title: Isolation of metallothionein from cells derived from aggressive form of high-grade prostate carcinoma using paramagnetic antibody-modified microbeads off-line coupled with electrochemical and electrophoretic analysis.
abstract: Prostate cancer with altered zinc(II) cell metabolism is the second most frequently diagnosed cancer in developed countries. The alterations of zinc(II) metabolism can influence metabolism of other metal ions and can also be associated with the expression and translation of metal-binding proteins including metallothioneins. The aim of this article was to optimize immunoseparation protocol based on paramagnetic beads conjugated with protein G for the isolation of metallothionein. Isolated metallothionein was determined by differential pulse voltammetry Brdicka reaction and SDS-PAGE. Optimal conditions: antigen-binding time - 60 min, temperature - 70°C, and buffer composition and pH - acetate buffer, pH 4.3, were determined. Under the optimized conditions, lysates from 22Rv1 prostate cancer cells treated with various concentrations of cadmium(II) and copper(II) ions were analyzed. We observed strong correlation in all experimental groups and all lysate types (r>0.83 at p<0.041) between metallothionein concentration related to viability and concentration of copper(II) ions and cadmium(II) ions in medium. Moreover, the results were compared with standard sample preparation as heat treatment and SDS-PAGE analysis.
authors.affiliation: Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. masarik@med.muni.cz
authors.forename: Michal
authors.initials: M
authors.lastname: Masarik
authors.forename: Jaromir
authors.initials: J
authors.lastname: Gumulec
authors.forename: Marketa
authors.initials: M
authors.lastname: Sztalmachova
authors.forename: Marian
authors.initials: M
authors.lastname: Hlavna
authors.forename: Petr
authors.initials: P
authors.lastname: Babula
authors.forename: Sona
authors.initials: S
authors.lastname: Krizkova
authors.forename: Marketa
authors.initials: M
authors.lastname: Ryvolova
authors.forename: Michal
authors.initials: M
authors.lastname: Jurajda
authors.forename: Jiri
authors.initials: J
authors.lastname: Sochor
authors.forename: Vojtech
authors.initials: V
authors.lastname: Adam
authors.forename: Rene
authors.initials: R
authors.lastname: Kizek
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D017097:Electric Impedance; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods*; D016014:Linear Models; D059346:Magnets; D008297:Male; D008668:Metallothionein / Q000302:isolation & purification* / Q000378:metabolism; D019216:Metals, Heavy; D008856:Microscopy, Fluorescence; D008863:Microspheres; D011471:Prostatic Neoplasms / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D013696:Temperature; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D007074:Immunoglobulin G; D019216:Metals, Heavy; D008668:Metallothionein