Functional analysis and drug response to zinc and D-penicillamine in stable ATP7B mutant hepatic cell lines.
OBJECTIVE
To study the effect of anti-copper treatment for survival of hepatic cells expressing different ATP7B mutations in cell culture.


METHODS
The most common Wilson disease (WD) mutations p.H1069Q, p.R778L and p.C271*, found in the ATP7B gene encoding a liver copper transporter, were studied. The mutations represent major genotypes of the United States and Europe, China, and India, respectively. A human hepatoma cell line previously established to carry a knockout of ATP7B was used to stably express WD mutants. mRNA and protein expression of mutant ATP7B, survival of cells, apoptosis, and protein trafficking were determined.


RESULTS
Low temperature increased ATP7B protein expression in several mutants. Intracellular ATP7B localization was significantly impaired in the mutants. Mutants were classified as high, moderate, and no survival based on their viability on exposure to toxic copper. Survival of mutant p.H1069Q and to a lesser extent p.C271* improved by D-penicillamine (DPA) treatment, while mutant p.R778L showed a pronounced response to zinc (Zn) treatment. Overall, DPA treatment resulted in higher cell survival as compared to Zn treatment; however, only combined Zn + DPA treatment fully restored cell viability.


CONCLUSIONS
The data indicate that the basic impact of a genotype might be characterized by analysis of mutant hepatic cell lines.
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Gursimran
G
Chandhok
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Judit
J
Horvath
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Annu
A
Aggarwal
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Mohit
M
Bhatt
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Andree
A
Zibert
Gursimran Chandhok, Andree Zibert, Hartmut HJ Schmidt, Klinik für Transplantationsmedizin, Universitätsklinikum Münster, D-48149 Münster, Germany.
Hartmut Hj
HH
Schmidt
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology*; D002712:Chlorides / Q000494:pharmacology*; D003300:Copper / Q000633:toxicity; D000073840:Copper-Transporting ATPases; D004305:Dose-Response Relationship, Drug; D005838:Genotype; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006527:Hepatolenticular Degeneration / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009154:Mutation; D010396:Penicillamine / Q000494:pharmacology*; D010641:Phenotype; D021381:Protein Transport; D014162:Transfection; D017967:Zinc Compounds / Q000494:pharmacology*
D027682:Cation Transport Proteins; D002614:Chelating Agents; D002712:Chlorides; D017967:Zinc Compounds; D003300:Copper; C016837:zinc chloride; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D010396:Penicillamine; C029892:cupric chloride
ATP7B; D-penicillamine; Mutations; Therapy; Wilson disease; Zinc