| Recent progress in vector development has made gene therapy a promising approach to cancer therapy. Transduced genes may be used to block activated oncogene expression, to express tumor supressor genes, to protect normal stem cells from high dose chemotherapy, or to introduce "suicide" gene into tumor cells. Suicide gene therapy is a new experimental form of cancer chemotherapy that is currently being evaluated in human trials. Cytosine deaminase (CD) is an enzyme found in a variety of bacteria and fungi where is capable of deaminating cytosine to uracil. CD is also able to deaminating 5-fluorocytosine (5-FC) to 5-fluorouracil(5-FU). The existence of CD in a prokaryotic cell renders the cell selectively sensitive to 5-FC. Selective expression of CD within neoplastic cells when combined with systemic 5FC administration may result in locally high concentration of 5-FU while minimizing systemic 5-FU toxicity. Pancreatic adenocarcinoma tends to be advanced at the time of presentation, and 70 ~ 90% of patients at the time of diagnosis have inoperable disease. 5-Fluorouracil(5-FU) is the agent most commonly used, alone or with radiotherapy, in the adjuvant treatment of resectable pancreatic cancer and in the palliation of unresectable pancreatic cancer. Unfortunately, the efficacy of 5-FU is limited by its narrow therapeutic index. The requirement for more effective strategies to treat pancreatic cancer is manifested by the present dismal clinical outcome of this disease. Prior efforts using retroviruses require tranduction of tumor cells in vitro or intratumoral inoculation of packaging cells. Compared to retroviruses, recombinated adenoviruses have high viral titers, high transfection efficiencies in vivo, and apparent safety in clinical trials This study evaluated the transfer of the CD gene into human pancreatic tumor cells utilizing an adenovirus vector and tested the efficacy of the CD/5-FC suicide gene in the treatment of pancreatic cancer.1．Constructions of recombinant adenovirusesThe 1.5kb CD gene was removed from pG1NaCEA-CD by a BamH I digestion and cloned into the pTrack-CMV with ligase, generating pAdTrack-CMV-CD. pTrack-CMV is a shuttle vector that contains CMV promoter, a multiple cloning site for insertion of genes, and green fluorescent protein(GFP). pAdTrack-CMV-CD and pAdEasy-1 were homologous recombinated in bacteria BJ5183, the newly-recombinated plasmid pAd-CD were tested by restriction endonulcease digestions. The pAd-CD was linearized with Pac I and transferred into 293 cells to form Ad-CD. After six cycles of freezing and thawing, cell debris was removed by centrifugation, then virus supernate was purified by cesium chloride gradient centrifugation. Concentrated virus was titered as 2×1011pfu/ml by TCID50(Tissue Culture Infectious Dose 50).2. Cell lines and adenovirus infection in vitroAIM: To evaluate in vitro killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic carcinoma cell lines.Methods: In vitro gene transduction: PaTu8988,SW1990, two human pancreatic carcinoma cell lines were obtained from Dr.Elsasser (Phillips university of German), Cells were plated 24h prior to infection, then infected with virus at a MOI of 100 for 1h at 37℃,followed by the addition of growth media. 24h later, GFP expression in pancreatic carcinoma cells was detected by fluorescence microscopy and flow cytometer. Then 5-FC was added. Cellular proliferation was measured by XTT assay.RT-PCR analysis: Total mRNA was prepared from pancreatic carcinoma cell lines infected with Ad-CD\Ad-Null and parental cells. Reverse transcription was performed starting from 2(g of total RNA, using oligo(dT)primer, other reagents, and procedures contained in the MmuLV RT-PCR Kit. Each primer, 1(l dNTPs, and 1 (l Taq DNA polymerase were used for PCR. The amplification was obtained with 30 cycles at 94℃,52℃,and 72℃ and with a final extension of 5 min at 72℃.Western blot analysis: Total protein was prepared from pancreatic carcinoma cell lines infected...
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