Semyon Rubinchik

Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells

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/ARR2PB.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/ARR2PB.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. Gene Therapy (2001) 8, 1363–1371.

Acid ceramidase inhibition: a novel target for cancer therapy

During the last decade, sphingolipid deregulation, namely the balance between the pro-apoptotic molecule ceramide and the anti-apoptotic sphingolipid sphingosine-1-phosphate, has emerged as an important factor in cancer pathology and resistance to therapy. Thus, our research has been focused on developing drugs that are able to restore normal sphingolipid balance, precisely through increasing the levels of ceramide and decreasing sphingosine-1-phosphate. Particularly, inhibition of the ceramide metabolizing enzyme acid ceramidase, whose over-expression in cancer cells has been implicated in resistance to treatment, is proving to be an efficient and promising strategy. In this review, we consider our recent work with acid ceramidase inhibitors, in combination with radiation or gene therapy as a sensitizer that enhance cancer therapy.

Creation of a new transgene cloning site near the right ITR of Ad5 results in reduced enhancer interference with tissue-specific and regulatable promoters.

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.

Enhanced apoptosis of glioma cell lines is achieved by co-delivering FasL-GFP and TRAIL with a complex Ad5 vector

Brain tumors (BTs) are among the most malignant forms of human cancer. Unfortunately, current treatments are often ineffective and produce severe side effects. Cytotoxic gene therapy is an alternative treatment strategy, with the potential advantages of reduced toxicity to normal brain tissue. Apoptosis-inducing “death ligands” Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) are genes with substantial cytotoxic activity in susceptible tumor cells. Here, we compared the effectiveness of Ad vector-mediated delivery of Fas ligand-green fluorescent protein (FasL-GFP) fusion protein, human TRAIL, and both genes simultaneously. We examined a panel of 13 cell lines (eight derived from primary isolates) for susceptibility to Ad5-based vector infection and for sensitivity to FasL- and TRAIL-mediated apoptosis. All cell lines were efficiently transduced, but, as expected, varied in their sensitivity to ligand-induced apoptosis. Generally, sensitivity to FasL-GFP correlated with cell surface FasR levels, but no such correlation was seen for TRAIL and its functional receptors, DR4 and DR5. The vector expressing both FasL-GFP and TRAIL was more effective than either of the single-gene vectors at comparable transduction levels, and it was effective against a broader range of cell lines. In five cell lines, coexpression resulted in apoptosis levels greater than those predicted for strictly additive activity of the two death ligands. We believe that Ad vector-mediated delivery of multiple death ligands may be developed as a potential BT therapy, either alone or in conjunction with surgical resection of the primary tumor.

New complex Ad vectors incorporating both rtTA and tTS deliver tightly regulated transgene expression both in vitro and in vivo.

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.

Transformation, Translation and TRAIL: An unexpected intersection

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with roles in tumor surveillance and tolerance. TRAIL selectively induces apoptosis in many malignant but not normal cells but the underlying cause for spontaneous TRAIL sensitivity remains elusive. We propose a novel hypothesis that links TRAIL sensitivity to translational arrest following stresses that inactivate eukaryotic elongation factor 2 (EF2). Affected cells experience a reduction in apoptotic threshold because, due to their short half-lives, levels of anti-apoptotic proteins quickly drop off once translation elongation is inhibited leaving pro-apoptotic proteins unchallenged. This change in protein profile renders affected cells sensitive to TRAIL-mediated apoptosis and places EF2 into the role of a sensor for cellular damage.

Novel system uses probasin-based promoter, transcriptional silencers and amplification loop to induce high-level prostate expression

BackgroundDespite several effective treatment options available for prostate cancer, it remains the second leading cause of cancer death in American men. Thus, there is a great need for new treatments to improve outcomes. One such strategy is to eliminate cancer through the expression of cytotoxic genes specifically in prostate cells by gene therapy vectored delivery. To prevent systemic toxicity, tissue- and/or cancer-specific gene expression is required. However, the use of tissue- or cancer-specific promoters to target transgene expression has been hampered by their weak activity.ResultsTo address this issue, we have developed a regulation strategy that includes feedback amplification of gene expression along with a differentially suppressible tetracycline regulated expression system (DiSTRES). By differentially suppressing expression of the tetracycline-regulated transcriptional activator (tTA) and silencer (tTS) genes based on the cell origin, this leads to the activation and silencing of the TRE promoter, respectively. In vitro transduction of LNCaP cells with Ad/GFPDiSTRES lead to GFP expression levels that were over 30-fold higher than Ad/CMV-GFP. Furthermore, Ad/FasL-GFPDiSTRES demonstrated cytotoxic effects in prostate cancer cells known to be resistant to Fas-mediated apoptosis.ConclusionProstate-specific regulation from the DiSTRES system, therefore, serves as a promising new regulation strategy for future applications in the field of cancer gene therapy and gene therapy as a whole.

Highly specific transgene expression mediated by a complex adenovirus vector incorporating a prostate-specific amplification feedback loop

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.

1058. Novel Prostate-Specific Vector Regulation System Incorporating a Probasin-Based Promoter, Tet-Regulated Transcriptional Silencer Protein, and a Positive Feedback Loop

Prostate cancer is now the second leading cause of cancer death among men in the United States. Although current treatments are initially effective, 25-50% of patients experience recurrence within 15 years. In addition, once the disease has progressed to a hormone refractory stage, no curative treatment options are available. Thus, there is a great need for new treatment strategies to improve these outcomes. One such strategy is to specifically eliminate prostate cancer cells through induction of the natural pathway of programmed cell death known as apoptosis. This may be accomplished by introduction of a pro-apoptotic gene, such as Fas ligand (FasL) into prostate cancer cells using a recombinant adenovirus (Ad) vector. Previously, our lab constructed an Ad vector which employed the ARR2PB promoter (a synthetically derived promoter utilizing the androgen response regions (ARR) of the Probasin promoter) to drive prostate-specific expression of a FasL-green fluorescent protein (FasL-GFP) fusion protein specifically in prostate cancer cells. By combining this specificity with the Tet-off regulatory system within a single Ad vector, we were able to increase the levels of prostate-specific expression significantly. This strategy, however, introduced a new problem|[mdash]|low but significant basal activity emanating from the tetracycline responsive element (TRE) promoter even under non-induced conditions. To address this new challenge, we have included an additional element, a regulatory element that expresses the tetracycline-regulated transcriptional silencer (tTS) protein specifically in non-prostate cells. This is accomplished by introducing two expression cassettes: (1) Lac repressor (LacR) under the direction of the ARR2PB promoter and (2) tTS with a LacR-responsive promoter, derived by the introduction of two Lac operators (lacO) flanking the TATA box of the hCMVie (human Cytomegalovirus intermediate/early) promoter. In non-prostate cells, LacR would not be expressed but tTS would be and would subsequently suppress TRE-directed transgene expression. On the other hand, in prostate cells, LacR would be expressed and thus would inhibit tTS expression, thereby allowing TRE-induced transgene expression in prostate cells. By combining into a single Ad vector both TRE-regulated, prostate-specific induction of FasL-GFP expression and expression of tTS exclusively in non-prostate cells, we hope to increase the specificity of our vector. Preliminary results suggest that this new vector does restrict transgene expression to cells of prostate origin. In addition, this prostate-specific expression is amplified by the positive feedback loop system included in the vector design.