Preprint Article Version 1 This version is not peer-reviewed

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

Version 1 : Received: 9 November 2018 / Approved: 12 November 2018 / Online: 12 November 2018 (10:05:01 CET)

A peer-reviewed article of this Preprint also exists.

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. 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.

Journal reference: Cells 2019, 8, 322
DOI: 10.3390/cells8040322

Abstract

Copper, the highly toxicity 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, 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 cytosol domain which is required for copper transfer to the Cu-chaperons and following to cuproenzymes. Here we assume that DMT1 can mediate copper way required for regulatory copper pool. To verify this thought, 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. In subcellular compartments, copper concentration decreased dramatically in DKO cells. CTR1-KO cells, but not DMT1-KO, demonstrated reduced sensitivity to cisplatin and silver ions, agents that enter the cell through CTR1. The expression of genes, whose protein products require copper: HIF1α, XIAP, COMMD1, CCS, Cp, but not SOD1 and NF-kB, changed their level. Perhaps these data will help to understand how the disturbances of copper homeodynamics lead to the development of neurodegenerative and oncological disorders. Possibility of using CTR1 KO and DMT1 KO cells to study homeodynamics of catalytic and signaling copper selectively is discussed.

Subject Areas

copper importers CTR1 and DMT1; CRICPR-Cas9; cisplatin; silver; signaling; copper homeodynamics

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