Arylamine N-acetyltransferase 1 protects against reactive oxygen species during glucose starvation: Role in the regulation of p53 stability

Abstract

Human arylamine N-acetyltransferase 1 (NAT1) has been associated with cancer cell growth and invasion, but the underlying molecular mechanisms remain unknown. NAT1 is located on the short arm of chromosome 8 (8p21), a region that is commonly deleted in colon cancer. Previously, it was reported that HT-29 colon cancer cells, which have a large deletion at 8p21-22, show marked morphological changes, increased E-cadherin expression and altered cell-cell contact inhibition following down-regulation of NAT1 with shRNA. By contrast, no effects on growth were observed in HeLa cells. In the present study, cellular changes following knockout of NAT1 with CRISPR/Cas9 in HT-29 and HeLa cells were compared in the presence and absence of glucose. Cell growth decreased in both cell-lines during glucose starvation, but it was enhanced in HT-29 cells following NAT1 deletion. This was due to an increase in ROS production that induced cell apoptosis. Both ROS production and cell death were prevented by the glutathione precursor N-acetylcysteine. NAT1 knockout also resulted in a loss of the gain-of-function p53 protein in HT-29 cells. When p53 expression was inhibited with siRNA in parental HT-29 cells, ROS production and apoptosis increased to levels seen in the NAT1 knockout cells. The loss of p53 may explain the decreased colony formation and increased contact inhibition previously reported following NAT1 down-regulation in these cells. In conclusion, NAT1 is important in maintaining intracellular ROS, especially during glucose starvation, by stabilizing gain-of-function p53 in HT-29 cells. These results suggest that NAT1 may be a novel target to decrease intracellular gain-of -function p53.

Loss of human arylamine N-acetyltransferase I regulates mitochondrial function by inhibition of the pyruvate dehydrogenase complex

“…Previously, we reported that deletion of NAT1 in various human cells including MDA-MB-231 cells, using CRISPR/Cas9 technology decreased oxidative phosphorylation (Lichter et al, 2017). Moreover, in human HT-29 cells, glucose utilisation decreased and ROS production increased following NAT1 deletion (Wang et al, 2018). These studies suggested that NAT1 affected mitochondrial function in a similar manner to that seen following Nat1 knockdown in mice.…”

“…However, this was not seen in BT-549 cells (Tiang et al, 2015). Deletion of the NAT1 gene increased reactive oxygen (ROS) production and apoptosis, especially under nutrient stress, in HT-29 cells but not in HeLa cells (Wang et al, 2018).…”

“…Human cancer cell lines MDA-MB-231 (breast adenocarcinoma) and HT-29 (colon adenocarcinoma) were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI 1640 medium (Thermo Fisher Scientific, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone; In Vitro Technologies, VIC, Australia), 100 units/ml penicillin/streptomycin and 2 mM L-glutamine in a humidified 5% CO2 atmosphere at 37 °C. The HT-29 CRISPR/Cas9 NAT1 knockout cell line has been described previously (Wang et al, 2018). The MDA-MB-231 NAT1 knockout cell line was generated by disrupting the NAT1 gene using a human NAT1 gene knockout CRISPR/Cas9 kit (Origene Technologies, Rockville, MD; KN221042).…”

“…The MDA-MB-231 NAT1 knockout cell line was generated by disrupting the NAT1 gene using a human NAT1 gene knockout CRISPR/Cas9 kit (Origene Technologies, Rockville, MD; KN221042). NAT1 knockout was verified by PCR of genomic DNA, lack of NAT1 acetylation activity and Western blot for NAT1 protein, as previously described (Wang et al, 2018).…”

Effect arylamine N-acetyltransferase 1 on morphology, adhesion, migration, and invasion of MDA-MB-231 cells: role of matrix metalloproteinases and integrin αV

“…The NAT1 gene was disrupted using CRISPR/Cas9 system as previously described [37]. After selection with puromycin (5 µM), single colonies were selected, expanded, and NAT1 activities measured by a highperformance liquid chromatography assay to screen for the NAT1 knockout cell lines [38].…”