ADENOVIRAL VECTOR FOR FHIT GENE TRANSFER

胰腺癌仍然是最难治疗的恶性肿瘤之一。在美国，它是导致癌症相关死亡的第四大原因，1998年估计有29,000人死亡。完全手术切除是唯一可能的长期生存。然而，所有阶段手术上可接受的疾病的总体5年生存率仅为21%。这种疾病起源的分子线索正在出现。胰腺癌中癌基因的激活和抑癌基因（如p53、k-ras、p16、p21/WAF和FHIT）的失活在肿瘤发生中起着重要作用。在我们的研究中，我们将把重点放在位于染色体3p14.2的脆弱组氨酸三联体基因（FHIT）上，该基因在大约66%的人类胰腺癌中受影响。3p15.2位点损伤的性质最近得到了进一步的阐明，并奠定了FHIT作为肿瘤抑制基因的基础。1.1 kb的FHIT cDNA转录物编码一种具有体外二核苷5 ',5 ' - p1， P3-三磷酸（Ap3A）水解酶活性的蛋白（FHIT）。Fhit的肿瘤抑制活性被认为与Fhit的参与有关。控制细胞增殖和Fhit的细胞因子信号通路中的Ap3A复合物似乎参与了p53独立的凋亡通路。为了明确Fhit作为肿瘤抑制因子在胰腺癌中的作用，探索Fhit基因转移在胰腺癌中的潜在治疗作用，需要实验数据。我们将研究重组腺病毒载体（rAd-FHIT）介导的FHIT基因过表达对胰腺癌细胞系细胞增殖、凋亡和细胞周期过程的影响，以及对裸鼠肿瘤致瘤性和肿瘤生长的影响。通过RT-PCR检测Fhit cDNA的表达水平，Western blot和免疫化学检测Fhit蛋白的表达水平，体外模型将为腺病毒基因转移系统的治疗效果提供信息。为了评估细胞凋亡的诱导作用，我们将通过FACS分析来评估G0/G1转染细胞与对照细胞的水平。全身治疗对转导和非转导细胞体外肿瘤生长抑制的影响将通过MTT试验进行评估。通过观察裸鼠瘤内注射Ad-FHIT与空载体的皮下肿瘤生长情况，来评价其体内抑瘤效果。腺病毒已被广泛研究并安全用于人类疫苗制备，Rad载体已被证明是有效的基因传递载体，因此，本研究的结果很可能导致人类胰腺癌的治疗意义。

Pancreatic cancer remains one of the most difficult malignancies to treat. It ranks 4th as a cause of cancer related death in USA and accounted for an estimated 29,000 deaths in 1998. Complete surgical resection offers the only potential for long term survival. Nevertheless, the overall 5 year survival for all stages of surgically respectable disease is only 21 percent. The molecular clues to the origin of this disease are emerging. Activation of oncogenes and inactivation of tumor suppressor genes such as p53, k-ras, p16, p21/WAF and FHIT in pancreatic cancer have been shown to play an important role in tumorigenesis. In our study we will focus on the fragile histidine triade gene (FHIT) located at chromosome 3p14.2 and affected in approximately 66 percent of human pancreatic cancer. The nature of damages to the 3p15.2 locus has recently been further elucidated and underlie FHIT as a tumor suppressor gene. The 1.1-kb FHIT cDNA transcript encodes a protein (Fhit) with in vitro dinucleoside 5`,5"-P1, P3- triphosphate (Ap3A) hydrolase activity. Tumor suppressor activity of Fhit is assumed to be associated with involvement of the Fhit. Ap3A complex in cytokine signaling pathways(s) controlling cell proliferation and Fhit seems to be involved in a p53 independent apoptotic pathways. In order to clarify the function of Fhit as a tumor suppressor in pancreatic cancer and to explore the potential therapeutic role of FHIT gene transfer in pancreatic cancer, experimental data are needed. We will study the effects of FHIT gene overexpression mediated by a recombinant adenoviral vector (rAd-FHIT) on cell proliferation, apoptosis and cell-cycle processes in pancreatic cancer cell lines and on tumorigenicity and tumor growth in nude mice. The in vitro model will give us information about the therapeutic efficacy of the Adenoviral gene transfer system by measuring the levels of Fhit cDNA expression by RT-PCR and Fhit protein expression by Western blot and immunochemistry. To evaluate the induction of apoptosis, we will evaluate the level of transfected cells in G0/G1 compared to control cells by FACS analysis. The effect of systemic therapy on in vitro tumor growth inhibition in transduced and nontransduced cells will be evaluated by the MTT test. The in vivo effect on tumor suppression will be evaluated by observing the subcutaneous tumor growth in nude mice injected intratumorally with Ad-FHIT compare to the empty vector. Adenoviruses have been studied extensively and used safely for human vaccine preparations and Rad vectors have shown to be effective gene delivery vehicles, therefore, the results of this study may well result in therapeutic implications for human pancreatic cancer.