Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein.
The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.
Freedman
J H
JH
Institute for Structural and Functional Studies, University City Science Center, Philadelphia, PA.
Peisach
J
J
D000596:Amino Acids / Q000302:isolation & purification; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000032:analysis*; D002460:Cell Line; D003300:Copper / Q000633:toxicity*; D003545:Cysteine / Q000378:metabolism; D004351:Drug Resistance; D008113:Liver Neoplasms; D008668:Metallothionein / Q000302:isolation & purification*; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D051381:Rats; D014407:Tumor Cells, Cultured / Q000032:analysis / Q000187:drug effects*
D000596:Amino Acids; D009363:Neoplasm Proteins; C007121:copper thionein; D003300:Copper; D008668:Metallothionein; D003545:Cysteine