Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.
BACKGROUND
Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution.


METHODS
Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison.


RESULTS
Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution.


CONCLUSIONS
This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.
Arkhipov
Sergey N
SN
Department of Chemistry, York University, Toronto, Ontario, Canada.
Berezovski
Maxim
M
Jitkova
Julia
J
Krylov
Sergey N
SN
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D000818:Animals; D002463:Cell Membrane Permeability; D019075:Electrophoresis, Capillary; D010450:Endopeptidases / Q000378:metabolism*; D051231:Farnesyltranstransferase; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D008780:Methyltransferases / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D041681:NIH 3T3 Cells; D009842:Oligopeptides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*
C503950:2',7'-difluorofluorescein-5-carboxyl-glycyl-cysteinyl-valyl-isoleucyl-alanine; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D008780:Methyltransferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D010450:Endopeptidases; D018631:ras Proteins