Hunan Provincial Key Laboratory of Micro & Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, P. R. China 410083.
Analyst. 2020 May 21;145(10):3697-3704. doi: 10.1039/c9an02464f. Epub 2020 Apr 16.
The cyclin-dependent kinase inhibitor p21 protein is a critical regulator that mediates various biological activities, such as cell cycle progression, apoptosis, and cellular senescence. As a DNA damage-inducing agent, doxorubicin could reactivate the transcriptional activity of p53 and modulate the p21 protein level. In this work, sensitive and selective monitoring of the intracellular p21 protein in doxorubicin-treated breast cancer cells was conducted using surface plasmon resonance (SPR). The fluidic channels were pre-immobilized with double stranded (ds) DNA/proliferating cell nuclear antigen (PCNA) for the capture of the p21 protein. The incorporation of the anti-p21 antibody-streptavidin conjugate pre-formed between streptavidin and biotinylated anti-p21 antibody that specifically recognizes the p21 protein leads to signal amplification. The detection limit of 0.85 pM for the p21 protein was lower than that using the commercial enzyme-linked immunosorbent assay (ELISA) kit. The treatment of MCF-7 breast cancer cells with wild-type p53 by various doses of doxorubicin leads to differences in the extent of DNA damage. Low-level DNA damage by low-dose doxorubicin up-regulates the p21 level, and p21 exerts its anti-apoptotic function, causing p53-dependent cell cycle arrest and DNA repair. However, massive DNA damage by high-dose doxorubicin represses the expression of the p21 protein through increased proteasome activity, leading to cell apoptosis. The proposed method is sensitive, selective and label-free, holding great promise for the assay of the DNA damage-induced intracellular p21 protein and understanding of p21 protein-mediated cell cycle arrest, DNA repair, and cell apoptosis.
细胞周期蛋白依赖性激酶抑制剂 p21 蛋白是一种关键的调节因子，介导多种生物学活性，如细胞周期进程、细胞凋亡和细胞衰老。作为一种诱导 DNA 损伤的药物，阿霉素可以重新激活 p53 的转录活性，并调节 p21 蛋白水平。在这项工作中，使用表面等离子体共振（SPR）对阿霉素处理的乳腺癌细胞内的 p21 蛋白进行了灵敏和选择性的监测。流体通道预先固定双链（ds）DNA/增殖细胞核抗原（PCNA），用于捕获 p21 蛋白。抗 p21 抗体-链霉亲和素缀合物的掺入，该缀合物在链霉亲和素和生物素化抗 p21 抗体之间预先形成，特异性识别 p21 蛋白，导致信号放大。p21 蛋白的检测限为 0.85 pM，低于商业酶联免疫吸附测定（ELISA）试剂盒的检测限。用不同剂量的阿霉素处理 MCF-7 乳腺癌细胞，导致 DNA 损伤程度的差异。低剂量阿霉素引起的低水平 DNA 损伤上调了 p21 水平，p21 发挥其抗凋亡功能，导致 p53 依赖性细胞周期停滞和 DNA 修复。然而，高剂量阿霉素引起的大量 DNA 损伤通过增加蛋白酶体活性抑制 p21 蛋白的表达，导致细胞凋亡。该方法灵敏、选择性好、无需标记，为测定 DNA 损伤诱导的细胞内 p21 蛋白以及理解 p21 蛋白介导的细胞周期停滞、DNA 修复和细胞凋亡提供了新的思路。
