our surprise we found that all placenta samples, in contrast to brain, expressed PTN mRNA with 5Ј exons that are homologous to a human endogenous retrovirus (HERV) and are spliced onto the intact open reading frame (ORF) of PTN. Upon analysis of human genomic DNA, we located the insertion of an HERV fragment into the intron region upstream of the ORF of the human PTN gene expanding this region relative to the ancestral PTN gene share...