MP45-08 STABILIZATION OF INVADOPODIA BY PLECTIN- MEDIATED CONJUNCTION TO VIMENTIN INTERMEDIATE FILAMENT IS A CRITICAL MOLECULAR STEP OF INVASION AND EXTRAVASATION FOR METASTASIS IN BLADDER CANCER.

INTRODUCTION AND OBJECTIVES: The two-step transcriptional amplification (TSTA) system was previously reported to enhance the tissue-specific gene expression driven by weak promoters, but the enhancement of the gene expression is limited to use in in vitro and in vivo experimental situations. METHODS: To achieve robust tissue-specific gene expression using the TSTA system, we developed an advanced TSTA system which includes polyglutamines and rat glucocorticoid receptor (GR) sequences between the GAL4 and VP16 sequences in the region of the first step of transcription. We herein evaluated this advanced TSTA system as a method to enhance the human telomerase reverse transcriptase (hTERT) promoter-driving cancer-specific transcription in various cancer cells. RESULTS: We constructed a luciferase gene encoding plasmid vector, where hTERT promoter-driving transcription is enhanced by the advanced TSTA (advanced TSTA-hTERT-Luc). The advanced TSTA enhanced cancer-specific luciferase gene expression in all of the examined 18 cancer cell lines. We then constructed a green fluorescent protein (GFP) gene encoding plasmid vector with the hTERT promoter and advanced TSTA, to utilize the system for the detection of viable bladder cancer cells. The advanced TSTA-hTERTGFP plasmid successfully induced cancer-specific gene expression, showing robust GFP expression in human bladder cancer cell lines, but no visible GFP expression in normal bladder urothelial cells. The advanced TSTA-hTERT-GFP plasmid allowed selective visualization of viable human bladder cancer cells in mixed cell culture containing 10and 100-fold more normal bladder urothelial cells. These findings indicate that the advanced TSTA-hTERT expressional system is a valuable tool for detecting viable bladder cancer cells. To further demonstrate the utility of the advanced TSTA system for cancerspecific gene expression and imaging, we constructed a luciferase gene encoding adeno-associated virus (AAV) vector in which the hTERT promoter-mediated expression was driven by the advanced TSTA systems. In an orthotopic liver tumor model, mice were treated with the vector via tail vein injection. An optical imaging device was used to visualize the in vivo luciferase expression in the orthotopic tumor. The advanced TSTA system significantly enhanced the luciferase expression compared with the one-step and conventional TSTA systems. CONCLUSIONS: This system can be applied for not only in vitro detection of the bladder and other types of cancer cells but also for the tumor imaging in vivo. Source of Funding: none