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

Chandhok G, Schmitt N, Sauer V, Aggarwal A, Bhatt M, Schmidt HH - PLoS ONE (2014)

Bottom Line: Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells.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.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Transplantation Medicine, Münster University Hospital, Münster, Germany; Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India.

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.

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Related in: MedlinePlus

Genotype of the ATP7B KO cell line after ZFN directed mutagenesis.Top line shows the schematic structure of the entire WD gene (exons boxed). Middle lines represent a blow-up of exon 8 and specify the sequence of the proposed ZFN binding sites (boxed) and the FOKI restriction site (arrow). The four bottom lines specify the nucleotide sequences and codons of wild type (upper two lines) and the nucleotide sequence of both chromosomes observed in KO cell (last two lines). Dashes indicate the observed deletions.
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pone-0098809-g001: Genotype of the ATP7B KO cell line after ZFN directed mutagenesis.Top line shows the schematic structure of the entire WD gene (exons boxed). Middle lines represent a blow-up of exon 8 and specify the sequence of the proposed ZFN binding sites (boxed) and the FOKI restriction site (arrow). The four bottom lines specify the nucleotide sequences and codons of wild type (upper two lines) and the nucleotide sequence of both chromosomes observed in KO cell (last two lines). Dashes indicate the observed deletions.

Mentions: We generated a stable human hepatoma ATP7B KO cell line to study Cu metabolism. HepG2 cells were transfected with plasmids encoding zinc-finger nuclease (ZFN) targeted to exon 8 of the ATP7B gene (Fig. 1). Sequence analysis of the genomic DNA revealed deletions around the FOKI cutting site and adjacent regions of the putative ZFN binding sites. Two deletions within exon 8 were found after cloning of individual sequences in E. coli. 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.


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

Chandhok G, Schmitt N, Sauer V, Aggarwal A, Bhatt M, Schmidt HH - PLoS ONE (2014)

Genotype of the ATP7B KO cell line after ZFN directed mutagenesis.Top line shows the schematic structure of the entire WD gene (exons boxed). Middle lines represent a blow-up of exon 8 and specify the sequence of the proposed ZFN binding sites (boxed) and the FOKI restriction site (arrow). The four bottom lines specify the nucleotide sequences and codons of wild type (upper two lines) and the nucleotide sequence of both chromosomes observed in KO cell (last two lines). Dashes indicate the observed deletions.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4044041&req=5

pone-0098809-g001: Genotype of the ATP7B KO cell line after ZFN directed mutagenesis.Top line shows the schematic structure of the entire WD gene (exons boxed). Middle lines represent a blow-up of exon 8 and specify the sequence of the proposed ZFN binding sites (boxed) and the FOKI restriction site (arrow). The four bottom lines specify the nucleotide sequences and codons of wild type (upper two lines) and the nucleotide sequence of both chromosomes observed in KO cell (last two lines). Dashes indicate the observed deletions.
Mentions: We generated a stable human hepatoma ATP7B KO cell line to study Cu metabolism. HepG2 cells were transfected with plasmids encoding zinc-finger nuclease (ZFN) targeted to exon 8 of the ATP7B gene (Fig. 1). Sequence analysis of the genomic DNA revealed deletions around the FOKI cutting site and adjacent regions of the putative ZFN binding sites. Two deletions within exon 8 were found after cloning of individual sequences in E. coli. 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.

Bottom Line: Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells.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.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Transplantation Medicine, Münster University Hospital, Münster, Germany; Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India.

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.

Show MeSH
Related in: MedlinePlus