Wm Gommans

Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation

BACKGROUND/AIMS To examine the extent and mechanisms of apoptosis in cholestatic liver injury and to explore the role of the transcription factor nuclear factor-kappa B in protection against bile acid-induced apoptosis. METHODS Cholestatic liver injury was induced by bile duct ligation in Wistar rats. Furthermore, primary cultures of rat hepatocytes were exposed to glycochenodeoxycholic acid (GCDCA), tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA) and to cytokines. Apoptosis was determined by TUNEL-staining, active caspase-3 staining, activation of caspase-8, -9 and -3. RESULTS Limited hepatocyte apoptosis and an increased expression of NF-kappaB-regulated anti-apoptotic genes A1 and cIAP2 were detected in cholestatic rat livers. Bcl-2 expression was restricted to bile duct epithelium. In contrast to TCDCA and TUDCA, GCDCA induced apoptosis in a Fas-associated protein with death domain (FADD)-independent pathway in hepatocytes. Although bile acids do not activate NF-kappaB, NF-kappaB activation by cytokines (induced during cholestasis) protected against GCDCA-induced apoptosis in vitro by upregulating A1 and cIAP2. CONCLUSIONS GCDCA induces apoptosis in a mitochondria-controlled pathway in which caspase-8 is activated in a FADD-independent manner. However, bile acid-induced apoptosis in cholestasis is limited. This could be explained by cytokine-induced activation of NF-kappaB-regulated anti-apoptotic genes like A1 and cIAP2.

An ex vivo human model system to evaluate specificity of replicating and non-replicating gene therapy agents

Inefficiency, aspecificity and toxicity of gene transfer vectors hamper gene therapy from showing its full potential. On this basis significant research currently focuses on developing vectors with improved infection and/or expression profiles. Screening assays with validity to the clinical context to determine improved characteristics of such agents are not readily available since this requires a close relationship to the human situation. We present a clinically relevant tissue slice technology to preclinically test improved vector characteristics.

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment

Zinc finger protein transcription factors (ZFP‐TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA‐binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene‐specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA‐binding motifs. In this study, we engineered ZFP‐TFs to downregulate the activity of the epithelial glycoprotein‐2 (EGP‐2) promoter. The protein EGP‐2 is overexpressed in a wide variety of cancer types and EGP‐2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP‐2 expression by ZFP‐TFs provides a novel anti‐cancer therapeutic. Using a straightforward strategy, we engineered a 3‐ZFP that could bind a 9 bp sequence within the EGP‐2 promoter. After the addition of a repressor domain, this 3‐ZFP‐TF could efficiently downregulate EGP‐2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP‐2 promoter activity. To inhibit the endogenous EGP‐2 promoter, we engineered 6‐ZFP‐TFs. Although none of the constructed ZFP‐TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP‐TFs are versatile bi‐directional modulators of gene expression and downregulation of EGP‐2 promoter activity using ZFP‐TFs can ultimately result in a novel anti‐cancer treatment.

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.

314. The Epithelial Glycoprotein-2 Promoter for Highly Specific and Efficient Cancer Gene Therapy for a Broad Range of Tumors

Top of pageAbstract Adenoviral cancer gene therapy is hampered by the lack of specific and efficient transgene expression in tumor cells. Employing tissue specific promoters to restrict transgene expression will increase specificity towards the tumor tissue. In addition, specific promoters can be used to increase the efficiency of adenoviral gene therapy by controlling adenoviral replication. However, many specific promoters are restricted to specific tumor types. The tumor specific promoter epithelial glycoprotein-2 (EGP-2) is known to be active in various epithelial-derived cancers including ovarian, colon, and breast cancer and might therefore be a good candidate to target a broad range of tumor types. In this study, the EGP-2 promoter was employed to improve the therapeutic potential of adenoviral cancer gene therapy. The EGP-2 promoter demonstrated an outstanding specificity and activity profile in vivo. In mouse liver tissue, 3 log lower activity compared to the strong constitutively active CMV promoter was shown compared with only 1 log lower activity in EGP-2 positive tumor tissue. Subsequently, the EGP-2 promoter was employed to control the expression of the suicide gene thymidine kinase (AdEGP-2 SR39TK). In vitro selective cell killing after infection with AdEGP-2 SR39TK and GCV administration was seen in EGP-2 positive cell lines, but not in EGP-2 negative cells. After iv injection of AdEGP-2 SR39TK in mice an absence of liver toxicity was demonstrated after administration of GCV, in contrast to AdCMV SR39TK which showed clear liver damage. These promising findings are currently being explored in tumor bearing mice. To successfully treat cancer, all tumor cells have to be eradicated. However, even after local administration adenoviral vectors will infect only a limited amount of tumor cells. Therefore, to increase the efficiency of the adenovirus the EGP-2 promoter was utilized to control the E1A gene, which is essential for adenoviral replication. This conditionally replicating adenovirus (CRAd) should replicate selectively in EGP-2 positive tumor cells, causing cell lysis and spread into the tumor mass. This cycle of selective replication and cell lysis is continued until no further tumor cells can be infected. The EGP-2 based CRAd was as efficient in cell killing as wild-type adenovirus in EGP-2 positive cell lines. In contrast, in EGP-2 negative cell lines replication of the EGP-2 based CRAd was attenuated up to 2 log when compared with the wild-type adenovirus. Currently, the EGP-2 based CRAd is further investigated in tumor bearing mice. Overall, it can be concluded that the EGP-2 promoter demonstrates high specificity and efficiency in adenoviral context and can therefore be a powerful tool in cancer gene therapy for the treatment of a broad range of tumor types.