Despite local and systemic therapies, the National Cancer Institute estimates that prostate cancer will cause over 30,000 deaths in 2006. This suggests that additional therapeutic approaches are ...needed. The chicken anemia viral protein Apoptin causes tumor-selective apoptosis in human tumor lines independent of p53 and Bcl-2 status. Tet-regulated expression of Apoptin from an adenoviral vector showed cytotoxicity in DU145, PC-3, and LNCaP tumor cells regardless of expression of p53, Bcl-2, Bcl-xL, Bax, survivin, FLIP(S), XIAP, or CIAP. Apoptin expression caused an increase in the tumor suppressor lipid ceramide, which regulates the cellular stress response. Interestingly, 10 of 15 primary prostate cancers examined by Western blotting overexpressed acid ceramidase (AC), suggesting that ceramide deacylation might serve to negate elevated levels of ceramide, creating a more antiapoptotic phenotype. This was confirmed in AC-overexpressing cells in which we observed decreased sensitivity to apoptosis following treatment with Apoptin. Addition of the AC inhibitor LCL204, in combination with Apoptin, augmented cell killing. This effect was also demonstrated in vivo in that Apoptin and LCL204 cotreatment significantly reduced tumor growth in DU145 xenografts (P<0.05). Taken together, our data demonstrated that Apoptin is a promising therapeutic agent for prostate cancer and that its function is improved when combined with acid ceramidase inhibitors.
Adhesion to host cells is an important step in pathogenesis of Campylobacter jejuni, which is the most prevalent bacterial cause of human gastroenteritis worldwide. In contrast to other bacteria such ...as E. coli and Salmonella, adherence of C. jejuni is not mediated by fimbria or pili. A number of C. jejuni adhesion-related factors have been described. However, the results obtained by different researchers in different laboratories are often contradictory and inconclusive, with only some of the factors described being confirmed as true adhesins. In this review, we present the current state of studies on the mechanisms of attachment of C. jejuni to host cells.
As of January 2005, there were 1020 gene therapy clinical trials ongoing worldwide with 675 or 66.2% devoted to cancer gene therapy. The majority are occurring in the US and Europe ...(http://www.wiley.co.uk/genetherapy/clinical/). At the present time, to our knowledge there are no trials that employ gene delivery of Fas Ligand (FasL). As an important note, and in contrast to somatic cell therapy trials, there are no reported deaths due to therapeutic vector administration in any cancer gene therapy trial. That said, from our studies and from the published literature, the issue of gene delivery remains the major obstacle to successfully employing gene therapy for cancer treatment. Numerous laboratories are studying this with many different approaches. My co-workers and I have focused on the delivery issue by using various approaches that address tumor targeting and transgene expression. In addition, we are focusing on enhancing tumor cell killing via the bystander effect and through use of small molecules to enhance bystander activity.
In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of ...Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR2PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression of Bax in prostate carcinoma, even in the context of high levels of Bcl-2 protein, resulted in apoptosis. These results suggest that a second-generation adenovirus-mediated, prostate-specific Bax gene therapy is a promising approach for the treatment of prostate cancer.
Fas ligand (FasL) is a member of the tumor necrosis family and when bound to its receptor, Fas, induces apoptosis. It plays important roles in immune response, degenerative and lymphoproliferative ...diseases, development and tumorigenesis. It is also involved in generation of immune privilege sites in the eye and testis. Harnessing the power of this molecule is expected to lead to a powerful chemotherapeutic. We describe the construction and characterization of replication-deficient adenoviral vectors that express a fusion of murine FasL and green fluorescent protein (GFP). FasL-GFP retains full activity of wild-type FasL, at the same time allowing for easy visualization and quantification in both living and fixed cells. The fusion protein is under the control of a tetracycline-regulated gene expression system. Tight control of expression is achieved by creating a novel 'double recombinant' Ad vector, in which the tet-responsive element and the transactivator element are built into the opposite ends of the same vector to avoid enhancer interference. Expression can be conveniently regulated by tetracycline or its derivatives in a dose-dependent manner. The vector was able to deliver FasL-GFP gene to cells in vitro efficiently, and the expression level and function of the fusion protein was modulated by the concentration of doxycycline. This regulation allows us to produce high titers of the vector by inhibiting FasL expression in an apoptosis-resistant cell line. Induction of apoptosis was demonstrated in all cell lines tested. These results indicate that our vector is a potentially valuable tool for FasL-based gene therapy of cancer and for the study of FasL/Fas-mediated apoptosis and immune privilege.
Development of novel therapeutic agents is needed to address the problems of locally recurrent, metastatic, and advanced hormone-refractory prostate cancer. We have constructed a novel complex ...adenovirus (Ad) vector regulation system that incorporates both the prostate-specific ARR2PB promoter and a positive feedback loop using the TRE promoter to enhance gene expression. This regulation strategy involves the incorporation of the TRE upstream of the prostate-specific ARR2PB promoter to enhance its activity with Tet regulation. The expressions of both GFP and tTA were placed under the control of these TRE-ARR2PB promoters, so that in the cells of prostate origin a positive feedback loop would be generated. This design greatly enhanced GFP reporter expression in prostate cancer cells, while retaining tight control of expression in nonprostate cancer cells, even at an MOI as high as 1000. This novel positive feedback loop with prostate specificity (PFLPS) regulation system we have developed may have broad applications for expressing not only high levels of toxic proteins in cancer cells, but alternatively could also be manipulated to regulate essential genes in a highly efficient conditionally replicative adenovirus vector specifically directed to prostate cancer cells. The PFLPS regulation system, therefore, serves as a promising new approach in the development of both a specific and effective vector for cancer gene therapy.
Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand ...(FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.
Tissue-specific transgene expression is a valuable research tool and is of great importance in delivering toxic gene products with adenovirus vectors to tumors. Limiting cytotoxic gene expression to ...the target cells is highly desirable. While a number of successful applications of tissue- and tumor-specific gene expression using Ad vectors has been reported, cloning of some promoters into Ad vectors resulted in modulation or loss of tissue specificity. This phenomenon is likely the result of the interaction of E1A enhancer (and possibly other Ad sequences) with the promoter cloned in the E1 region. We have compared performance parameters of prostate-specific and tet-regulatable promoters in plasmids containing the terminal repeat sequences of Ad5 with or without the E1A enhancer. Subsequently, adenoviral vectors were constructed containing identical expression units either in the E1 region or near the right ITR, and tested in several cell lines. Here, we report that promoters placed near the right ITR of Ad5 retain higher selectivity and lower background expression in both plasmid and adenovirus vectors. We confirm that the E1A enhancer can interfere with the desired activity of nearby promoters, and describe an alternative transgene insertion site for construction of Ad vectors.
Prostate cancer is the second leading cause of cancer death in the United States. Treatment options for confined disease are generally successful in prolonging life but long-term cures (10-15 years) ...are elusive for the majority of patients. The prognosis for advanced extra-capsular prostate cancer is grim. However, we are now entering the era of gene therapy options for treatment of prostate cancer. The human genome project coupled with genomics and protemics are providing information that will lead to selection of genes for treatment of prostate cancer. The problem is the science of delivery lags behind knowledge of gene function. Thus, it is important to develop therapies that do not require delivery to 100% of tumor cells but which nevertheless kills the entire cancer by virtue of the bystander effect or other means. This review covers the use, in gene therapy, of apoptotic inducing molecules such as Fas Ligand, and TRAIL which are believed to induce bystander killing activity and Bax which also may function in a similar way.
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