The effect of zinc and D-penicillamine in a stable human hepatoma ATP7B knockout cell line.

Gursimran Chandhok,Vanessa Sauer,Mohit Bhatt,Nadine Schmitt,Annu Aggarwal,Hartmut H. J. Schmidt,James F. Collins

doi:10.1371/journal.pone.0098809

Gursimran Chandhok, Vanessa Sauer + Show 5 more

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https://doi.org/10.1371/journal.pone.0098809

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Abstract

Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

Highlights

Wilson disease (WD), an orphan disease, is caused by mutations in the ATP7B gene on chromosome 13 leading to an imbalance in copper homeostasis [1,2]
Establishment of ATP7B knockout cell line HepG2 cells were purchased from American Type Culture Collection (ATCC) and cultured in RPMI media supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin (PAA). 2.5 mg of custom made CompoZr knockout Zinc Finger Nucleases (ZFN) plasmids were transfected into HepG2 (Sigma; #CKOZFND3740-1KT)
Sequence analysis of regions outside this area confirmed that other exons of ATP7B as well as exon/intron boundaries were identical to parental HepG2 cells

Summary

Introduction

Wilson disease (WD), an orphan disease, is caused by mutations in the ATP7B gene on chromosome 13 leading to an imbalance in copper homeostasis [1,2]. Excessive copper (Cu) accumulation in the liver and brain are the hallmarks of this disease. ATP7B plays a central role in Cu homeostasis in the liver [8]. This transmembrane protein is primarily expressed in hepatocytes and mediates incorporation of Cu into ceruloplasmin and excretion of toxic Cu via bile. Impairment of ATP7B in WD leads to progressive Cu accumulation in the liver and is believed to be followed over time by spillage to other organs like brain, kidney, and cornea. Individual ATP7B mutations have been associated with various phenotypes [9]

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