High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123

Citation
Ca. Buser et al., High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123, ANALYT BIOC, 290(1), 2001, pp. 126-137
Citations number
33
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
290
Issue
1
Year of publication
2001
Pages
126 - 137
Database
ISI
SICI code
0003-2697(20010301)290:1<126:HLCSCO>2.0.ZU;2-X
Abstract
Cellular transformation by Ras oncoproteins requires the posttranslation mo dification of farnesylation in a reaction catalyzed by farnesyl protein tra nsferase (FPTase). Thus, inhibitors of FPTase have been developed as potent ial 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 tr ansferase I (GGPTase-I) in human tumor cells. Ne have developed a high-perf ormance 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 m utation) treated with the prenyltransferase inhibitor, L-778,123, Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro I C50 = 2 nM) with some activity against GGPTase-I tin vitro IC50 = 98 nM). W e find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mi xture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated wit h nanomolar concentrations of L-778,123, and a mixture of unprocessed, farn esylated, 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. (C) 2001 Academic Press.