title: High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123.
abstract: Cellular transformation by Ras oncoproteins requires the posttranslation modification of farnesylation in a reaction catalyzed by farnesyl protein transferase (FPTase). Thus, inhibitors of FPTase have been developed as potential anticancer agents. However, recent studies with selective inhibitors of FPTase have shown that Ki4B-Ras retains its ability to transform cells by undergoing alternative prenylation by the related geranylgeranyl protein transferase I (GGPTase-I) in human tumor cells. We have developed a high-performance liquid chromatography/mass spectrometry assay for the detection and quantitation of the different processing states of Ki4B-Ras isolated from PSN-1 cells (a human pancreatic cell line with an activating Gly12 to Arg mutation) treated with the prenyltransferase inhibitor, L-778,123. Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro IC50 = 2 nM) with some activity against GGPTase-I (in vitro IC50 = 98 nM). We find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mixture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated with nanomolar concentrations of L-778,123, and a mixture of unprocessed, farnesylated, and geranylgeranylated-Ki4B-Ras in cells treated with micromolar concentrations of compound. Of importance, this technique does not require metabolic labeling and may be used as a pharmacodynamic assay for Ki4B-Ras processing in mouse models.
authors.affiliation: Department of Cancer Research, Merck Research Laboratories, West Point, Pennsylvania 19486, USA. carolyn_buserdoepner@merck.com
authors.forename: C A
authors.initials: CA
authors.lastname: Buser
authors.forename: C J
authors.initials: CJ
authors.lastname: Dinsmore
authors.forename: C
authors.initials: C
authors.lastname: Fernandes
authors.forename: I
authors.initials: I
authors.lastname: Greenberg
authors.forename: K
authors.initials: K
authors.lastname: Hamilton
authors.forename: S D
authors.initials: SD
authors.lastname: Mosser
authors.forename: E S
authors.initials: ES
authors.lastname: Walsh
authors.forename: T M
authors.initials: TM
authors.lastname: Williams
authors.forename: K S
authors.initials: KS
authors.lastname: Koblan
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D051231:Farnesyltranstransferase; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000201:enzymology; D017368:Protein Prenylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects* / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D011994:Recombinant Proteins; C425231:L 778,123; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase; D004122:Dimethylallyltranstransferase; D051231:Farnesyltranstransferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)