Host-cell reactivation of cisplatin-damaged pRSVcat in a human lymphoid cell line.
A method has been developed for studying host-cell reactivation of cisplatin-damaged plasmid DNA in human T lymphocytes. Parameters of electroporation were established for transfection of the shuttle vector pRSV cat into H9 cells, and a rapid single-vial assay was used for measurement of chloramphenicol acetyltransferase (CAT) activity in extracts of transfected cells. pRSVcat was modified with cisplatin to defined levels ranging from 5 to 40 platinum molecules per plasmid, and then transfected into H9 cells. Graded reductions in CAT activity were observed with increasing levels of platination of the plasmid. At 40 platinum molecules per plasmid, CAT activity was reduced to levels of the negative controls. The efficiency of electroporation-mediated transfer of plasmid into H9 cells was not reduced by high levels of cisplatin modification. The level of modification effecting a 63% reduction in CAT gene expression (the B0), was found to be 17 cisplatin molecules per plasmid. This assay system offers a rapid and sensitive method for assessing the capability of human lymphoid cells to reactivate cisplatin-damaged plasmid DNA.
Dabholkar
M
M
Medicine Branch, National Cancer Institute, Bethesda, MD.
Eastman
A
A
Reed
E
E
D001358:Avian Sarcoma Viruses / Q000235:genetics; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D004249:DNA Damage; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D010957:Plasmids / Q000187:drug effects*; D013601:T-Lymphocytes; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase; D002945:Cisplatin