(P13-049-23) N-Acetyltransferase 1 Knockout in Immortalized Human Bronchial Cells Results in a Reduction of Cellular Growth

Objectives: Arylamine N-acetyltransferases are polymorphic xenobiotic metabolizing enzymes and there are two isoenzymes found in humans, N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2). Both can perform N-acetylation and O-acetylation of arylamine carcinogens. Recent literature has implicated NAT1 in an endogenous role (aside from substrate acetylation) in colon and breast cancer cells, illustrated by the loss of NAT1 resulting in reduced cell growth and tumor cell properties as well as mitochondrial metabolism dysfunction. The role of NAT1 in human lung cells and its importance in lung biology remains largely understudied. The objective of this study is to understand the endogenous role of NAT1 in immortalized (“normal”) human bronchial cells, both epithelial (BEP2D) and fibroblast (WTHBF-6), and establish how that loss of NAT1 impacts lung, breast, and colon cancer cells.

Methods: First, we constructed cell lines stably expressing Cas9 to utilize CRISPR/Cas9 to knockout NAT1, prior to the insertion of two different guide-RNA (gRNA) sequences for NAT1 into BEP2D and WTHBF-6 cell types. Following antibiotic selection, we isolated single cell colonies of both cell lines for each gRNA and confirmed the loss of NAT1 by measuring the N-acetylation of a selective substrate for NAT1 (p-aminobenzoic acid). We then compared the growth rate and colony formation of NAT1 KO cells to the wild type (parental) lung cells.

Results: We observed that for both BEP2D and WTHBF-6, there was a reduction in the growth of NAT1 KO cells compared to parental cells. Interestingly, there was a reduction in the visual number of cells per colony in the NAT1 KO cells compared to parental cells in colony formation assays. Lastly, we screened the karyotype of the NAT1 KO cells to confirm that both the insertion of Cas9 and deletion of NAT1 did not induce genomic instability.

Conclusions: These data provide additional evidence that NAT1 has an endogenous role aside from substrate acetylation. The loss of NAT1 results in reduced cell growth not only in cancer cells but in “normal” cells as well. Future work is aimed at investigating the potential mitochondrial metabolism changes in these NAT1 KO lung cells.

Funding Sources: This work is supported by NIEHS grants R35-ES032876, P42-ES023716, and P30-ES030283.