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ATF-2 and p300 cooperate in the activation of transcription of the c-jun gene during the differentiation of F9 cells. We show here that a repressor of AP-1, JDP2 (Jun dimerization protein 2), inhibits the transactivation of the c-jun gene by ATF-2 and p300, by recruitment of a histone deacetylase complex, thereby repressing the retinoic acid (RA)-induced transcription of the c-jun gene and inhibiting the RA-mediated differentiation of F9 cells. Furthermore, JDP2/HDAC complex was replaced by p300 complex on the DRE (differentiation response element) of the c-jun promoter within 24 h after the start of exposure of cells to RA, with an accompanying change in the histone-acetylation status of the chromatin, indicating that the initiation of transcription of the c-jun gene was controled by sequential action of HDAC(s) and HAT(s) on c-jun promoter.

The DNA methylation plays a key role in the regulation of gene expression, genotnic imprinting and X chromosome inactivation and has been shown to be essential for mammalian development. The Dnmtl is one of the DNA methyltransferases that catalyzed DNA methylation on CpG dinucleotides. The Dnmtl is constitutively expressed and is required for the maintenance of global methylation after DNA replication. In this study, we investigated the effects of histone deacetylase (HDAC) inhibitor and DNA demethylation agent on promoter activity of mouse Dnmtl gene in somatic cells. The promoter activity of Dnmtl gene was increased approximately 2-fold in the treatment of cells by Trichostatin A (TSA) at lx 10"* M, as compared with that without of treatment of TSA. By contrast, treatment with 5-azacytidine (5aza-C) did not affect the promoter activity of the Dnmtl gene. These results indicate that the Dnmtl gene is possibly to regulated by histone acetylation.

Metastasis, a poor prognostic indicator causing increased mortality, occurs in an organ specific pattern with lungs and lymph nodes most commonly involved. Cancer cells expressing ‘CXC'-chemokine family Receptor-4 (CXCR4) are chemo-attracted to the pulmonary tissue expressing its cognate ligand Stromal Derived Factor-1 (SDF-1), which aid in formation of metastasis. In this regard, we propose to modulate this axis through knock down of SDF-1 specifically in pulmonary vasculature to prevent lung metastasis. Our laboratory has previously shown that human adenovirus (Ad), when injected via the tail vein into human Coxsackie Adenovirus Receptor (hCAR) transgenic mice, mice that ubiquitously express the hCAR native Ad receptor, transduces pulmonary vasculature preferentially. To this end, we plan to knock- down SDF-1 using shRNA against SDF-1 employing Ad as the vector to deliver them in vivo to achieve pulmonary targeting. To establish a pulmonary metastatic model in the “hCAR” mouse, we injected B16-F10 melanoma cells stably expressing CXCR4 (to cause metastasis) and firefly luciferase (to track tumor cells) via tail vein. Presence of lung metastasis was confirmed by histopathological analysis, immunohistochemistry for CXCR4 and luciferase assay of the tissues. In order to identify a shRNA to deliver to the pulmonary vasculature to decrease SDF-1, we evaluated several plasmid shRNAs for their ability to suppress SDF-1 expression at the mRNA level using qRT-PCR in vitro. From this experiment, we identified one shRNA which gave significant decrease in mRNA of SDF-1 measured by qRT-PCR (>70%). We created an Ad vector expressing this particular shRNA and measured its efficiency of inhibiting SDF-1 expression of approximately 75% for mRNA by qRT-PCR and approximately 90% for protein by Western Blot. Future studies include demonstrating the functional knock-down of SDF-1 in vitro by transwell chamber assay and subsequently analyzing the biological effect of knocking down SDF-1 in vivo in the pulmonary metastatic model established in “hCAR” mouse. In conclusion, therapies available for prevention of metastasis are limited despite different treatment modalities available for cancer. Our approach of targeted knock down of SDF-1 would reduce the metastatic burden to the lungs with potential for clinical translation into a therapeutic approach to prevent lung metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1508.

Viral vectors have been developed as therapeutic agents for the introduction of exogenous genes into living cells ([1][1]), and clinical trials of gene therapy and the use of viral vectors in the laboratory have been reported with increasing frequency during the past decade ([2][2]–[4][3]). We

Human adenovirus serotype 5 (HAdV-5) attaches to its primary receptor, the coxsackie and adenovirus receptor (CAR) as the first step of infection. However, CAR expression decreases as tumors progress, thereby diminishing the utility of HAdV-5-based vectors for cancer therapy. In contrast, many aggressive tumor cells highly express CD46, a cellular receptor for HAdV-3. We hypothesized that a mosaic HAdV vector, containing two kinds of fiber proteins, would provide extensive transduction in a heterogeneous population of tumor cells with varying expression levels of HAdV receptors. We therefore generated a fiber-mosaic HAdV vector displaying both a chimeric HAdV-3 fiber and the HAdV-5 fiber protein. We verified the structural integrity of purified viral particles and confirmed that the fiber-mosaic HAdV vector has expanded tropism. We conclude that the use of fiber-mosaic HAdV vectors is a promising approach for transducing a heterogeneous cell population with different expression levels of adenovirus receptors.

Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout.