CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences

Copper, the highly toxic micronutrient, plays two essential roles: it is a catalytic and structural cofactor for Cu-dependent enzymes, and it acts as a secondary messenger. In the cells, copperis imported by CTR1 (high-affinity coppertransporter 1), a transmembrane high-affinity copperimporter, and DMT1(divalent metal transporter). In cytosol, enzyme-specificchaperones receive copperfrom CTR1 C-terminus and deliver it to their apoenzymes. DMT1cannot be a donor of catalytic copperbecause it does not have a cytosol domain which is required for coppertransfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1can mediate copperway required for a regulatory copperpool. To verify this hypothesis, we used CRISPR/ Cas9to generate H1299 cell line with CTR1 or DMT1single knockout (KO) and CTR1/ DMT1double knockout (DKO). To confirm KOs of the genes qRT-PCR were used. Two independent clones for each gene were selected for further studies. In CTR1 KO cells, expression of the DMT1gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copperconcentration dropped, however, in nuclei basal level of copperdid not change dramatically. CTR1 KO cells, but not DMT1KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1KO, we were able to show that both, CTR1 and DMT1, provided the formation of vital intracellular cuproenzymes ( SOD1, COX), but not secretory ceruloplasmin. The loss of CTR1 resulted in a decrease in the level of COMMD1, XIAP, and NF-κB. Differently, the DMT1deficiency induced increase of the COMMD1, HIF1α, and XIAPlevels. The possibility of using CTR1 KO and DMT1KO cells to study homeodynamics of catalytic and signaling copperselectively is discussed.