| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Purpose : Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies in humans. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced RCC. In this regard, gene therapy with adenoviral vectors is a promising new modality for cancer. However, a primary limiting factor for the use of adenoviral vectors for cancer gene therapy is critical dependence on cellular expression of the primary adenoviral receptor, the coxsackie and adenovirus receptor (CAR), known to be down-regulated in many cancer types. The addition of the histone deacetylase inhibitors (FR901228) after adenovirus infection is known to increase the expression of viral proteins and transgene expression. The objectives of this study are to evaluate the role of CAR in human renal cancer progression, and potential application of CAR on human renal cancer therapy.
Materials and Methods : The expression of CAR in RCC cell lines (AC
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HN, Caki-1, A498) was determined using fluorescence activated cell scanning (FACS). To examine the status of CAR mRNA associated with clinical specimens in 20 patients, total cellular RNA was prepared from normal and cancer tissue derived from the same patient and subjected to quantitative-reverse transcription polymerase chain reaction assay (RT PCR).
Cytotoxicity studies were performed to determine a minimally cytotoxic FR901228 concentration for RCC cell lines. The levels of CAR expression were determined by FACS and/or RT-PCR analysis of CAR in control and FR901228-treated RCC cell lines. The efficiency of adenovirus mediated transgene expression was also studied.
Result : Loss or decreased CAR expression is detected in RCC cell lines (ACHN : 12%, Caki-1 : 9%, A498 : 3%) using FACS. We observed that the loss or reduced expression of CAR levels is frequently detected in renal cancer tissue as compared with their normal tissue (p<0.01).
In addition, we found a stage-dependent decrease in CAR expression.
The drug concentration showing no or minimal cytotoxicity selected for these studies was 1 ng/ml FR901228 for RCC cells lines. Treatment of cancer cells with a low concentration (1 ng/ml) of the histone deacetylase inhibitor FR901228 increased CAR (ACHN : 68%, Caki-1 : 78%, A498 : 64%). This increase was associated with a 10 - 50 fold increase in adenovirus infection as evidenced by increased transgene expression from a beta-galactosidase containing adenoviral vector.
Conclusion : This is first detailed evaluation of the susceptibility of clinical RCC to adenoviral gene therapy. The down-regulation has a major impact on developing gene therapy modalities. We demonstrated that nontoxic doses of the histone deacetylase inhibitor FR901228, a histone deacetylase inhibitor, can result in marked increases in expression of CAR and RCC cells. This increase mediates enhanced transgene expression after adenovirus infection. These studies suggest a simple, clinically practical method for increasing the sensitivity of tumor cells to adenoviral gene therapy vectors. Less
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