DETECT OF DOXORUBICIN AND MITOXANTRONE INDUCED DRUG-DNA ADDUCTS IN TUMOR CELLS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定为研究者机构。我们的总体目标是提高阿霉素的抗癌活性，阿霉素是当今临床使用最广泛的抗癌药物之一。我们最近已经表明，多柔比星被甲醛（由甲醛释放前药提供，其本身在临床试验中作为单一药剂）活化，并且这导致培养物中肿瘤细胞中多柔比星-DNA加合物水平的显著增加，以及两种药物之间的协同相互作用（Cutts等人，癌症研究所，61，8194，2001），导致极大增强的拓扑异构酶II非依赖性形式的细胞死亡（Swift等人，癌症研究所，66，4863，2006）。因为多柔比星通过甲醛活化与DNA共价结合，所以它不再是主动外排的底物（对多柔比星获得性抗性的常见形式），并且我们已经证明这些加合物的形成可以在很大程度上克服P-糖蛋白和拓扑异构酶II介导的对多柔比星的抗性（Cutts等人，Oncol.结果：15，199，2005）。 我们目前通过两种实验程序测量多柔比星-DNA加合物，这两种方法都有局限性：（1）总基因组加合物水平通过14 C标记的多柔比星的闪烁计数常规地测定（每10 kb DNA的加合物（其在从药物处理的细胞中分离后保持与DNA复合），但是该方法极其昂贵并且还缺乏灵敏度，从而需要比生长抑制所需的更高的药物剂量。(2)基因特异性加合物水平也可以从药物处理的细胞中分离DNA后DNA的表观交联程度来确定（Cutts等人，癌症研究所，61，8194，2001），然而，该方法非常费力，并且在Southern杂交方法中涉及的许多实验步骤期间损失了大量加合物。 AMS的使用在很大程度上克服了这两个限制，并提供了测量多柔比星-DNA加合物水平的三个主要优点：（1）灵敏度大幅提高（估计至少有10，000倍的灵敏度），这将使我们能够在临床上检测加合物（和亚临床）药物剂量（2）测量细胞中多柔比星-DNA加合物修复速率的能力（3）能够监测活检中药物-DNA加合物水平的潜力（血液，实体瘤），以优化药物治疗方案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our overall aim is to enhance the anticancer activity of doxorubicin, one of the most widely used anticancer drugs in clinical use today. We have recently shown that doxorubicin is activated by formaldehyde (supplied by formaldehyde-releasing prodrugs which themselves are in clinical trials as single agents), and this results in a dramatic increase of the level of doxorubicin-DNA adducts in tumor cells in culture, together with a synergistic interaction between the two drugs (Cutts et al., Cancer Res., 61, 8194, 2001), resulting in a greatly enhanced, topoisomerase II ¿ independent form of cell death (Swift et al., Cancer Res., 66, 4863, 2006). Because the doxorubicin is bound covalently to DNA by the formaldehyde activation, it is no longer a substrate for active efflux (a common form of acquired resistance to doxorubicin) and we have demonstrated that formation of these adducts can largely overcome P-glycoprotein and topoisomerase II-mediated resistance to doxorubicin (Cutts et al., Oncol. Res.,15, 199, 2005). We currently measure doxorubicin-DNA adducts by two experimental procedures, both of which have limitations: (1)Total genomic adduct levels (adducts per 10 kb of DNA) are routinely determined from scintillation counting of 14C labeled doxorubicin (which remains complexed to DNA following isolation from drug-treated cells), but this procedure is extremely expensive and also lacks sensitivity, thereby necessitating higher drug doses than required for growth inhibition. (2) Gene-specific adduct levels can also be determined from the extent of apparent cross-linking of DNA following isolation of the DNA from drug-treated cells (Cutts et al., Cancer Res., 61, 8194, 2001), however this procedure is very laborious and suffers from a large loss of adducts during the many experimental steps involved in the Southern hybridization procedure. The use of AMS largely overcomes both of these limitations and provides three major advantages for measuring doxorubicin-DNA adduct levels: (1) a dramatic increase of sensitivity (estimated as at least a 10,000 fold greater sensitivity) that would enable us to detect adducts at clinical (and sub-clinical) drug doses (2) the ability to measure the rate of repair of doxorubicin-DNA adducts in cells (3) potential to be able to monitor drug-DNA adduct levels in biopsies (blood, solid tumor) of patients undergoing chemotherapy in order to optimize drug treatment schedules.