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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的总体目标是提高阿霉素的抗癌活性，阿霉素是目前临床上使用最广泛的抗癌药物之一。我们最近已经证明阿霉素可被甲醛激活(由甲醛释放前体药物提供，这些药物本身也作为单一药物在临床试验中)，这导致肿瘤细胞中多柔比星-DNA加合物的水平急剧增加，以及两种药物之间的协同作用(Cutts等，癌症研究，61,8194,2001)，导致大大增强的，拓扑异构酶II不依赖的细胞死亡的形式(Swift等，癌症研究，66,4863,2006)。因为阿霉素通过甲醛活化与DNA共价结合，它不再是主动外排的底物(阿霉素获得性耐药的一种常见形式)，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介导的对阿霉素的耐药性(Cutts等人，Oncol.研究报告，15,199,2005)。 我们目前通过两种实验程序来测量阿霉素-DNA加合物，这两种方法都有局限性： (1)总的基因组加合物水平(每10kb DNA的加合物)通常是通过14C标记的阿霉素(从药物处理的细胞中分离出来后与DNA保持络合)的闪烁计数来测定的，但这种方法极其昂贵，而且缺乏敏感性，因此需要比抑制生长所需的药物剂量更高的药物剂量。 (2)基因特定的加合物水平也可以根据从药物处理的细胞中分离DNA后DNA的明显交联度来确定(Cutts等人，癌症研究，61,8194,2001)，然而这一过程非常费力，并且在Southern杂交过程中的许多实验步骤中遭受了大量加合物的损失。 AMS的使用在很大程度上克服了这两个限制，并为测量阿霉素-DNA加合物水平提供了三个主要优势： (1)敏感度大幅提高(估计敏感度至少提高10,000倍)，使我们能够在临床(和亚临床)药物剂量下检测加合物 (2)测量阿霉素-DNA加合物在细胞中修复速度的能力 (3)有可能监测正在接受化疗的患者的活检组织(血液、实体肿瘤)中的药物DNA加合物水平，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.