ADENOVIRAL VECTOR FOR FHIT GENE TRANSFER

胰腺癌仍然是最难治疗的恶性肿瘤之一。它在美国与癌症相关的死亡原因中排名第四，1998年估计有29,000人死亡。完全手术切除是长期存活的唯一可能。尽管如此，所有外科可接受疾病阶段的5年总存活率只有21%。这种疾病起源的分子线索正在浮现。癌基因的激活和抑癌基因如p53、k-ras、p16、p21/WAF和FHIT的失活在胰腺癌的发生发展中起重要作用。在我们的研究中，我们将重点研究位于染色体3p14.2的脆性组氨酸三联体基因(FHIT)，该基因在大约66%的人类胰腺癌中受到影响。最近进一步阐明了3p15.2基因座的损伤性质，并将其作为肿瘤抑制基因的基础。1.1kb的FHIT基因转录本编码一种具有体外二核苷5‘，5“-P1，P3-三磷酸(Ap3A)水解酶活性的蛋白质(FHIT)。FHIT的肿瘤抑制活性被认为与FHIT的参与有关。Ap3A复合体中的细胞因子信号通路(S)控制着细胞的增殖，而FHIT似乎参与了一条不依赖于P53的细胞凋亡通路。为了阐明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，还需要实验数据。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增殖、凋亡和细胞周期进程的影响，以及对裸鼠致瘤性和肿瘤生长的影响。体外模型通过RT-PCR检测FHIT基因的表达水平，免疫印迹和免疫化学方法检测FHIT蛋白的表达，从而为腺病毒基因转移系统的治疗效果提供信息。为了评价细胞凋亡的诱导作用，我们将通过流式细胞仪检测G0/G1期细胞与对照细胞的表达水平。系统治疗对转导和非转导细胞体外肿瘤生长的抑制作用将通过四甲基偶氮唑盐比色法进行评价。通过观察瘤内注射Ad-FHIT与空载体对裸鼠皮下肿瘤生长的影响，评价其体内抑瘤效果。腺病毒已被广泛研究并安全地用于人类疫苗制备，Rad载体已被证明是有效的基因输送载体，因此，本研究的结果可能对人类胰腺癌的治疗具有重要意义。

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.