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

1
Laboratory of Trace elements metabolism, ITMO University, Kronverksky av. 49, 197101 St.-Petersburg, Russia
2
Department of Molecular Genetics, Research Institute of Experimental Medicine, Acad. Pavlov str. 12, 197376 St.-Petersburg, Russia
3
Department of Biophysics, Peter the Great St. Petersburg Polytechnic University, Politekhnicheskaya str. 29, 195251 St.-Petersburg, Russia
4
Laboratory of molecular pharmacology, Istituto di Ricerche Farmacologiche “Mario Negri”, IRCCS, Via La Masa 19, 20156 Milan, Italy
*
Author to whom correspondence should be addressed.
Cells 2019, 8(4), 322; https://doi.org/10.3390/cells8040322
Received: 9 November 2018 / Revised: 28 March 2019 / Accepted: 3 April 2019 / Published: 6 April 2019
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Abstract

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, copper is imported by CTR1 (high-affinity copper transporter 1), a transmembrane high-affinity copper importer, and DMT1 (divalent metal transporter). In cytosol, enzyme-specific chaperones receive copper from CTR1 C-terminus and deliver it to their apoenzymes. DMT1 cannot be a donor of catalytic copper because it does not have a cytosol domain which is required for copper transfer to the Cu-chaperons that assist the formation of cuproenzymes. Here, we assume that DMT1 can mediate copper way required for a regulatory copper pool. To verify this hypothesis, we used CRISPR/Cas9 to generate H1299 cell line with CTR1 or DMT1 single knockout (KO) and CTR1/DMT1 double 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 DMT1 gene was significantly increased and vice versa. In subcellular compartments of the derived cells, copper concentration dropped, however, in nuclei basal level of copper did not change dramatically. CTR1 KO cells, but not DMT1 KO, demonstrated reduced sensitivity to cisplatin and silver ions, the agents that enter the cell through CTR1. Using single CTR1 and DMT1 KO, 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 DMT1 deficiency induced increase of the COMMD1, HIF1α, and XIAP levels. The possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed. View Full-Text
Keywords: copper importers CTR1 and DMT1; CRISPR-Cas9; cisplatin; silver; signaling; cuproenzymes; copper homeodynamics copper importers CTR1 and DMT1; CRISPR-Cas9; cisplatin; silver; signaling; cuproenzymes; copper homeodynamics
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Ilyechova, E.Y.; Bonaldi, E.; Orlov, I.A.; Skomorokhova, E.A.; Puchkova, L.V.; Broggini, M. CRISP-R/Cas9 Mediated Deletion of Copper Transport Genes CTR1 and DMT1 in NSCLC Cell Line H1299. Biological and Pharmacological Consequences. Cells 2019, 8, 322.

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