GEMZAR--MECHANISMS OF CYTOTOXICITY & RADIOSENSITIZATION

吉西他滨是一种新的核苷类似物，对包括胰腺癌和非小细胞肺癌在内的几种实体瘤具有临床活性。虽然吉西他滨作为单一药物或与其他化疗药物联合使用在这些肿瘤类型的患者中显示出良好的活性，但我们最近已经证明，吉西他滨也提高了实体瘤细胞对电离辐射的敏感性。临床前研究表明，吉西他滨对人胰腺癌、结直肠癌、头颈部癌、乳腺癌和胶质母细胞瘤等肿瘤细胞具有放射增敏作用。将这些研究转化为对不能切除的头颈癌患者进行的I期试验表明，吉西他滨也可以在体内使肿瘤组织增敏，大多数患者对治疗取得完全反应。有了这些令人鼓舞的结果，我们现在建议扩大这些体外和体内的研究，以确定吉西他滨辐射增敏肿瘤细胞的机制，并确定它是否与细胞毒性机制不同。这些研究将在我们令人鼓舞的I期初步结果的基础上在人头颈部癌细胞中进行，我们还将在体外评估人胶质母细胞瘤细胞对基于吉西他滨的放射增敏的反应。初步数据表明，吉西他滨的放射增敏需要dATP的大幅减少，这是由于抑制了核苷酸还原酶，而不受吉西他滨三磷酸的量或DNA中药物的量的影响。此外，新的数据表明，在表达野生型p53的细胞系中，放射增敏不太成功。这里提出的研究将评估dATP耗竭和DNA中吉西他滨核苷酸在细胞毒性和放射增敏中的作用。将使用匹配的野生型和突变型p53细胞株以及使用人乳头瘤病毒E6蛋白消除wtp53的方法来评估p53的作用。这些研究将在体外和在裸鼠体内进行。此外，在手术切除肿瘤之前对患者输注吉西他滨，将可以测量吉西他滨代谢所需的关键蛋白质、吉西他滨的磷酸化、对核苷酸还原酶的影响以及P53状态。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的放射增敏治疗。

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.