Lee Kevin J, Mann Elise, da Silva Luciana Madeira, Scalici Jennifer, Gassman Natalie R
University of South Alabama College of Medicine, Mobile, AL 36688 USA.
University of South Alabama Mitchell Cancer Institute, Mobile, AL 36604, USA.
Curr Res Biotechnol. 2019 Nov;1:78-86. doi: 10.1016/j.crbiot.2019.11.001. Epub 2019 Nov 15.
Exposures to genotoxic carcinogens and reactive species result in strand breaks and a spectrum of covalent modifications to DNA that can induce mutations and contribute to the initiation and progression of cancer. Measurements of DNA damage within tissue or tumor samples can serve as a biomarker for exposures or assess changes in DNA repair capacity relevant in cancer development and treatment. Numerous methods to characterize DNA damage exist. However, these methods are primarily applicable to isolated DNA or cultured cells, often require a substantial amount of material, and may be limited to the detection and quantification of only a handful of DNA adducts. Here, we used the Repair Assisted Damage Detection (RADD) assay to detect and excise DNA adducts using a cocktail of DNA repair enzymes, then the damage site within the genome are tagged for detection using a modified nucleotide. We previously demonstrated the RADD assay can detect lesions within isolated DNA and fixed cells, and now RADD can be used to detect DNA adducts and DNA strand breaks in formalin-fixed paraffin-embedded (FFPE) tissue samples. We verified the ability of the RADD assay to detect DNA damage in tissue by exogenously inducing DNA damage with X-rays and restriction enzymes. We also showed that RADD can be multiplexed with antibodies to detect cell cycle markers or other proteins of interest. Finally, we showed that RADD can detect DNA damage within clinically relevant ovarian tumor samples. RADD is a flexible and easy-to-use assay that allows relative damage levels to be determined within FFPE samples and allows the heterogeneity of DNA adducts and strand breaks within clinically relevant samples to be measured.
接触基因毒性致癌物和活性物质会导致DNA链断裂以及一系列对DNA的共价修饰，从而诱发突变并促进癌症的发生和发展。对组织或肿瘤样本中的DNA损伤进行测量，可作为接触的生物标志物，或评估与癌症发生和治疗相关的DNA修复能力的变化。目前存在多种表征DNA损伤的方法。然而，这些方法主要适用于分离的DNA或培养细胞，通常需要大量材料，并且可能仅限于检测和定量少数几种DNA加合物。在此，我们使用修复辅助损伤检测（RADD）分析法，利用DNA修复酶混合物来检测和切除DNA加合物，然后使用修饰的核苷酸对基因组内的损伤位点进行标记以便检测。我们之前证明RADD分析法可检测分离的DNA和固定细胞内的损伤，现在RADD可用于检测福尔马林固定石蜡包埋（FFPE）组织样本中的DNA加合物和DNA链断裂。我们通过用X射线和限制酶外源诱导DNA损伤，验证了RADD分析法检测组织中DNA损伤的能力。我们还表明，RADD可与抗体多重联用，以检测细胞周期标志物或其他感兴趣的蛋白质。最后，我们证明RADD可检测临床相关卵巢肿瘤样本中的DNA损伤。RADD是一种灵活且易于使用的分析法，可在FFPE样本中确定相对损伤水平，并可测量临床相关样本中DNA加合物和链断裂的异质性。
