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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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Cellular copper of KO cell line.(A) Total cellular Cu was determined after 24 h or after chase (48 h). Cells were subjected to AAS analysis. Data are recorded for viable 106 cells. (B) Cu concentrations of cell lysates from KO (black) and HepG2 cells (open) are shown. Subcellular fractions were derived by differential centrifugation after 48 h Cu exposure. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
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pone-0098809-g004: Cellular copper of KO cell line.(A) Total cellular Cu was determined after 24 h or after chase (48 h). Cells were subjected to AAS analysis. Data are recorded for viable 106 cells. (B) Cu concentrations of cell lysates from KO (black) and HepG2 cells (open) are shown. Subcellular fractions were derived by differential centrifugation after 48 h Cu exposure. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).

Mentions: The cellular Cu concentration was determined in KO and parental cells after Cu loading by AAS (Fig. 4A). Cellular Cu concentrations were almost identical in KO and HepG2 cells. The efflux of Cu from the cells was also assessed following a chase period consisting of several washings of the cells with standard cell culture media. Significant lower Cu concentrations were observed in both cell lines after the chase period. The subcellular Cu concentrations were analyzed after differential centrifugation. Most of the Cu was found in the supernatant of the 15,000xg fraction suggesting that Cu is mostly in the compartment enriched for cytosolic proteins (Fig. 4B). The observed differences between KO and HepG2 cells with regard to the subcellular Cu concentration did not reach significance.


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)

Cellular copper of KO cell line.(A) Total cellular Cu was determined after 24 h or after chase (48 h). Cells were subjected to AAS analysis. Data are recorded for viable 106 cells. (B) Cu concentrations of cell lysates from KO (black) and HepG2 cells (open) are shown. Subcellular fractions were derived by differential centrifugation after 48 h Cu exposure. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098809-g004: Cellular copper of KO cell line.(A) Total cellular Cu was determined after 24 h or after chase (48 h). Cells were subjected to AAS analysis. Data are recorded for viable 106 cells. (B) Cu concentrations of cell lysates from KO (black) and HepG2 cells (open) are shown. Subcellular fractions were derived by differential centrifugation after 48 h Cu exposure. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
Mentions: The cellular Cu concentration was determined in KO and parental cells after Cu loading by AAS (Fig. 4A). Cellular Cu concentrations were almost identical in KO and HepG2 cells. The efflux of Cu from the cells was also assessed following a chase period consisting of several washings of the cells with standard cell culture media. Significant lower Cu concentrations were observed in both cell lines after the chase period. The subcellular Cu concentrations were analyzed after differential centrifugation. Most of the Cu was found in the supernatant of the 15,000xg fraction suggesting that Cu is mostly in the compartment enriched for cytosolic proteins (Fig. 4B). The observed differences between KO and HepG2 cells with regard to the subcellular Cu concentration did not reach significance.

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