MCF-7 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) via UCSF Cell Culture Facility (San Francisco, CA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (UCSF Cell Culure Facility). Custom-made DMEM with 0.22 g/L inorganic phosphate (UCSF Cell Culture Facility) was used for all MRS experiments. For inhibition of Hsp90, 3 μM 17-AAG (LC Laboratories, Woburn, MA, USA) was added to the culture medium for a period of 48 hours. Medium was replenished with fresh 17-AAG every 24 hours during treatment. The doubling time of logarithmic-phase MCF-7 cells growing in tissue culture flasks or on beads was determined by seeding at a density of 1 × 106 cells per 75-cm2 flask or at a density of 3 × 106 cells per 0.5 mL of beads, trypsinizing at 24-hour intervals and counting the number of cells by using a hemocytometer.
Cell proliferation assay
The effect of 17-AAG treatment on cell proliferation was determined by using the WST-1 cell proliferation assay (Roche, Indianapolis, IN, USA). Cells were seeded in 96-well microplates (1.5 × 106 cells per well in 150 μL of culture medium), and control and 17-AAG-treated cells were probed at different time periods between 4 and 48 hours. For this, cells were incubated for 1 hour with WST-1 reagent, and cell viability was determined by quantification of the absorbance at 440 nm by using a plate reader spectrophotometer (Tecan, Grödig, Austria).
Protein concentrations per cell were determined in cells lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) by using a Coomassie Plus-The Better Bradford™ Assay Kit (Pierce, Rockford, IL, USA), and bovine serum albumim was used as a protein standard. Absorbance was measured at 595 nm by using a plate reader spectrophotometer (Tecan).
The effect of 17-AAG on levels of client proteins was analyzed by Western blotting. Cellular lysates were extracted in Cell Lysis Buffer (Cell Signaling Technology) with added Protease Inhibitor (Calbiochem, now part of EMD Biosciences, Inc., San Diego, CA, USA). Protein concentration was determined by using a Coomassie Plus Protein Assay (Pierce). Approximately 30 μg of protein was loaded into 4% to 20% SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA). Gels were run for 30 minutes and then proteins were electrotransferred onto nitrocellulose membranes. Membranes were blocked for 1 hour in 5% milk in TBST (Tris-buffered saline Tween 20) and then incubated overnight with primary antibodies against Akt, c-Raf, and Actin (Cell Signaling Technology), and Actin was used as a loading control. After incubation for 2 hours with secondary antibody anti-IgG horseradish peroxidase-linked antibody (Cell Signaling Technology), the immunocomplexes were visualized with ECL Western Blotting Substrate (Pierce).
Cells (1 × 106) were seeded in 75-cm2 tissue culture flasks in 10 mL of culture medium. Following treatment, approximately 2 × 106 cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and fixed in 70% ethanol. Fixed cells were incubated with RNase A at 100 units/mL (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes, washed with PBS, and then stained with propidium iodide at 20 μg/mL (Sigma-Aldrich) for 30 minutes. Cells were counted with a fluorescence-activated cell sorting (FACS) Calibur flow cytometer and CellQuest Pro software (BD Biosciences, Mountain View, CA, USA) and single cells were gated away from clumped cells by using forward light scattering on an FL2-width versus FL2-area dot plot. The percentages of cells in the G1, S, and G2/M phases were determined by plotting a histogram of FL2-A. The relative cell sizes of the G1, S, and G2/M phase populations of control and 17-AAG-treated cells were determined by using forward scattering height (FSC-H).
Perfused cell magnetic resonance spectroscopy studies
Approximately 4 × 107
MCF-7 cells were grown on Biosilon microcarrier beads (Nunc, Rochester, NY, USA) in order to be loaded into a perfusion system for MRS studies as described previously [37
]. Cells were seeded on the microcarrier beads, allowed to attach for 24 hours, and then treated for 48 hours prior to the MR experiment. To monitor a similar number of cells during MRS acquisition, control cells were seeded at a density of approximately 5 × 106
cells/mL of beads and 17-AAG-treated cells were seeded at a density of approximately 9 × 106
cells/mL of beads. To obtain cell counts for both the start and end of the experiment, an additional set of cells was seeded on beads for each experiment and treated in a fashion identical to those loaded into the perfusion system.
MRS studies were performed on a 500-MHz INOVA spectrometer (Varian, Santa Clara, CA, USA). Composite pulse proton-decoupled 31P spectra were first acquired to confirm cell viability and quantify metabolite levels. 31P spectra were acquired with a 30° pulse and 3-second relaxation delay and 1,000 transients for a total acquisition time of 70 minutes. Cells were then perfused with fresh culture medium containing only 56 μM [1,2-13C]-choline (Cambridge Isotope Laboratories, Andover, MA, USA) and no unlabeled choline for a period of 14 hours. Proton-decoupled (Waltz 16) 13C spectra were acquired during that time in 2-hour intervals by using a 60° pulse and 6-second relaxation delay and 1,024 transients. Spectra were quantified with ACD/Spec Manager version 9.15 software (Advanced Chemistry Development, Toronto, ON, Canada). Peak integrals obtained by deconvolution were normalized to cell number and to a metabolite of known concentration in the culture medium (1.87 μM inorganic phosphate [Pi] for 31P spectra and 5 mM [1-13C]-glucose [Cambridge Isotope Laboratories] for 13C spectra). Data were corrected for saturation effects by using correction factors obtained from a fully relaxed spectrum of perfused cells (fully relaxed 13C spectra were acquired with a 90°, 20-microsecond pulse and 30-second relaxation delay for 1,024 transients, and fully relaxed 31P spectra were acquired with a 90°, 22-microsecond pulse and 30-second relaxation delay for 1,000 transients).
Cells were grown in culture with medium in which all glucose and choline were completely replaced by 5 mM [1-13
C]-glucose and 56 μM [1,2-13
C]-choline (Cambridge Isotope Laboratories) for a period of 48 hours prior to extraction. Approximately 7 × 107
cells were extracted by means of the dual-phase extraction method, as described previously [26
]. Briefly, cultured cells were collected by trypsinization, counted to obtain cell number, and fixed in 10 mL of methanol. Chloroform (10 mL) was added and then water (10 mL) was as well. The solution was vortexed and centrifuged to separate the lipid and aqueous phases and then each phase was collected separately. Solvents were lyophilized off each sample. Aqueous samples were re-dissolved in a volume of 400 μL of deuterium oxide, and lipid samples were re-dissolved in 500 μL of deuterated chloroform-methanol mixture.
13C, 1H, and 31P spectra were acquired on the lipid and aqueous portions of the cell extracts by using a 600-MHz INOVA spectrometer (Varian) equipped with a 5-mm broadband observe probe. Proton-decoupled (Waltz 16) 13C and 31P spectra were acquired with a 30° pulse and 3-second relaxation delay. 13C aqueous spectra were acquired for 5,000 transients and a total acquisition time of 6 hours. 13C lipid spectra were acquired for 2,500 transients and a total acquisition time of 3 hours. 31P aqueous and lipid spectra were acquired for 2,500 transients and a total acquisition time of 3 hours. 1H spectra were acquired with a 90° pulse and 3-second relaxation delay and 256 transients for a total acquisition time of 25 minutes. Spectra were quantified with ACD/Spec Manager version 9.15 software (Advanced Chemistry Development). Peak integrals obtained by deconvolution were normalized to an external standard (100 mM trimethylsilyl propionate [TSP] [Sigma-Aldrich] for 13C spectra and 10 mM methylene diphosphoric acid [MDPA] [Sigma-Aldrich] for 31P spectra) and to cell number and corrected for saturation effects by using data obtained from fully relaxed spectra of cell extracts as described above. In addition, studies were performed with choline concentration at the normal concentration in human plasma. For this, cells were grown with a 10 μM choline concentration in the medium, and cell extracts and MRS experiments were performed as above.
Microarray analysis of gene expression
Total cellular RNA was isolated from approximately 1 × 107 cells after 48 hours of treatment with 17-AAG or vehicle (dimethyl sulfoxide) by using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA) in accordance with the instructions of the manufacturer. RNA quality was determined by Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and RNA integrity number (RIN) values of at least 8.0 were considered acceptable (most values were 9.5 or higher). Microarray hybridization was performed at the UCSF Genomics Core Laboratories (UCSF/Gladstone, San Francisco, CA, USA) by using the Human Gene 1.0 ST. Human Gene 1.0ST was analyzed by fluorescence detection by using the Agilent GeneArray Scanner (Agilent Technologies). Data acquisition was performed with Micro Array Suite 5.0 software (Affymetrix, Santa Clara, CA, USA). Microarray experiments were performed with four repeats of each condition. Microarray data are available through the ArrayExpress public repository [EMBL:E-MTAB-339] in compliance with the standards of the Microarray Data Gene Expression Society.
Reverse transcription-polymerase chain reaction analysis of gene expression
An RNeasy Mini Kit (Qiagen) was used to extract total cellular RNA from samples. Total RNA concentration of samples was determined by using an ND1000 Fluorospectrometer (NanoDrop Technologies, Wilmington, DE, USA). The QuantiTect Reverse Transcription kit (Qiagen) was used to perform reverse transcription. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on the resulting cDNA on a Taqman 7900 (Applied Biosystems, Carlsbad, CA, USA). Expressions of ChoKα, ChoKβ, and SLC44A1 were examined with Assays-on-Demand (Applied Biosystems) and normalized to the expression of the 18S ribosomal subunit (Integrated DNA Technologies, Coralville, IA, USA).
Choline kinase activity assay
ChoK activity was measured in cell extracts by 1
H MRS as previously described [40
]. In brief, approximately 1 × 107
cells were resuspended in 500 μL of lysis buffer containing 100 mM Tris (pH 8.0), 1 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM dithiothreitol (DTT) (all chemicals from Sigma-Aldrich). Lysate was repeatedly passaged through a fine-tipped needle (27.5-gauge) for homogenization and was sonicated 10 × 1 s at 20 kHz for membrane solubilization. The homogenate was centrifuged at 16,000 rpm for 30 minutes at 4°C, and 100 μL of reaction buffer containing 100 mM Tris (pH 8.0), 60 mM choline chloride, 120 mM ATP, and 120 mM magnesium chloride (all chemicals from Sigma-Aldrich) at the final concentration were added to the lysate supernatant. MRS experiments were performed at 25°C on a 600-MHz INOVA spectrometer (Varian). The conversion of choline to PC was measured in an array of 1
H spectra over a 1-hour period. 1
H spectra were obtained with a 90° pulse and 3-second relaxation delay. Choline and PC concentrations were determined as peak areas, and ChoK activity was determined by linear regression analysis of points in the linear portion of the curve representing the time course of PC formation.
CTP:PC cytidylyltransferase activity assay
This assay was based on the method outlined by Vance and colleagues [41
] in 1981 but with substantial modifications for compatibility with MRS. In brief, 2 × 107
cells were resuspended in 540 μL of lysis buffer containing 50 mM HEPES (pH 7.0) (Sigma-Aldrich), 5 mM EDTA (Sigma-Aldrich), 5 mM EGTA (ethylene glycol tetraacetic acid) (Sigma-Aldrich), 5.5 mM sodium bisulfite (Sigma-Aldrich), and 1 μL/mL protease inhibitor cocktail (Calbiochem). Lysate was repeatedly passaged through a fine-tipped needle (27.5-gauge) for homogenization and was sonicated 10 × 1 s at 20 kHz for membrane solubilization. The homogenate was centrifuged at 16,000 rpm for 30 minutes at 4°C. The cytidylyltransferase activity of the supernatant fraction was measured immediately after the addition of 60 μL of reaction mixture (final concentrations: 50 mM Tris-HCl [pH 8.0], 10 mM cytidine triphosphate [CTP], 5 mM PC, 5 mM DTT, and 25 mM MgCl2
) (all chemicals from Sigma-Aldrich). MRS experiments were performed at 33°C on the 600-MHz INOVA spectrometer (Varian). Proton-decoupled (Waltz 16) 31
P MR spectra were obtained with 30° pulse and 2.6-second repetition time. PC and CDP-choline concentrations were determined from peak areas. Cytidylyltransferase activity was determined by linear regression analysis of points in the linear portion of the curve representing the time course of CDP-choline formation.
Phospholipase C activity assay
PtdCho-specific PLC activity was determined in cell lysates by using the EnzChek Direct Phospholipase C Assay (Invitrogen Corporation, Carlsbad, CA, USA). The assay measures PLC activity by addition of a proprietary substrate (glycero-phosphoethanolamine with a dye-labeled sn-2 acyl chain), which is cleaved by PtdCho-specific PLC. The cleavage releases the dye-labeled diacylglycerol, which produces a positive fluorescence signal that can be measured. Fluorescence (485 nm excitation and 535 nm emission) was measured by a SpectroFluor Plus spectrofluorometer (Tecan).
Phospholipase A1/A2 activity assay
The activity of PtdCho-specific PLA1 and PLA2 enzymes was determined in cell lysates by using the EnzCheck Phospholipase A1 and Phospholipase A2 Assay kits (Invitrogen Corporation). The assay measures PLA1 activity by the addition of a proprietary substrate (dye-labeled glycerophosphoethanolamines with BODIPY FL dye-labeled acyl chain at the sn-1 position and dinitrophenyl quencher-modified head group). The sn-1 ester linkage of the substrate is cleaved by PLA1, which releases the dye-labeled acyl chain and produces a positive fluorescence signal that can be measured. The PLA2 activity assay measures the sn-2 ester link hydrolysis of the proprietary substrate 1-O-(6-BODIPY 558/568-aminohexyl)-2-BODIPY FL C5-Sn-glycero-3-phosphocholine by PLA2 enzyme, which releases the dye-labeled portion and produces a positive fluorescent signal that can be measured. Fluorescence (505 nm excitation and 515 nm emission) was measured by a SpectroFluor Plus spectrofluorometer (Tecan).
The two-tailed Student t test was used to determine the statistical significance of the results, and a P value of not more than 0.05 was considered significant. All results are expressed as mean ± standard deviation.