ADENOVIRAL VECTOR FOR FHIT GENE TRANSFER

用于 FHIT 基因转移的腺病毒载体

• 批准号: 6135030
• 负责人: KRISTOFFEL R DUMON
• 金额: $ 4.63万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6135030
• 关键词: Adenoviridae; athymic mouse; cell line; gene therapy; neoplasm /cancer therapy; nonhuman therapy evaluation; pancreas neoplasms; transfection /expression vector; tumor suppressor genes

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.

胰腺癌仍然是最难治疗的恶性肿瘤之一。 它在美国癌症相关死亡原因中排名第四，1998 年估计有 29,000 人死亡。完整的手术切除是长期生存的唯一可能。 然而，外科手术相关疾病的所有阶段的总体 5 年生存率仅为 21%。 关于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发挥着重要作用。 在我们的研究中，我们将重点关注位于染色体 3p14.2 的脆弱组氨酸三联体基因 (FHIT)，该基因在大约 66% 的人类胰腺癌中受到影响。 3p15.2 基因座损伤的性质最近已得到进一步阐明，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 (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。控制细胞增殖的细胞因子信号传导途径中的 Ap3A 复合物和 Fhit 似乎参与了 p53 独立的细胞凋亡途径。为了阐明Fhit作为胰腺癌肿瘤抑制因子的功能并探索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基因过表达对细胞增殖、凋亡和细胞周期的影响 胰腺癌细胞系中的过程以及裸鼠的致瘤性和肿瘤生长。体外模型将通过 RT-PCR 测量 Fhit cDNA 表达水平以及通过蛋白质印迹和免疫化学测量 Fhit 蛋白表达水平，为我们提供有关腺病毒基因转移系统治疗功效的信息。 为了评估细胞凋亡的诱导，我们将评估转染细胞的水平 通过 FACS 分析将 G0/G1 与对照细胞进行比较。 将通过MTT测试评估全身治疗对转导和非转导细胞的体外肿瘤生长抑制的效果。 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情况来评价体内肿瘤抑制效果。 腺病毒已被研究 广泛且安全地用于人类疫苗制剂，Rad载体已被证明是有效的基因传递载体，因此，这项研究的结果很可能对人类胰腺癌产生治疗意义。