Cross-linking of cytokeratins to DNA in vivo by chromium salt and cis-diamminedichloroplatinum(II).
The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.
Wedrychowski
A
A
Schmidt
W N
WN
Ward
W S
WS
Hnilica
L S
LS
D000818:Animals; D002840:Chromates / Q000494:pharmacology*; D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism*; D007633:Keratins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D017680:Potassium Compounds; D051381:Rats; D011919:Rats, Inbred Strains
D002840:Chromates; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D017680:Potassium Compounds; C027373:potassium chromate(VI); D007633:Keratins; D002945:Cisplatin