ADENOVIRAL VECTOR FOR FHIT GENE TRANSFER

胰腺癌仍然是最难治疗的恶性肿瘤之一，在美国排在癌症相关死亡原因的第四位，1998年估计有29，000人死于胰腺癌。 完整的手术切除提供了长期生存的唯一可能性。 尽管如此，手术治疗的所有阶段的总体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重要作用。 在我们的研究中，我们将集中在脆性组氨酸三联体基因（FHIT）位于染色体3p14.2和影响约66%的人胰腺癌。 3p15.2位点的损伤性质最近得到进一步阐明，并成为FHIT作为肿瘤抑制基因的基础。 1.1-kb FHIT cDNA转录物编码具有体外二核苷5 '，5”-P1，P3-三磷酸（Ap 3A）水解酶活性的蛋白质（Fhit）。 Fhit的肿瘤抑制活性被认为与Fhit的参与有关。 Ap 3A复合物在控制细胞增殖和Fhit的细胞因子信号传导途径中似乎参与p53非依赖性凋亡途径。为了阐明FHIT在胰腺癌中作为抑癌基因的作用，并探讨FHIT基因转移在胰腺癌中的潜在治疗作用，需要实验数据。 我们将研究由重组腺病毒载体（rAd-FHIT）介导的FHIT基因过表达对胰腺癌细胞系的细胞增殖、凋亡和细胞周期过程以及对裸鼠成瘤性和肿瘤生长的影响。体外模型将通过RT-PCR测量Fhit cDNA表达水平和通过Western印迹和免疫化学测量Fhit蛋白表达水平，为我们提供关于腺病毒基因转移系统的治疗功效的信息。 为了评价细胞凋亡的诱导，我们将通过FACS分析评价G 0/G1期转染细胞与对照细胞相比的水平。 将通过MTT试验评价全身治疗对转导和非转导细胞中体外肿瘤生长抑制的影响。 通过观察瘤内注射Ad-FHIT的裸鼠与空载体相比的皮下肿瘤生长来评价对肿瘤抑制的体内作用。 腺病毒已被广泛研究，并安全地用于人类疫苗制剂和Rad载体已被证明是有效的基因递送载体，因此，本研究的结果可能会导致对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.