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When NIH 3T3 fibroblasts were transduced with a retroviral vector containing a cDNA for porcine pancreatic elastase 1 and cultured in the presence of affinity-purified human plasminogen, the exogenously added plasminogen was digested to generate the kringle 1–3 segment known as angiostatin, a potent angiogenesis inhibitor. This was evidenced by immunoblot analysis of the plasminogen digests using a monoclonal antibody specifically reacting with the kringle 1–3 segment, and by efficient inhibition of proliferation of human umbilical vein endothelial cells by the plasminogen digests isolated from the culture medium of 3T3 fibroblasts. However, when Lewis lung carcinoma cells were transduced with the same vector and injected subcutaneously into mice in their back or via the tail vein, their growth at the injection sites or in the lungs was markedly suppressed compared with the growth of similarly treated nontransduced Lewis lung carcinoma cells. Nevertheless, the transduced cells were able to grow as avidly as the control cells in vitro. Assuming that the elastase 1 secreted from the transduced cells is likely to be exempt from rapid inhibition by its physiological inhibitor, α1-protease inhibitor, as shown in the inflammatory tissues, the elastase 1 secreted from the tumor cells may effectively digest the plasminogen that is abundantly present in the extravascular spaces and generate the kringle 1–3 segment in the vicinity of implanted tumor cell clusters. Although the selection of more profitable virus vectors and cells to be transduced awaits further studies, such a protease gene transfer strategy may provide us with a new approach to anti-angiogenesis gene therapy for malignant tumors and their metastasis in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Regions required for chicken glycine decarboxylase gene transcription were examined. A region between −82 and +22 (−82/+22) with motifs similar to binding sites for Sp1, NF-Y and CP2 was assigned to the proximal promoter active in both chicken hepatoma cell line, LMH, and hepatocytes in primary culture. In LMH cells, a genomic region, KX, between KpnI (−4155) and XbaI (−2113) sites changed promoter activity with the aid of four additional genomic regions termed upstream regulator regions for suppression (UpRS) and activation (UpRA) of transcription. Those precise segments are UpR1S (−376/−346), UpR1A (−345/−291), UpR2S (−137/−108) and UpR2A (−107/−83). Within KX, −4155/−3605 activates and −3604/−3367 suppresses the promoter. −3366/−3024 activates or suppresses the promoter, probably with different UpR counterparts. −2197/−2113 restores the actions of −3366/−3024. While in LMH cells, the upstream UpRs abrogate the functions of immediately downstream UpRs, UpR1S or UpR2S or both may be at least less active in hepatocytes than in LMH cells. Nuclear extracts from various chicken tissues and LMH cells had UpR2A binding proteins in different populations, suggesting that together with the UpRs, the segments in KX are involved in the regulation of cell type-specific transcription of this gene. [Copyright &y& Elsevier]

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.Molecular Therapy (2006) 13, 738–746; doi: 10.1016/j.ymthe.2005.11.010 [ABSTRACT FROM AUTHOR]

Background: The transduction of cancer cells using rAAV occurs with low efficiency, which limits its utility in gene therapy. Treatment with a histone deacetylase (HDAC) inhibitor is known to cause the gene expression from a silenced rAAV genome that has been integrated into the host's genome. However, rAAV exists mostly as an extrachromosomal genome rather than as an integrated genome. Here we show that HDAC inhibitors markedly enhance the rAAVmediated transduction of tumor cells in vitro as well as in vivo with improved therapeutic benefit. Our data also suggest that episomal AAV vector genome in the tumor cells is in the histone-associated chromatin form that is capable of superior transcription.Methods: Cell lines U251MG, 9L, HEp-2, HepG-2 and NKO-1 were transduced with the AAV1-5 vectors expressing eGFP gene at 1 × 10(4) genome copies/cell along with the HDAC inhibitor FK228. Effects on the expression of co-receptors for the AAV were also estimated by the FACS or Q-PCR. Copy number of the rAAV genome in the infected cells was estimated with Q-PCR analysis. Histone association of episomal AAV vector genome or proviral plasmid was characterized by using the chromatin immunoprecipitation (ChIP) assay. To analyze the effect of the FK228 on the transduction efficiency in vivo, U251MG cells were transduced with rAAV2Luciferase at 1 × 10(4) genome copies/cell, and then inoculated subcutaneously into the BALB/c mice along with the inraperitoneal injection of the FK228 at 1 mg/kg. Furthermore, 9L tumor cells were transduced with an AAV5HSV-tk and then inoculated subcutaneously into the BALB/c mice. The tumor- bearing animals received intraperitoneal injection of the FK228 and exposed to GCV for 14 days.Results: The FK228 treatment significantly improved the expression of the transgene in cancer cells in a dose-dependent manner. The highest enhancement was observed in the U-251MG cells with AAV2eGFP. The enhancement of the integrin level as well as coreceptors was limited. Copy number of the rAAV in the transduced cells was also modestly affected by the FK228 treatment. Interestingly, association of the acetylated histone H3 in episomal AAV vector genome was suggested by the ChIP assay. In the analysis using the optical bioluminescence imaging of the subcutaneous tumors, the luciferase expression in animals treated with the FK228 (n=5) was 37.4 fold higher than that in control animals (n=3). Estimation of the tumor growth in the athymic nude mice demonstrated that the combination of AAV-mediated transduction for HSV-tk/GCV therapy and FK228 treatment (n=10) resulted in significant improvement relative to HSV-tk/GCV therapy without FK228 treatment (P < 0.05, n=6).Conclusion: Our study suggests that the improved rAAV-mediated transduction induced by HDAC inhibitor might be related to proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. The use of the HDAC inhibitor would greatly enhance the utility of rAAV-mediated transduction strategies for future cancer gene therapy.Molecular Therapy (2006) 13, S45–S45; doi: 10.1016/j.ymthe.2006.08.129 [ABSTRACT FROM AUTHOR]