Sensitive and selective monitoring of the DNA damage-induced intracellular p21 protein and unraveling the role of the p21 protein in DNA repair and cell apoptosis by surface plasmon resonance

摘要

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.