GEMZAR--MECHANISMS OF CYTOTOXICITY & RADIOSENSITIZATION

吉西他滨是一种新的核苷类似物，在几种实体肿瘤类型中具有临床活性，包括胰腺癌和非小细胞肺癌。虽然吉西他滨作为单一药物或与其他化疗药物联合在这些肿瘤类型的患者中显示出良好的活性，但我们最近证明，吉西他滨也增强了实体肿瘤细胞对电离辐射的敏感性。临床前研究表明，吉西他滨对胰腺癌、结直肠癌、头颈癌、乳腺癌和胶质母细胞瘤等人类肿瘤细胞具有放射致敏作用。将这些研究转化为不可切除头颈癌患者的I期试验表明，吉西他滨也可以在体内使肿瘤组织敏感，大多数患者对治疗有完全反应。有了这些令人鼓舞的结果，我们现在建议在体外和体内扩展这些研究，以确定吉西他滨放射致敏肿瘤细胞的机制，并确定它是否与细胞毒性机制不同。这些研究将在人类头颈部癌细胞中进行，基于我们令人鼓舞的初步I期结果，我们还将评估人类胶质母细胞瘤细胞对体外吉西他滨放射增敏的反应。初步数据表明，吉西他滨的放射增敏需要dATP的大幅降低，这是由于对核糖核苷酸还原酶的抑制，而不受三磷酸吉西他滨的量或DNA中药物的量的影响。此外，新的数据表明，放射增敏在表达野生型p53的细胞系中不太成功。本文提出的研究将评估DNA中dATP缺失和吉西他滨核苷酸在细胞毒性和放射致敏中的作用。将使用匹配的野生型和突变型p53细胞系评估p53的效果，并使用人乳头瘤病毒的E6蛋白消除wt p53。这些研究将在裸鼠体内和体外对这些细胞系进行。此外，在手术切除肿瘤前给患者输注吉西他滨，可以测量吉西他滨代谢、吉西他滨磷酸化、对核糖核苷酸还原酶的影响和p53状态所需的关键蛋白质。这些研究将弥合体外和体内研究之间的差距，并有助于优化吉西他滨放射增敏治疗。

Gemcitabine is a new nucleoside analog with clinial activity in several solid tumor types, including pancreatic and non-small cell lung cancer. While gemcitabine has shown good activity as a single agent or in combination with other chemotherapeutic agents in patients with these tumor types, we have demonstrated recently that gemcitabine also enhances the sensitivity of solid tumor cells to ionizing radiation. Preclinical studies demonstrate that gemcitabine can radiosensitize human tumor cells derived from pancreatic cancer, colorectal carcinoma, head and neck cancer, breast cancer and glioblastoma. Translation of these studies to a Phase I trial in patients with unresectable head and neck cancer demonstrated that gemcitabine can sensitize tumor tissue in vivo as well, with most patients achieving a complete response to treatment. With these encouraging results, we now propose to extend these studies both in vitro and in vivo to determine the mechanism by which gemcitabine radiosensitizes tumor cells and determine whether it is distinct from the mechanism of cytotoxicity. These studies will be performed in human head and neck cancer cells, based on our encouraging preliminary Phase I results, and we will also evaluate response of human glioblastoma cells to radiosensitization with gemcitabine based radiosensitization in vitro. Preliminary data suggests that radiosensitization with gemcitabine requires a substantial decrease in dATP, due to inhibition of ribonucleotide reductase, and not affected by the amount of gemcitabine triphosphate or the amount of drug in DNA. Furthermore, new data indicates that radiosensitization is less successful in cell lines that express wild-type p53. The studies proposed here will evaluate the roles of dATP depletion and gemcitabine nucleotide in DNA in cytotoxicity versus radiosensitization. The effect of p53 will be assessed using matched wild type and mutant p53 cell lines, as well as eliminating wt p53 using the E6 protein of human papillomavirus. These studies will be performed these cell lines in vitro and in vivo in nude mice. In addition, infusion of gemcitabine in patients prior to surgical removal of tumors will allow measurement of critical proteins required for gemcitabine metabolism, phosphorylation of gemcitabine, effect on ribonucleotide reductase and p53 status. These studies will bridge the gap between in vitro and in vivo studies and help to optimize radiosensitizing therapy with gemcitabine.