Effects of cis-diamminedichloroplatinum (CDDP) on HeLa cell non-histone nuclear proteins.

Abstract

Two-dimensional isoelectric focusing and sodium dodecyl sulfate (SDS) gel electrophoretic analyses of successive salt extracts of purified HeLa cell nuclei were used to study the effects of cis-diamminedichloroplatinum (CDDP) (cisplatin) on the synthesis and extractability of nuclear non-histone proteins. Nuclei were extracted sequentially with 0.025 M NaCl-0.075 M EDTA, 0.01 M Tris, and 0.6 M NaCl. Each fraction contained 100-400 polypeptide spots, only a few of which were affected by a 3.5-h CDDP pretreatment of the cells. The biosynthesis and/or metabolism of four polypeptide spots was significantly affected by the CDDP treatment. These polypeptides included: (a) 36K/5.8 (designated by MW/PI) in the 0.025 M NaCl-0.075 M EDTA extract, which decreased in intensity with treatment at 10 micrograms CDDP/ml; (b) polypeptide spots 50K/6.0 and 45K/5.3 in the Tris extract, which increased in intensity over a range of CDDP concentrations of 0-5 microgram CDDP/ml; and (c) a polypeptide complex at 110K/7.7 in the 0.6 M Na Cl extract, which decreased in intensity at CDDP concentrations of 0-5 microgram CDDP/ml. Scanning densitometry of the protein spots of the 0.6 M NaCl extract demonstrated that the 110K/7.7 complex decreased to half its intensity compared with non-drug-treated controls at a CDDP concentration of 0.9 microgram CDDP/ml. We have found that high-resolution two-dimensional gel electrophoretic analysis of nuclear proteins is a valuable technique for studying the effects of cytotoxic agents on the synthesis and/or extractability of specific cellular proteins.