DNA DAMAGE AND REPAIR IN BREAST CANCER

一些证据表明，DNA损伤的积累加上DNA修复缺陷在乳腺癌易感性中起重要作用，可能在肿瘤进展中起重要作用。这些证据包括显示人类乳腺肿瘤和周围正常组织中氧化DNA损伤水平增加的数据。研究还表明，乳腺癌易感基因BRCA 1是突变啮齿动物模型中氧化损伤的转录偶联修复所必需的。我们之前的工作已经表明，核苷酸切除修复在Li-Fraumeni综合征中是有缺陷的，Li-Fraumeni综合征是一种与乳腺癌易感性增加相关的遗传性癌症倾向综合征。最后，几个研究小组已经表明，在新诊断的散发性乳腺癌患者及其健康的一级女性亲属中存在去除UV和X射线照射诱导的DNA损伤的缺陷。这些数据表明，核苷酸切除和碱基切除修复途径可能参与乳腺癌的发展。乳腺癌细胞中的DNA修复机制是否有缺陷以及哪些特定的修复途径是最重要的，这些都有待确定。我们假设乳腺癌发生的关键步骤之一是对DNA损伤的正常反应的丧失，并且核苷酸切除修复中的特定缺陷可能在恶性表型的发展和进展中至关重要。肿瘤发生过程中获得性核苷酸切除DNA修复缺陷可能涉及许多基因，包括众所周知的DNA修复相关基因以及乳腺癌易感基因BRCA 1和BRCA 2。我们检测了正常人乳腺上皮细胞（HMEC）的转录偶联核苷酸切除修复以及紫外线诱导的整体修复能力。AG 11134和乳腺癌细胞系MCF 7和MDA-MB-468。通过非程序性DNA合成对总体基因组修复的检查揭示，MCF-7和MDA-MB-468细胞在总体基因组中对UV诱导的损伤具有缺陷修复。此外，这两种乳腺癌细胞系表现出RNA合成恢复减少，表明活跃转录序列的修复可能存在缺陷。与正常人乳腺上皮细胞和正常皮肤成纤维细胞相比，使用基因特异性修复试验（玻尔试验）检测的环丁烷嘧啶二聚体的转录偶联修复在两种肿瘤细胞系中均降低。这些结果表明，这些乳腺癌细胞系是有缺陷的核苷酸切除修复，然而，机制和遗传变化，占这种修复表型尚不清楚。我们目前正在研究核苷酸切除修复相关基因（ERCC 1，XPA，，XPB，，XPF，XPC和RPA）在紫外线照射前后的mRNA和蛋白质表达水平。初步结果表明，ERCC 1和XPA基因在蛋白质水平上的表达发生了变化，这可能是核苷酸切除修复途径的两个步骤中的缺陷。转录偶联修复也在其他乳腺癌肿瘤细胞系中进行测定，以辨别其他乳腺癌细胞系是否也有修复缺陷。正在进行的工作是检查机制的解释，重点是修复相关的基因功能的改变。- DNA损伤，DNA修复，乳腺癌，氧化损伤，激素，转录，染色质

Several lines of evidence suggest that accumulation of DNA damage coupled with defects in DNA repair play an important role in breast cancer susceptibility and perhaps in tumor progression. This evidence includes data showing that levels of oxidative DNA damage are increased in human breast tumors and in surrounding normal tissue. It has also been shown that the breast cancer susceptibility gene, BRCA1, is required for transcription coupled repair of oxidative damage in mutant rodent models. Our own previous work has shown that nucleotide excision repair is defective in the Li-Fraumeni syndrome, a heritable cancer prone syndrome associated with increased susceptibility to breast cancer. Finally, several groups have shown that defects in the removal of UV- and X-irradiation induced DNA damage are present in newly diagnosed sporadic breast cancer patients and in their healthy first- degree female relatives. These data suggest that both the nucleotide excision and base excision repair pathways may be involved in breast cancer development. It remains to be established whether DNA repair mechanisms are defective in breast cancer cells and which specific repair pathways are of primary importance. We hypothesize that one of the critical steps in mammary carcinogenesis is the loss of the normal response to DNA damage and that specific defects in nucleotide excision repair may be critical in development and progression of the malignant phenotype. Acquired defects in nucleotide excision DNA repair during the process of tumorigenesis may involve a number of genes including well known DNA repair related genes as well as breast cancer susceptibility genes BRCA1 and BRCA2.In the present study, we examine transcription coupled nucleotide excision repair as well as ultraviolet- light induced overall repair capacity in normal human mammary epithelial cells (HMEC) AG11134 and the breast cancer cell lines MCF7 and MDA-MB-468. Examination of overall genome repair by unscheduled DNA synthesis reveals that both MCF-7 and MDA-MB-468 cells have defective repair of UV-induced lesions in the overall genome. In addition, the two breast cancer cell lines exhibit reduced RNA synthesis recovery suggesting a possible defect in repair of actively transcribing sequences. Transcription coupled repair of cyclobutane pyrimidine dimers examined using the gene specific repair assay (Bohr assay) is reduced in both tumor cell lines when compared to normal human mammary epithelial cells and normal skin fibroblasts. These results suggest that these breast cancer cell lines are defective in nucleotide excision repair; however, the mechanism and genetic changes that account for this repair phenotype are unclear. We are currently examining the mRNA and protein expression levels of nucleotide excision repair related genes (ERCC1, XPA,, XPB, ,XPF, XPC and RPA) before and after UV irradiation. Preliminary results suggest an alteration in the expression of the ERCC1 and XPA gene at the protein level possible pinpointing defects in two steps pf the nucleotide excision repair pathway. Transcription coupled repair is also being assayed in other breast cancer tumor cell lines to discern whether other breast cancer cell lines are also repair deficient. On going work is examining mechanistic explanations focused on alterations in repair related gene function. - DNA damage, DNA repair, breast cancer, oxidative damage, hormones, transcription, chromatin