Search

Cell type- and tissue-specific promoters play an important role in the development of site-selective vectors for gene therapy. A large number of highly specific promoters has been described, but their applicability is often hampered by their inefficient transcriptional activity. In this study, we describe a new strategy for enhancing the activity of weak promoters without loss of specificity. The basic principle of this strategy is to establish a positive feedback loop which is initiated by transcription from a cell type-specific promoter. This was achieved by using a cell type-specific pro- moter to drive the simultaneous expression of the desired effector/reporter gene product and a strong artificial transcriptional activator which stimulates transcription through appropriate binding sites in the promoter. Using a VP16-LexA chimeric transcription factor, we show that this approach leads to a 14- to >100-fold enhancement of both the endothelial cell-specific von Willebrand factor promoter and the gastrointestinal-specific sucrase-isomaltase promoter while maintaining approximately 30- to >100-fold cell type specificity. [ABSTRACT FROM AUTHOR]

A correction to the article "Transcriptional Targeting of Adenoviral Vector Through the CXCR4 Tumor-Specific Promoter" that was previously published in the journal is presented.

Anchorage-independence is a hallmark of metastatic cancer cells. In previous studies we characterized a novel model for anchorage-independence employing dynamic matrix detachment (DMD) using rotation in low shear stress conditions. We observed that in contrast to the classical apoptosis-inducing static matrix detachment (SMD) model, the venous circulation-mimicking DMD model induced necrosis in transformed cells. In the current study we revisited the mechanism of DMD-induced cell death and evaluated the contribution of alphavbeta3 integrin overexpression in human melanoma cells to anchorage-independence in DMD. DMD cell culture induced primarily necrosis in the melanoma cells studied. alphavbeta3, but not the control related alphaIIbbeta3 integrin, could confer survival advantage in DMD. While apoptosis was unaffected, constitutive, unligated alphavbeta3 overexpression was associated with attenuation of necrosis in DMD. alphavbeta3 overexpressing melanoma cells manifested AKT activation that was independent of DMD conditions. Furthermore, while a small molecular inhibitor of AKT phosphorylation induced apoptosis in adherent cells, in DMD conditions it had no effect on cell outcome. Thus, alphavbeta3-overexpressing melanoma cells are partially protected from DMD-induced cell death in an apoptosis-independent mechanism. This finding may be one of the factors accounting for anchorage-independence in circulating metastatic melanoma cells., (2009 Elsevier Ireland Ltd. All rights reserved.)

Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of scFv in situ. To circumvent these limitations, we have developed an approach to express scFv in vivo. An anti-erbB2 scFv was engineered for secretion by eukaryotic cells. The secreted scFv could bind to its target and specifically suppress cell growth of erbB2-positive cells in vitro. Adenoviral vectors expressing the cDNA for the secretory scFv likewise could induce target cells to produce an anti-tumor anti-erbB2 scFv. In vivo gene transfer via the anti-erbB2 scFv encoding adenovirus also showed anti-tumor effects. Thus, by virtue of engineering a secreted version of the anti-tumor anti-erbB-2 scFv, and in vivo expression via adenoviral vector, effective concentrations of scFv were achieved. In vivo gene transfer clearly represents a powerful means to realize effective scFv-based approaches. This method will likely have applicability for a range of disorders amenable to targeted therapeutic approaches.

Ovarian carcinoma is a leading cause of cancer death in women. Though advances in conventional therapies have been achieved, long-term survival rates for most patients diagnosed with ovarian cancer are still low. Therefore, novel molecular therapeutic strategies such as gene therapy are being intensively pursued. Such approaches are based on the enormous progress that has been achieved in the elucidation of the molecular foundations of ovarian cancer. In this regard transcriptional control elements (promoters) of genes frequently upregulated or specifically expressed in tumors can be applied in a heterologous context to drive expression of therapeutic genes in targeted gene therapy strategies. This review discusses transcriptional targeting strategies in ovarian cancer gene therapy and gives an overview of tumor-specific promoters (TSPs) that have been applied for this purpose., (Copyright 2001 Academic Press.)

Purpose: We hypothesized that adenovirus-mediated soluble fms-like tyrosine kinase receptor (sFLT-1) gene therapy can inhibit the ovarian tumor growth and increase survival of mice in the context of ovarian carcinoma., Experimental Design: We constructed an infectivity-enhanced recombinant adenovirus (AdRGDGFPsFLT-1) expressing soluble FLT-1 and green fluorescent protein (GFP). An adenovirus AdRGDGFP expressing GFP alone was used as control. The functional validation of adenovirus-mediated sFLT-1 was determined by an in vitro human umbilical vein endothelial cell proliferation inhibition assay. To evaluate the therapeutic potential of adenovirus-expressed sFLT-1 to inhibit the growth of ovarian tumors and to increase the survival duration of mice with ovarian tumors, two tumor models were used. First, SKOV3.ip1 ovarian carcinoma cells were infected ex vivo with either AdRGDGFPsFLT-1 or AdRGDGFP or uninfected and then inoculated s.c. into BALB/c nude mice, and tumor growth was monitored. Second, SKOV3.ip1 cells were inoculated i.p. into CB17 SCID mice and then treated with two doses of either AdRGDGFPsFLT-1 or AdRGDGFP or with PBS on days 1 and 14 after inoculation of cells, and the survival duration was monitored., Results: Treatment with adenovirus-expressed sFLT-1 significantly inhibited the proliferation of human umbilical vein endothelial cells. The s.c. tumor nodules in mice derived from cells infected with AdRGDGFPsFLT-1 were significantly smaller than those infected with either AdRGDGFP or uninfected. In addition, i.p. administration of the AdRGDGFPsFLT-1 resulted in a significant increase in the survival times of mice compared with AdRGDGFP- or PBS-treated mice., Conclusions: Our results suggest that adenovirus-mediated sFLT-1 gene therapy can effectively inhibit ovarian tumor growth and increase survival in a murine model of ovarian carcinoma.

The use of adenoviruses for antivascular cancer gene therapy is limited by their low transduction efficiency for endothelial cells. We have developed a recombinant bispecific antibody as a molecular bridge, linking the adenovirus capsid to the endothelial cell surface protein endoglin, for vascular targeting of adenoviruses. Endoglin (CD105), a component of the transforming growth factor beta receptor complex, represents a promising target for antivascular cancer therapy. Endoglin is expressed predominantly on endothelial cells and is upregulated in angiogenic areas of tumors. We isolated single-chain Fv fragments directed against human endoglin from a human semisynthetic antibody library. One of the isolated scFv fragments (scFv C4) bound specifically to various proliferating primary endothelial cells or cell lines including HUVEC, HDMEC, HMVEC, and HMEC. ScFv C4 was therefore used to construct a bispecific single-chain diabody directed against endoglin and the adenovirus fiber knob domain (scDb EDG-Ad). This bispecific molecule mediated enhanced and selective adenovirus transduction of HUVECs, which was independent from binding to the coxsackievirus and adenovirus receptor (CAR) and alpha(v)-integrins. Thus, adenovirus infection was redirected to a new cellular receptor (CD105) and cell entry pathway. These results demonstrate the utility of bispecific single-chain diabodies, which can be produced in large quantities in bacteria, for the retargeting of adenoviruses in cancer gene therapy.

The anti-tumor drug Flavopiridol is a potent inhibitor of cyclin-dependent kinases (cdks). As a consequence, Flavopiridol-treated cells arrest in both G1 and G2, but Flavopiridol has also been shown to be cytotoxic for some tumor cell lines. The underlying molecular events are, however, unclear. We now show that Flavopiridol induces apoptosis in human umbilical vein endothelial cells (HUVECs), as judged by the occurrence of classical apoptotic markers, including chromatin condensation, internucleosomal cleavage, DNA fragmentation (TUNEL assay), annexin V binding and poly(ADP-ribose) polymerase (PARP)-cleavage. Such induction of apoptosis occurs with equal efficiency in both proliferating and G0/G1-arrested cells. Because growth-arrested HUVECs lack cdk2 activity and contain high levels of the cdk inhibitor p27, our observations suggest that cell cycle regulated cdks may not be the only critical target for Flavopiridol-induced apoptosis. Surprisingly, A549 lung carcinoma cells were clearly dependent on cell proliferation for the induction of cell death, pointing to cell type-related differences in the mechanism of Flavopiridol action.