Panoply™ Human DAO Knockdown Stable Cell Line

D-amino acid oxidase (DAO) is a flavin adenine dinucleotide (FAD)-dependent oxidase that metabolizes neutral and polar D-amino acids. Unlike L-amino acids, D-amino acids are present in very low amounts in mammalian tissues, thus the physiological functions of DAO have been poorly studied. Recently, it has been found that DAO expression is upregulated during cellular senescence. Cellular senescence is a permanent cell cycle arrest induced by various stresses, such as persistent DNA damage and oxidative stress. Since DAO produces reactive oxygen species (ROS) as a byproduct during substrate oxidation, and the accumulation of ROS mediates the induction of cellular senescence, researchers have investigated the relationship between DAO and cellular senescence. They found that inhibiting DAO attenuates DNA damage-induced cellular senescence, while ectopic expression of wild-type DAO (rather than mutants lacking enzyme activity) enhances this senescence in a ROS-dependent manner. Furthermore, the addition of D-amino acids and riboflavin (a metabolic precursor of FAD) to the culture medium enhances the pro-senescence effect of DAO. These results indicate that DAO promotes cell senescence by enzymatically generating reactive oxygen species (ROS), and its activity is regulated by substrate and coenzyme availability.

To investigate whether DAO is involved in cellular senescence, researchers used two human tumor cell lines expressing wild-type p53-osteosarcoma U2OS cells and hepatocellular carcinoma HepG2 cells (Figure 1)-to assess the effect of DAO knockdown on DNA damage-induced cellular senescence. Researchers treated DAO-knockdown U2OS and HepG2 cells with the anticancer drug etoposide (a drug that induces DNA double-strand breaks), and then verified DAO expression levels using quantitative PCR (qPCR) and Western blot analysis (Figures 1A and 1B). They observed that in both U2OS and HepG2 cells, the proportion of SA-β-gal positive cells increased after etoposide treatment (Figures 1C and 1D), consistent with previous observations. More importantly, etoposide-induced SA-β-galactosidase activation was inhibited in DAO-knockdown cells. Furthermore, DAO knockdown partially restored the etoposide-induced loss of proliferation (Figure 1E). Immunoblotting analysis showed that DAO knockdown also inhibited the upregulation of p21, a key aging mediator induced by etoposide, although DAO knockdown may have caused only mild cellular stress, as p21 levels were slightly elevated even in the absence of etoposide (Figure 1F), suggesting that DAO may play a role in inducing aging.

Figure 1. Knockdown of DAO inhibits DNA damage-induced senescence. (Nagano T, et al., 2019)