NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap(4)A) and Down-Regulates Immune Response and Cancer Promotion Genes

Marriott, Andrew S.; Vasieva, Olga; Fang, Yongxiang; Copeland, Nikki A.; McLennan, Alexander G.; Jones, Nigel J.

The parent KBM-7 line used in this study contains the BCR-ABL1 gene fusion and potentially inactivating mutations in TP53 and NOTCH1, but lacks the other common genetic aberrations found in myeloid malignancies [38] . It expresses the majority of annotated proteins from a wide range of signaling pathways, making it a suitable cell line for this study. The complete absence of NUDT2 protein from the NuKO NUDT2 disruptant was confirmed by Western blotting (Fig 1) . The steady-state concentration of intracellular Ap 4 A in unstressed mammalian cells is typically in the range 0.1-1.0 pmol/10 6 cells (0.05-0.5 μM), the exact amount being species-and cell type-dependent [17, 47] . Log phase KBM-7 cells had a level of 0.21±0.02 (n = 3) pmol/10 6 cells. However, the NuKO derivative had a 175-fold increased level of 36.9±0.3 (n = 3) pmol/10 6 cells, providing the clearest evidence yet that Ap 4 A is an important NUDT2 substrate in vivo and that this enzyme plays an essential role in maintaining the low background level of Ap 4 A. Note that an Ap 4 A content of 1 pmol/10 6 cells equates roughly to an intracellular concentration of 0.5 μM if uniformly distributed [17] so the level in NuKO cells will be around 20 μM. Regarding whether this high level and the resulting changes in the cells reported here are biologically relevant, we have previously measured up to 20 μM Ap 4 A in DNA repair-defective cells treated with mitomycin C [17] while a concentration as high as 775 μM has been reported in FCεR1-activated mast cells [31] . Chromatographic analysis of the Ap 4 A from NuKO cells showed that about 35% was present in the form of ADP-ribosylated derivatives (ADPR-Ap 4 A), mainly mono-ADPR-Ap 4 A (Fig 1) . We have previously shown that ADP-ribosylation of Ap 4 A by PARP1 and PARP2 in Chinese hamster EM9 cells and mouse embryo fibroblasts occurs in response to DNA damage [17] ; however, it appears that the high level of Ap 4 A here is subject to constitutive ADP-ribosylation.