Cancer Research AACR Conference on Molecular Diagnostics - 2008 AACR Conference on Molecular Diagnostics - 2008
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beloueche-Babari, M.
Right arrow Articles by Ronen, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beloueche-Babari, M.
Right arrow Articles by Ronen, S. M.
[Cancer Research 65, 3356-3363, April 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Magnetic Resonance Spectroscopy Monitoring of Mitogen-Activated Protein Kinase Signaling Inhibition

Mounia Beloueche-Babari1, L. Elizabeth Jackson1, Nada M.S. Al-Saffar1, Paul Workman2, Martin O. Leach1 and Sabrina M. Ronen1

1 Cancer Research UK Clinical Magnetic Resonance Research Group, Institute of Cancer Research and Royal Marsden NHS Foundation Trust, Sutton and 2 Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, United Kingdom

Requests for reprints: Mounia Beloueche-Babari, Cancer Research UK Clinical Magnetic Resonance Research Group, Institute of Cancer Research, and Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: 44-208-661-3738; Fax: 44-208-661-0846; E-mail: Mouniab{at}icr.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several mitogen-activated protein kinase (MAPK) signaling inhibitors are currently undergoing clinical trial as part of novel mechanism-based anticancer treatment strategies. This study was aimed at detecting biomarkers of MAPK signaling inhibition in human breast and colon carcinoma cells using magnetic resonance spectroscopy. We investigated the effect of the prototype MAPK kinase inhibitor U0126 on the 31P-MR spectra of MDA-MB-231, MCF-7 and Hs578T breast, and HCT116 colon carcinoma cells. Treatment of MDA-MB-231 cells with 50 µmol/L U0126 for 2, 4, 8, 16, 24, 32, and 40 hours caused inhibition of extracellular signal–regulated kinases (ERK1/2) phosphorylation from 2 hours onwards. 31P-MR spectra of extracted cells indicated that this was associated with a significant drop in phosphocholine levels to 78 ± 8% at 8 hours, 74 ± 8% at 16 hours, 66 ± 7% at 24 hours, 71 ± 10% at 32 hours, and 65 ± 10% at 40 hours post-treatment. In contrast, the lower concentration of 10 µmol/L U0126 for 40 hours had no significant effect on either P-ERK1/ 2 or phosphocholine levels in MDA-MB-231 cells. Depletion of P-ERK1/2 in MCF-7 and Hs578T cells with 50 µmol/L U0126 also produced a drop in phosphocholine levels to 51 ± 17% at 40 hours and 23 ± 12% at 48 hours, respectively. Similarly, in HCT116 cells, inhibition with 30 µmol/L U0126 caused depletion of P-ERK1/2 and a decrease in phosphocholine levels to 80 ± 9% at 16 hours and 61 ± 4% at 24 hours post-treatment. The reduction in phosphocholine in MDA-MB-231 and HCT116 cells correlated positively with the drop in P-ERK1/2 levels. Our results show that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in cellular phosphocholine levels. Thus, phosphocholine has potential as a noninvasive pharmacodynamic marker for monitoring MAPK signaling blockade.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mitogen activated protein kinases (MAPKs) are a family of protein kinases involved in many key cellular functions including cell proliferation, apoptosis and motility. MAPKs fall into four main categories: extracellular signal–regulated kinases (ERK1/2), c-Jun NH2-terminal kinases (JNK1/2/3), and p38 kinases (p38{alpha}/ß/{gamma}/{delta}), in addition to the more recently identified ERK5 (1). The ERK1/2 (or the classic MAPK) pathway constitutes a well-characterized component of the Ras signaling cascade (2). Activated Ras catalyses recruitment and activation of C-Raf which in turn activates MAPK kinases (MEK1/2). Once activated, MEK1/2 phosphorylates and activates ERK1/ERK2, which results in activation of several target proteins, including transcription factors that regulate gene expression and promote cellular proliferation (3).

Accumulating evidence suggests that ERK1/2 signaling could be involved in the pathology of human cancers. Ras signal transduction proteins are activated in nearly 30% of all human cancers (4, 5). For example, K-ras mutations are observed in around 70% of human colorectal tumors (4, 6) and the mutation has been shown to correlate with poor clinical outcome (6). Raf is activated in tumor cells harboring mutated growth factor receptors (7, 8) and mutations in the B-raf gene have been reported in several human cancers and in particular in malignant melanomas (7–10). In addition, MEK signaling is activated by oncogenic growth factors and reports have shown that MEK activation can induce cellular transformation (as reviewed in ref. 7). Because of its importance in malignant transformation, Ras-Raf-ERK1/2 signaling has emerged as a key target for the development of novel mechanism-based antitumor agents targeted at signal transduction pathways that drive the malignant phenotype of tumor cells (7, 8).

Preclinical studies have shown the potential therapeutic benefits of MEK1/2 signaling inhibition, including suppression of growth and induction of apoptosis in various types of animal tumor models including the colon, the lung, and melanoma (11–13). Moreover, blockade of this pathway is expected to affect cell invasion and angiogenesis, as indicated by the involvement of MAPK signaling in these processes (7).

Several inhibitors of the Ras-Raf-ERK1/2 pathway are now in clinical trial. These include antisense oligonucleotides targeting the mRNA transcripts of H-ras (ISIS 2503) and C-raf (ISIS 5132), in addition to small molecule inhibitors of Ras farnesylation (e.g., R115777), C-Raf (BAY 43-9006), and MEK1/2 (CI-1040, formerly known as PD184352) as reviewed in refs. (7, 8, 14).

Monitoring the molecular response to these novel therapies is crucial for correlating antitumor effects with inhibition of the intended biochemical target and assessing the efficacy of treatment (15, 16). Thus, detecting markers of response to ERK1/2 signaling inhibition will be very important in the clinical evaluation of this novel type of anticancer treatment. Measurement of signaling pathway inhibition in human tissue by molecular biological techniques is surgically invasive, requiring a tissue biopsy. For example, measurement of ERK1/2 phosphorylation has been used to show proof of principle for MEK inhibition by CI-1040 (11). Thus, surgically noninvasive pharmacodynamic endpoints involving molecular spectroscopy and imaging methodology would have a significant advantage (15, 16).

Magnetic resonance spectroscopy (MRS) is a valuable technique for studying cell and whole tissue biochemistry as it allows noninvasive detection and monitoring of a variety of metabolites in one single measurement (17). The applications of MRS to biological systems are diverse and include monitoring of tissue energy and metabolism and drug pharmacokinetics (18). MRS is increasingly used for monitoring changes in tumor cell metabolism in response to anticancer therapies in cells, animal models, and patients (18–20). Studies have shown that in vivo 31P-MR spectra of tumors exhibit alterations in the level of intermediates originating from phospholipid metabolism compared with normal tissue. Tumors often show larger signals from phosphomonoesters (including phosphocholine and phosphoethanolamine) and phosphodiesters (including glycerophosphocholine and glycerophosphoethanolamine). MRS has also shown that response to traditional anticancer treatments, such as radiotherapy and chemotherapy, often correlates with a drop in the levels of phosphomonoesters (18).

Despite the widespread presence of ras mutations in human cancers, only a handful of studies have used MRS to assess the effect of activation of Ras and, to a lesser extent, specific downstream signaling pathways, on tumor cell metabolism. Using 1H-MRS, it has been shown that simultaneous immortalization and transformation of rat Schwann cells with SV40 large T antigen and H-ras, respectively, induced a significant increase in phosphocholine and a decrease in glycerophosphocholine levels relative to primary cells (21). Another 1H-MRS study showed that H-ras-transfected NIH3T3 murine fibroblasts contained lower levels of unsaturated fatty chains relative to the parent line (22). Using 31P-MRS, we have shown that Ras activation in NIH3T3 fibroblasts correlated with an increase in phosphocholine levels which was reversed upon treatment with novel Ras signaling inhibitors (23). In this study, we have investigated four human carcinoma cell lines, three breast, and one colorectal, to determine whether MRS could detect metabolic biomarkers associated with selective inhibition of MAPK signaling with the prototype MEK inhibitor U0126 (24). Our results indicate that MAPK signaling inhibition with U0126 is associated with a time-dependent decrease in phosphocholine levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell culture. MDA-MB-231 and MCF-7 breast and HCT116 colorectal carcinoma cells were maintained in DMEM containing 10% (v/v) heat inactivated FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin. Hs578T breast carcinoma cells were grown in RPMI containing 10% (v/v) FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine. Tissue culture materials were purchased from Life Technologies (Paisley, United Kingdom). All cells were maintained in 175-cm2 tissue culture flasks at 37°C in a 5% CO2 humidified atmosphere and growth medium replenished every 48 hours.

Mitogen-activated protein kinase signaling inhibition. MDA-MB-231 cells were treated for 2, 4, 8, 16, 24, 32, and 40 hours with 50 µmol/L of the MEK1/2 inhibitor U0126 or for 40 hours with 10 µmol/L U0126 (refs. 24, 25; Promega, Southampton, United Kingdom) to achieve ca. 50% drop in cellular proliferation at 40 hours. MCF-7 and Hs578T breast carcinoma cells were treated with 50 µmol/L U0126 for 40 and 48 hours, respectively. HCT116 human colon carcinoma cells were treated with 30 µmol/L U0126 for 6, 16, and 24 hours. All control cells received growth medium containing DMSO at concentrations equivalent to those given to treated cells [i.e., 0.02% (v/v) for 10 µmol/L U0126 or 0.1% (v/v) for 50 µmol/L and 30 µmol/L U0126].

Analysis of P-ERK1/2 levels. To monitor the effect of U0126 on MAPK signaling, the levels of phosphorylated ERK1/2 were assessed using Western blotting. For this, cells were lysed in lysis buffer (150 mmol/L NaCl, 28.2 mmol/L Tris-HCl, 1.1 mmol/L Tris base, 0.2% SDS, 1% NP40, 10% glycerol, 0.5 mmol/L sodium orthovanadate, 10 mmol/L sodium pyrophosphate, 0.1 mol/L sodium fluoride, and 1 mmol/L EDTA). Equal amounts of total protein (as measured using the Bio-Rad assay method and bovine serum albumin as a standard) were loaded onto 10% polyacrylamide gels and protein transferred onto immobilon-P membranes (Millipore, Bedford MA). Blots were then blocked in 5% nonfat milk for 2 hours and incubated with a primary anti-P-ERK1/2 antibody (Sigma, Dorset, United Kingdom) for 1 hour. This was followed by incubating the blots with horseradish peroxidase–linked secondary anti-mouse antibody (Amersham Biosciences, Buckinghamshire, United Kingdom) for 1 hour. Equal protein loading was verified by stripping the blots in 1x Re-Blot Plus Strong Solution (Chemicon, Hampshire, United Kingdom) for 15 minutes and incubating with an anti-glyceraldehyde-3-phosphate dehydrogenase primary antibody for 1 hour (Chemicon). This was followed by incubation with an anti-mouse secondary antibody. Detection of specific antibody-antigen interactions was achieved using Enhanced Chemiluminescence Plus reagents (Amersham Biosciences) and exposure to X-OMAT Kodak autoradiography film (Rochester, NY). Bands representing P-ERK1/2 were quantified by densitometry using Image Quant version 5 (Molecular Dynamics, Sunnyvale, CA).

Cell cycle analysis. Flow cytometry was used to assess the effect of MAPK signaling inhibition on the cell cycle distribution of treated cells. For this, ~2 x 106 cells were fixed in 70% ice-cold ethanol and stained with a solution containing 40 µg/mL propidium iodide and 100 µg/mL RNase A (Sigma) in PBS. Cells were then sorted using an Elite ESP Beckman Coulter cell sorter at 488 nm and data analyzed using WinMDI computer software version 2.7 (Scripps Institute, La Jolla, CA) and Cylchred computer software version 1.0.2 (College of Medicine, University of Wales, United Kingdom).

Cell extracts. To determine the effect of MAPK signaling inhibition on cellular metabolism, control and U0126-treated cells were extracted using the dual phase extraction method (23, 26). Lyophilized samples of the water-soluble phase were reconstituted in 700 µL of a D2O solution containing 10 mmol/L EDTA and 0.86 mmol/L methylenediphosphonic acid (as internal reference) at pH 8.2.

31P magnetic resonance spectroscopy measurements. 31P-MR spectra were acquired at room temperature on a 500-MHz Bruker spectrometer using power gated composite pulse 1H decoupling, a 30° flip angle, a 2-second repetition time, a spectral width of 100 ppm and acquiring 32,000 data points. Spectra were processed using a 1-Hz line broadening and analyzed using MestRe-C version 2.3 (University of Santiago de Compostela, Spain). Metabolite concentrations were determined by peak integration, normalized relative to the internal standard, and corrected for saturation and cell number in each sample.

Statistical analysis. An unpaired two-tailed Student's t test was used to determine the statistical significance of the results with Ps ≤ 0.05 considered significant. Data are shown as the mean ± SD. Linear regression analysis was done using SPSS version 11.5.1 (SPSS, Inc., Chicago, IL), providing the Pearson correlation coefficient (r) and the level of significance (P).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mitogen-Activated Protein Kinase Signaling Inhibition in Human Breast Carcinoma Cells
Effect of treatment with 50 µmol/L U0126 in MDA-MB-231 cells. Western blot analysis indicated that treatment of MDA-MB-231 cells with 50 µmol/L U0126 induced total suppression of ERK1/2 phosphorylation that was detectable as early as 2 hours post-treatment (Fig. 1A and B). P-ERK1/2 levels dropped to below detection level at 2, 4, and 8 hours post-treatment. Some recovery of P-ERK1/2 levels was seen at later times with values (as a percentage of control) of 10 ± 10% at 16 hours (P = 0.0006), 24 ± 24% at 24 hours (P = 0.01), 14 ± 16% at 32 hours (P = 0.02), and 24 ± 8% at 40 hours (P = 0.006). No change was observed in the levels of total ERK1/2 (data not shown). In addition, this U0126 exposure produced a time-dependent reduction in cell number per flask that was significant from 32 hours (60 ± 3% relative to control; n = 4, P = 0.0001) and which reached 50 ± 10% relative to control by 40 hours (n = 5, P = 0.0006; Fig. 1B). Analysis by flow cytometry showed that treatment with 50 µmol/L U0126 also triggered significant alterations in cell cycle distribution (Fig. 1C). A significant increase in G1 (49 ± 3% to 70 ± 2%, P = 0.002) as well as a drop in S phase (40 ± 3% to 23 ± 2%, P = 0.002) were both visible from 16 hours post-treatment onwards. A decrease in G2 (11 ± 2% to 7 ± 1%, P = 0.05) was also detected at 24 hours and sustained for up to 40 hours following treatment.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. The effect of inhibition with 50 µmol/L U0126 on MAPK signaling, cellular proliferation, and cell cycle distribution in MDA-MB-231 cells. A, Western blots of depletion of P-ERK1/2 at 2, 4, and 8 hours post-treatment with evidence of partial recovery from 16 hours onwards. B, quantitative analysis of P-ERK1/2 levels ({square}) and cell number/flask ({blacksquare}) following treatment at the different time points. C, cell cycle analysis showing no significant alterations at 2, 4, and 8 hours but an increase in G1 and a drop in S-phase populations from 16 to 40 hours after U0126 treatment. A drop in the G2-phase population is also detected at 24, 32, and 40 hours following treatment. *indicates a statistically significant difference from the control i.e. P ≤ 0.05.

 
We next acquired 31P-MR spectra from control and treated cells. A 31P-MR spectrum from the aqueous fraction of control MDA-MB-231 cell extracts is illustrated in Fig. 2A. 31P-MRS of U0126 treated cells indicated that cellular phosphocholine levels did not change significantly at 2 hours (102 ± 19%, P = 0.9) and 4 hours (83 ± 13%, P = 0.2) but decreased to 78 ± 8% at 8 hours (P = 0.05) and remained at an average level of 70 ± 10% (P ≤ 0.04) relative to control for up to 40 hours post-treatment (Fig. 2B). The drop in phosphocholine correlated strongly and positively with the reduction in P-ERK1/2 levels following U0126 treatment (r = 0.845, P = 0.008) with exclusion of the 2-hour time point, which exhibited a lag in the effect on phosphocholine presumably as a result of intermediate signaling processes. Data from the 40-hour time point are also summarized in Table 1. Following 40 hours of U0126 treatment phosphocholine levels were 65 ± 10% relative to control (P = 0.0006). A significant increase in glycerophosphoethanolamine levels to 157 ± 27% (P = 0.01) was also detected, whereas no significant changes occurred in glycerophosphocholine or nucleotide triphosphate (NTP) levels. Thus, the phosphocholine/NTP ratio, important in in vivo studies, decreased significantly to 80 ± 11% relative to control (P = 0.01).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. The effect of inhibition with 50 µmol/L U0126 on phosphocholine (PC) levels in MDA-MB-231 cells. A, 31P MR spectrum from a MDA-MB-231 cell extract showing an expansion of the PC region in control and U0126-treated cells at 40 hours (inset). Spectra are normalized for cell number per sample. Inorganic phosphate (Pi); glycerolphosphoethanolamine (GPE); glycerophosphocholine (GPC); {alpha}-NTP, ß-NTP, and {gamma}-NTP; uridine diphosphate sugars (UDPS). B, cellular phosphocholine levels dropped from 8 hours post-treatment and remained lower than control levels throughout the course of the experiment. *indicates a statistically significant difference from the control i. e. P ≤ 0.05.

 

View this table:
[in this window]
[in a new window]
  		Table 1. The effect of MAPK signaling inhibition with U0126 in MDA-MB-231 (50 µmol/L for 40 hours), MCF-7 and Hs578T (50 µmol/L for 40 and 48 hours, respectively), and HCT116 (30 µmol/L for 24 hours) cells on the 31P MRS-detectable water-soluble metabolites

 
Effect of treatment with 10 µmol/L U0126 in MDA-MB-231 cells. To confirm that modulation of phosphocholine levels was associated with P-ERK1/2 levels, we also treated MDA-MB-231 cells with a lower concentration of U0126 and investigated the cells at a time point when ERK1/2 phosphorylation was not affected. Treatment of MDA-MB-231 cells with 10 µmol/L U0126 for 40 hours had no effect on P-ERK1/2 levels (equal to 124 ± 32% relative to control, P = 0.4). We next assessed whether this lower concentration of 10 µmol/L U0126 had any effects on the 31P–containing metabolites. 31P-MRS analysis revealed no significant differences in levels of either phosphocholine (37 ± 6 versus 39 ± 8 fmol/cell), glycerophosphocholine (4 ± 1 versus 5 ± 2 fmol/cell), or NTP levels (10 ± 2 versus 11 ± 3 fmol/cell) between control and treated cells. Interestingly, and as with the higher concentration of 50 µmol/L U0126, treatment with 10 µmol/L U0126 was associated with a significant increase in glycerophosphoethanolamine levels from 1.7 ± 04 to 2.9 ± 0.6 fmol/cell (n = 5, P = 0.01).

Effect of inhibition with U0126 in MCF-7 and Hs578T cells. The effect of MAPK signaling inhibition on cellular 31P-MRS detectable metabolites was further investigated in two additional human breast cancer cell lines. Western blotting showed that treatment of MCF-7 and Hs578T cells with 50 µmol/L U0126, for 40 and 48 hours, respectively, caused depletion of P-ERK1/2 levels, confirming blockade of MAPK signaling (Fig. 3). The levels of total ERK1/2 were not altered following U0126 treatment (data not shown). Exposure to U0126 also caused a significant drop in cellular proliferation to 54 ± 4% (n = 5, P = 0.0001) and 66 ± 11% (n = 3, P = 0.05) relative to control, in MCF-7 and Hs578T cells, respectively.



View larger version (10K):
[in this window]
[in a new window]
  		Figure 3. The effect of inhibition with 50 µmol/L U0126 on MAPK signaling in MCF-7 and Hs578T human breast cancer cells. Western blots of the treatment of MCF-7 and Hs578T cells with 50 µmol/L U0126 for 40 and 48 hours, respectively, caused depletion of P-ERK1/2 levels.

 
Flow cytometry analysis showed that, similarly to MDA-MB-231 cells, treatment with U0126 caused a significant increase in the G1 phase population from 55 ± 5% to 66 ± 5% (n = 4, P = 0.04) in MCF-7 cells and from 73 ± 2% to 81 ± 1% (n = 3, P = 0.004) in Hs578T cells. U0126 also caused a drop in the S phase population in MCF-7 cells (36 ± 2% to 29 ± 4%, P = 0.04) and in Hs578T cells (20 ± 4% to 10 ± 1%, P = 0.01), whereas no significant effect was observed on the G2 phase population in either cell line.

31P-MR spectra from cell extracts indicated that, as with MDA-MB-231 cells, U0126 treatment caused a significant drop in the levels of phosphocholine to 53 ± 16% in MCF-7 (n = 5, P = 0.004) and 22 ± 12% in Hs578T cells (n = 3, P = 0.01) relative to control. Data obtained from the two cell lines are summarized in Table 1. Glycerophosphoethanolamine was only detectable in MCF-7 cells and its levels increased to 150 ± 10% following U0126 treatment (P = 0.01). No significant changes were observed in the other 31P-MRS-detectable metabolites in both lines. Consequently the phosphocholine/NTP ratio dropped significantly to 57 ± 28% in MCF-7 cells (P = 0.03) and to 26 ± 14% in Hs578T cells (P = 0.02).

Mitogen-Activated Protein Kinase Signaling Inhibition in HCT116 Human Colon Carcinoma Cells
The effect of inhibition of the MAPK pathway on the 31P-MR-detectable metabolites was also investigated in a human cancer cell line derived from a different tissue type (i.e., colon). Inhibition of MAPK signaling with 30 µmol/L U0126 for 6, 16, and 24 hours in HCT116 cells was associated with a time-dependent reduction in cell number per flask to 84 ± 3% relative to control (P = 0.003) by 24 hours post-treatment which was indicative of decreased cell proliferation (Fig. 4B). In addition, Western blot analysis showed that U0126 treatment induced a substantial drop in ERK1/2 phosphorylation that was detectable from 6 hours and sustained for up to 24 hours of exposure to U0126 (Fig. 4A and B). P-ERK1/2 levels dropped to 3 ± 4%, 7 ± 8%, and 1 ± 1% relative to control at 6, 16, and 24 hours post U0126 treatment, respectively (P ≤ 0.004). No change was recorded in total ERK1/2 levels (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 4. The effect of inhibition with 30 µM U0126 on MAPK signalling in HCT116 human colon cancer cells. A, western blots showing that treatment of HCT116 cells with 30 µmol/L U0126 led to depletion of P-ERK1/2 levels at 6 h, 16 h and 24 h post-treatment. B, inhibition with 30 µmol/L U0126 caused depletion of P-ERK1/2 levels (X) from 6 h onwards (as quantified by densitometric analysis), a time-dependent drop in cell number per flask ({bullet}) and in cellular PC levels ({circ}) from 16 h onwards (as measured by 31P MRS of cell extracts). C, cell cycle analysis showing a G1 arrest and a drop in the S phase population at 16 h and 24 h and a decrease in the G2 phase population at 24 h. * Indicates a statistically significant difference from the control i.e. P ≤ 0.05.

 
Flow cytometry analysis showed that, similarly to the three breast lines, inhibition of HCT116 cells with U0126 was associated with an increase in the G1 cell population (22 ± 6% to 58 ± 5%, P = 0.0001) and a drop in the S-phase cell population (57 ± 7 to 26 ± 6%, P = 0.0002) that were observed from 16 hours onwards. A drop in the G2-phase population (21 ± 3% to 11 ± 1%, P = 0.0002) was also recorded at 24 hours (Fig. 4C).

We next assessed whether inhibition with U0126 was associated with changes in the levels of 31P-containing metabolites of treated cells. 31P-MR analysis of the water-soluble fraction of cell extracts showed that phosphocholine levels were not significantly altered at 6 hours (86 ± 16% relative to control; n = 3, P = 0.22) but decreased significantly to 80 ± 8% (n = 3, P = 0.01) and 61 ± 4% (n = 4, P = 0.0004) at 16 and 24 hours post-treatment, respectively (Fig. 4B). A drop in the phosphocholine/NTP ratio to 78 ± 11% (P = 0.04) relative to control was also detectable at 24 hours but not earlier. Data from the 24-hour time point are summarized in Table 1. The decrease in phosphocholine showed a similar correlation with the decrease in P-ERK1/2 as that found in MDA-MB-231 cells. When the data from the MDA-MB-231 and HCT116 cell studies were combined they showed a similar positive correlation (r = 0.813, P = 0.001) between phosphocholine and P-ERK1/2 to that shown by MDA-MB-231 cells alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As part of the novel molecularly targeted approach to cancer treatment, several inhibitors of the receptor tyrosine kinase Ras-Raf-MEK1/2-ERK1/2 signaling pathway are currently undergoing clinical trial (7, 8, 14) including the MEK1/2 inhibitor CI-1040 (7, 11, 27). The ability to monitor molecular target inhibition is critically important for the development and evaluation of such novel agents in the clinic. Surgically noninvasive methods are particularly attractive (15, 16). In the present study, we have used a noninvasive technique (i.e., MRS), to detect potential biomarkers of MAPK signaling inhibition in human breast and colon cancer cells using the commercially available prototype MEK1/2 inhibitor U0126 (24). Like CI-1040 and PD98059, U0126 seems to act by preventing the activation of MEK1/2 by C-Raf more potently than it inhibits the catalytic activity of MEK1/2 (25). However, in addition to blocking ERK1/2 signaling U0126 can also inhibit the activation of another MAPK that might be involved in cell cycle entry and cell transformation (i.e., ERK5; refs. 28–31), at concentrations comparable with those required for inhibition of ERK1/2 phosphorylation (32). In MDA-MB-231 cells, inhibition with 50 µmol/L U0126 caused total abrogation of ERK1/2 phosphorylation that was detectable at 2, 4, and 8 hours with evidence of partial recovery from 16 hours post-treatment and thereafter. In addition, a progressive antiproliferative effect was also observed. 31P-MRS showed that this U0126 treatment was associated with a significant drop in phosphocholine levels that commenced with a time lag following the decrease in P-ERK1/2 levels at 8 hours post-treatment and was sustained throughout the remaining time points of the experiment. Thus, inhibition of MAPK signaling was associated with changes in the cellular content of phosphocholine, although this change was temporally delayed, as might be expected for a downstream signaling process. This drop in phosphocholine correlated strongly and positively with the drop in P-ERK1/2 levels following inhibition with U0126.

Phosphocholine is the first intermediate in the Kennedy pathway for the de novo synthesis of the membrane phospholipid phosphatidylcholine and is formed following choline phosphorylation via choline kinase (33). Phosphocholine can also result from the breakdown of phospholipid phosphatidylcholine via phospholipase C (33). Reports have shown that phospholipid turnover can be regulated by the cell cycle (33, 34). Specifically, in MDA-MB-231 cells, phosphocholine levels were reported to be 50% lower in confluent cells relative to logarithmically growing cells (35). In addition, it was found that the size of the phosphocholine pool was reduced by a half in large T47D multicellular spheroids with a considerable population of quiescent cells when compared with smaller spheroids with a greater proportion of proliferating cells (36). A direct positive correlation between phosphocholine levels and the proportion of cells in S phase has also been reported (37). In our experiments with MDA-MB-231 cells, the drop in phosphocholine observed following treatment with 50 µmol/L U0126 preceded the antiproliferative effect of the MEK inhibitor and thus was not likely to result from it. To determine the involvement of cell cycle changes in the effect observed on phosphocholine, we analyzed the cell cycle distribution of control and treated cells at each time point of our experiments. Treatment with 50 µmol/L U0126 caused a drop in the S phase and an increase in the G1-phase cell populations that were detectable from 16 hours and sustained throughout the time course of treatment. This effect on cell cycling has also been observed with the more specific MEK1/2 inhibitor CI-1040 (11), further corroborating the importance of MAPK signaling for progression through G1 (38). Because the drop in phosphocholine occurred earlier than the changes in cell cycle distribution, we concluded that it could not result simply from the effects of cell cycle arrest.

To further test whether the change in phosphocholine was related to MAPK signaling, MDA-MB-231 cells were treated with a lower concentration of 10 µmol/L U0126 that did not affect ERK1/2 phosphorylation. 31P-MRS analysis indicated that phosphocholine levels were not significantly altered in 10 µmol/L treated cells relative to control. These observations supported our conclusion of an association between the effect of U0126 on P-ERK1/2 and phosphocholine.

We extended our investigation to two other human breast cancer cell lines (i.e., MCF-7 and Hs578T cells) together with the HCT116 human colon cancer cell line to further strengthen our findings and test their generality. Our results showed that MAPK signaling inhibition in HCT116 cells was also associated with a time-dependent drop in phosphocholine levels that was temporally preceded by the drop in P-ERK1/2 levels. A significant drop in phosphocholine levels and in phosphocholine/NTP ratio was also detected following inhibition of ERK1/2 phosphorylation in Hs578T and MCF-7 breast cancer cells. Comparison of results from all four cell lines indicated that the amplitude of the drop in phosphocholine and the phosphocholine/NTP was inversely proportional to the level of these variables in control cells with the biggest change being observed in the line with the lowest levels and vice versa. In addition to the effect on phosphocholine, an increase in glycerophosphoethanolamine levels was also observed in MDA-MB-231 as well as MCF-7 cells following treatment with U0126. Interestingly, and in the case of MDA-MB-231 cells at least, this increase in glycerophosphoethanolamine levels was recorded at both the 10 and 50 µmol/L concentrations (i.e., in the presence and absence of effects on ERK1/2 signaling at 40 hours). It is therefore possible that this increase could be attributed to off-target effects of U0126.

It has been shown that cellular phosphocholine levels are sensitive to the concentration of external precursor levels, and that these increase in response to increased concentration of choline in the medium (39, 40). Interestingly, despite the greater amount of choline in the medium available per cell following the drop in cell number triggered upon U0126 treatment, a clear decrease in phosphocholine was still detectable. This indicated that the actual net drop in phosphocholine could be greater than the apparent drop observed in our experiments.

Furthermore, it is known that phosphocholine is involved in a range of cellular activities including lipid membrane biosynthesis and mitogenic signaling (33). It is therefore plausible that many factors may contribute to the maintenance of its levels and consequently the following issues have to be taken into account. (a) The temporal displacement between the effect on P-ERK1/2 (immediate) and that on phosphocholine (later) as shown by this study, (b) the different basal levels of phosphocholine (and P-ERK1/ 2) in different cell lines, and (c) the fact that phosphocholine could be regulated by pathways other than MAPK. Indeed, the observation that following depletion of P-ERK1/2, cellular phosphocholine content decreased relative to the control but remained detectable supports the argument about the involvement of pathways other than MAPK in the maintenance of phosphocholine levels. Interestingly, and despite the potential variations across tumor cell lines in the extent to which MAPK signaling may be involved in phosphocholine metabolism, we found a similar positive correlation between phosphocholine and P-ERK1/2 levels in MDA-MB-231 breast and HCT116 colon cancer cells. Previous work has shown that Ras activation results in up-regulation of several key enzymes involved in the modulation of phospholipid metabolism. These include choline kinase and phospholipase D (41), phospholipases A and C (42–44), and CTP/phosphocholine cytidylyltransferase (ct; refs. 45, 46). Treatment with the less potent MEK1/2 inhibitor PD98059 caused a reduction in the level of ct gene expression in H-ras transformed C3H10T1/2 mouse fibroblasts (46). In addition, inhibition of MAPK signaling with U0126 has been shown to block phosphocholine formation in human colon carcinoma cells by interfering with choline uptake and phosphorylation via choline kinase (47). Interestingly, phosphocholine was found, in some cases, to be required for activation of C-Raf and ERK1/2 as well as DNA synthesis, and inhibition of choline kinase activity was sufficient to cause blockade of MAPK signaling (48). Indeed, more recent evidence has also shown that inhibition of choline kinase resulted in a drop in intracellular phosphocholine levels which correlated with inhibition of mitogenic signaling in human breast cancer cells (49). Thus, one possible mechanism behind the drop in phosphocholine in our cells is inhibition of choline uptake and phosphorylation following MAPK signaling blockade. Furthermore, the observation that the decrease in phosphocholine is detected later than the drop in P-ERK1/2 suggests that multiple steps are required between signaling downstream of ERK1/2 and choline metabolism. This hypothesis requires further investigation.

The findings of the present study are in keeping with our previous results showing that MAPK signaling inhibition with U0126 in ras-transformed NIH3T3 murine fibroblasts was also associated with a significant drop in phosphocholine levels and in the phosphocholine/NTP ratio (50). It is worth noting that we have recently reported an elevation in phosphocholine levels in human colon cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino, 17-demethoxygeldanamycin which is known to deplete many signaling proteins including C-Raf and P-Akt and the cell cycle kinase Cdk-4 (51). However, in this case, given the multitude of targets modulated simultaneously by this inhibitor, the increase in phosphocholine may not necessarily be linked solely to C-Raf depletion and the subsequent MAPK signaling inhibition.

The present study has shown that pharmacologic inhibition of MAPK signaling in human breast and colon cancer cells, induced by the prototype MEK inhibitor U0126, is associated with a drop in phosphocholine levels that precedes any effects on cell cycling or growth. Comparison of two different concentrations of U0126 at the investigated exposure times in MDA-MB-231 cells showed that the drop in phosphocholine occurred in the presence of P-ERK1/2 signaling suppression and was not seen in the absence of effects on P-ERK1/2 levels. In addition, this effect was confirmed in three different breast cancer cell lines and one colorectal cancer line, suggesting that it may be a general effect associated with inhibition of the MAPK pathway. However, it would be of interest to assess the effect on the 31P-MR spectra of treated cells of even more specific ERK1/2 inhibitors such as CI-1040, when these become more widely available, because U0126 is also known to block ERK5 signaling. This would in particular help determine whether the observed effect on phosphocholine was associated with specific inhibition of ERK1/2, ERK5, or both signaling pathways. These experiments as well as investigation of intact cells and tumor xenograft models will be necessary to further test the usefulness of phosphocholine as a noninvasive biomarker of MAPK signaling inhibition.

The results presented in this study indicate that phosphocholine has potential as a biomarker for noninvasive monitoring of MAPK signaling blockade. This is consistent with the need to implement biomarkers of target inhibition, particularly those that are noninvasive, in contemporary drug development (15, 16, 52–54). Our findings could open up new possibilities for using MRS to noninvasively monitor response to novel anti-MAPK signaling therapies in vivo. They also highlight the value of defining the appropriate time point for potential response assessment during clinical trials of novel MAPK signaling inhibitors.


   Acknowledgments
 
Grant support: Cancer Research UK [CUK] grants C1060/A808 (M. Beloueche-Babari, L.E. Jackson, M.O. Leach, and S.M. Ronen) and C309/A2187 (P. Workman), AICR grant 03-304 (N.M.S. Al-Saffar), and Cancer Research UK Life Fellow (P. Workman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank J. Titley for help with flow cytometry analyses.


   Footnotes
 
Note: S.M. Ronen is currently at the Department of Experimental Diagnostic Imaging, M.D. Anderson Cancer Center, Houston, TX 77030-4095.

Received 9/22/03. Revised 1/14/05. Accepted 2/10/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002;298:1911–2.[Abstract/Free Full Text]
  2. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene 1998;17:1395–413.[CrossRef][Medline]
  3. Adjei AA. Ras signaling pathway proteins as therapeutic targets. Curr Pharm Des 2001;7:1581–94.[CrossRef][Medline]
  4. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9.[Abstract/Free Full Text]
  5. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70.[CrossRef][Medline]
  6. Andreyev HJN, Norman AR, Cunningham D, Oates JR, Clarke PA. Kirsten ras mutations in patients with colorectal cancer: the multicenter "RASCAL" study. J Natl Cancer Inst 1998;90:675–84.[Abstract/Free Full Text]
  7. Sebolt-Leopold JS. Development of anticancer drugs targeting the MAP kinase pathway. Oncogene 2000;19:6594–9.[CrossRef][Medline]
  8. Hilger RA, Scheulen ME, Strumberg D. The Ras-Raf-MEK-ERK pathway in the treatment of cancer. Onkologie 2002;25:511–8.[CrossRef][Medline]
  9. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949–54.[CrossRef][Medline]
  10. Brose MS, Volpe P, Feldman M, et al. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 2002;62:6997–7000.[Abstract/Free Full Text]
  11. Sebolt-Leopold JS, Dudley DT, Herrera R, et al. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med 1999;5:810–6.[CrossRef][Medline]
  12. Collisson EA, De A, Suzuki H, Gambhir SS, Kolodney MS. Treatment of metastatic melanoma with an orally available inhibitor of the Ras-Raf-MAPK Cascade. Cancer Res 2003;63:5669–73.[Abstract/Free Full Text]
  13. Kramer BW, Gotz R, Rapp UR. Use of mitogenic cascade blockers for treatment of C-Raf induced lung adenoma in vivo: CI-1040 strongly reduces growth and improves lung structure. BMC Cancer 2004;4:24.[CrossRef][Medline]
  14. Nottage M, Siu LL. Rationale for ras and raf-kinase as a target for cancer therapeutics. Curr Pharm Des 2002;8:2231–42.[CrossRef][Medline]
  15. Gelmon KA, Eisenhauer EA, Harris AL, Ratain MJ, Workman P. Anticancer agents targeting signaling molecules and cancer cell environment: challenges for drug development? J Natl Cancer Inst 1999;91:1281–7.[Free Full Text]
  16. Workman P. Challenges of PK/PD measurements in modern drug development. Eur J Cancer 2002;38:2189–93.
  17. Gadian DG. NMR and its applications to living systems. New York: Oxford University Press Inc.; 1995.
  18. Negendank W. Studies of human tumors by MRS: a review. NMR Biomed 1992;5:303–24.[Medline]
  19. De Certaines JD, Larsen VA, Podo F, Carpinelli G, Briot O, Henriksen O. In vivo 31P MRS of experimental tumours. NMR Biomed 1993;6:345–65.[Medline]
  20. Leach MO, Verrill M, Glaholm J, et al. Measurements of human breast cancer using magnetic resonance spectroscopy: a review of clinical measurements and a report of localized 31P measurements of response to treatment. NMR Biomed 1998;11:314–40.[CrossRef][Medline]
  21. Bhakoo KK, Williams SR, Florian CL, Land H, Noble MD. Immortalization and transformation are associated with specific alterations in choline metabolism. Cancer Res 1996;56:4630–5.[Abstract/Free Full Text]
  22. Ferretti A, Knijn A, Iorio E, et al. Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts. Biochim Biophys Acta 1999;1438:329–48.[Medline]
  23. Ronen SM, Jackson LE, Beloueche M, Leach MO. Magnetic resonance detects changes in phosphocholine associated with Ras activation and inhibition in NIH 3T3 cells. Br J Cancer 2001;84:691–6.[CrossRef][Medline]
  24. Favata MF, Horiuchi KY, Manos EJ, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 1998;273:18623–32.[Abstract/Free Full Text]
  25. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351:95–105.[CrossRef][Medline]
  26. Tyagi RK, Azrad A, Degani H, Salomon Y. Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy. Magn Reson Med 1996;35:194–200.[Medline]
  27. Herrera R, Sebolt-Leopold JS. Unraveling the complexities of the Raf/MAP kinase pathway for pharmacological intervention. Trends Mol Med 2002;8:243–3.
  28. Chiariello M, Marinissen MJ, Gutkind JS. Multiple mitogen-activated protein kinase signaling pathways connect the Cot oncoprotein to the c-jun promoter and to cellular transformation. Mol Cell Biol 2000;20:1747–58.[Abstract/Free Full Text]
  29. English JM, Vanderbilt CA, Xu SC, Marcus S, Cobb MH. Isolation of MEK5 and differential expression of alternatively spliced forms. J Biol Chem 1995;270:28897–902.[Abstract/Free Full Text]
  30. Pearson G, English JM, White MA, Cobb MH. ERK5 and ERK2 cooperate to regulate NF-{kappa}B and cell transformation. J Biol Chem 2001;276:7927–31.[Abstract/Free Full Text]
  31. Hayashi M, Tapping RI, Chao TH, et al. BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. J Biol Chem 2001;276:8631–4.[Abstract/Free Full Text]
  32. Kamakura S, Moriguchi T, Nishida E. Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases. Identification and characterization of a signaling pathway to the nucleus. J Biol Chem 1999;274:26563–71.[Abstract/Free Full Text]
  33. Podo F. Tumour phospholipid metabolism. NMR Biomed 1999;12:413–39.[CrossRef][Medline]
  34. Jackowski S. Coordination of membrane phospholipid synthesis with the cell cycle. J Biol Chem 1994;269:3858–67.[Abstract/Free Full Text]
  35. Daly PF, Lyon RC, Faustino PJ, Cohen JS. Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy. J Biol Chem 1987;262:14875–8.[Abstract/Free Full Text]
  36. Ronen SM, Rushkin E, Degani H. Lipid metabolism in large T47D human breast cancer spheroids: 31P-NMR and 13C-NMR studies of choline and ethanolamine uptake. Biochim Biophys Acta 1992;1138:203–12.[Medline]
  37. Smith TAD, Eccles S, Ormerod MG, Tombs AJ, Titley JC, Leach MO. The phosphocholine and glycerophosphocholine content of an oestrogen-sensitive rat mammary tumour correlates strongly with growth rate. Br J Cancer 1991;64:821–6.[Medline]
  38. Pages G, Lenormand P, Lallemain G, Chambard JC, Meloche S, Pouyssegur J. Mitogen-activated protein-kinases p42(mapk) and p44(mapk) are required for fibroblast proliferation. Proc Natl Acad Sci U S A 1993;90:8319–23.[Abstract/Free Full Text]
  39. Aiken NR, Gillies RJ. Phosphomonoester metabolism as a function of cell proliferative status and exogenous precursors. Anticancer Res 1996;16:1393–7.[Medline]
  40. Franks SE, Kuesel AC, Lutz NW, Hull WE. P-31 MRS of human tumor cells: effects of culture media and conditions on phospholipid metabolite concentrations. Anticancer Res 1996;16:1365–74.[Medline]
  41. De Molina AR, Rodriguez-Gonzalez A, Lacal JC. Targeting new anticancer drugs within signalling pathways regulated by the Ras GTPase superfamily. Int J Oncol 2001;19:5–17.[Medline]
  42. Lacal JC, Moscat J, Aaronson SA. Novel source of 1,2-diacylglycerol elevated in cells transformed by Ha-ras oncogene. Nature 1987;330:269–72.[CrossRef][Medline]
  43. Lacal JC, De la Pena P, Moscat J. Rapid stimulation of diacylglycerol production in Xenopus oocytes by microinjection of H-ras p21. Science 1987;238:533–6.[Abstract/Free Full Text]
  44. Teegarden D, Taparowsky EJ, Kent C. Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene. J Biol Chem 1990;265:6042–7.[Abstract/Free Full Text]
  45. Geilen CC, Wieder T, Boremski S, Wieprecht M, Orfanos CE. c-Ha-ras oncogene expression increases choline uptake, CTP:phosphocholine cytidylyltransferase activity and phosphatidylcholine biosynthesis in the immortalized human keratinocyte cell line HaCaT. Biochim Biophys Acta 1996;1299:299–305.[Medline]
  46. Bakovic M, Waite K, Vance DE. Oncogenic Ha-Ras transformation modulates the transcription of the CTP:Phosphocholine cytidylyltransferase {alpha} gene via p42/44(MAPK) and transcription factor Sp3. J Biol Chem 2003;278:14753–61.[Abstract/Free Full Text]
  47. Liu D, Hutchinson OC, Osman S, Price P, Workman P, Aboagye EO. Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin. Br J Cancer 2002;87:783–9.[CrossRef][Medline]
  48. Jimenez B, Del Peso L, Montaner S, Esteve P, Lacal JC. Generation of phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases in growth factors-induced mitogenic stimulation. J Cell Biochem 1995;57:141–9.[CrossRef][Medline]
  49. De Molina AR, Banez-Coronel M, Gutierrez R, et al. Choline kinase activation is a critical requirement for the proliferation of primary human mammary epithelial cells and breast tumor progression. Cancer Res 2004;64:6732–9.[Abstract/Free Full Text]
  50. Beloueche M, Jackson LE, Leach MO, Ronen SM. Ras transformation and metabolism: an MRS investigation. Proc Intl Soc Magn Reson Med 9 2001;Glasgow, UK (Abstract).
  51. Chung Y-L, Troy H, Banerji U, et al. Magnetic resonance spectroscopic pharmacodynamic markers of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17AAG), in human colon cancer models. J Natl Cancer Inst 2003;95:1624–33.[Abstract/Free Full Text]
  52. Workman P. How much gets there and what does it do?: the need for better pharmacokinetic and pharmacodynamic endpoints in contemporary drug discovery and development. Curr Pharm Des 2003;9:891–902.[CrossRef][Medline]
  53. Frank R, Hargreaves R. Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2003;2:566–80.[CrossRef][Medline]
  54. Parulekar WR, Eisenhauer EA. Phase I trial design for solid tumor studies of targeted, non-cytotoxic agents: theory and practice. J Natl Cancer Inst 2004;96:990–7.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 JCOHome page
W. M. Claudino, A. Quattrone, L. Biganzoli, M. Pestrin, I. Bertini, and A. Di Leo
Metabolomics: Available Results, Current Research Projects in Breast Cancer, and Future Applications
J. Clin. Oncol., July 1, 2007; 25(19): 2840 - 2846.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
D. Sachdev and D. Yee
Disrupting insulin-like growth factor signaling as a potential cancer therapy
Mol. Cancer Ther., January 1, 2007; 6(1): 1 - 12.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
C. Guo, G. Wu, J. L. Chin, G. Bauman, M. Moussa, F. Wang, N. M. Greenberg, S. S. Taylor, and J. W. Xuan
Bub1 Up-Regulation and Hyperphosphorylation Promote Malignant Transformation in SV40 Tag-Induced Transgenic Mouse Models
Mol. Cancer Res., December 1, 2006; 4(12): 957 - 969.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
P. Workman, E. O. Aboagye, Y.-L. Chung, J. R. Griffiths, R. Hart, M. O. Leach, R. J. Maxwell, P. M. J. McSheehy, P. M. Price, and J. Zweit
Minimally invasive pharmacokinetic and pharmacodynamic technologies in hypothesis-testing clinical trials of innovative therapies.
J Natl Cancer Inst, May 3, 2006; 98(9): 580 - 598.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
M. Sankaranarayanapillai, W. P. Tong, D. S. Maxwell, A. Pal, J. Pang, W. G. Bornmann, J. G. Gelovani, and S. M. Ronen
Detection of histone deacetylase inhibition by noninvasive magnetic resonance spectroscopy
Mol. Cancer Ther., May 1, 2006; 5(5): 1325 - 1334.
[Abstract] [Full Text] [PDF]

Home page
 JAOA: Journal of the American Osteopathic AssociationHome page
N. Shah, A. Sattar, M. Benanti, S. Hollander, and L. Cheuck
Magnetic Resonance Spectroscopy as an Imaging Tool for Cancer: A Review of the Literature
J Am Osteopath Assoc, January 1, 2006; 106(1): 23 - 27.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
M. Beloueche-Babari, L. E. Jackson, N. M.S. Al-Saffar, S. A. Eccles, F. I. Raynaud, P. Workman, M. O. Leach, and S. M. Ronen
Identification of magnetic resonance detectable metabolic changes associated with inhibition of phosphoinositide 3-kinase signaling in human breast cancer cells
Mol. Cancer Ther., January 1, 2006; 5(1): 187 - 196.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beloueche-Babari, M.
Right arrow Articles by Ronen, S. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beloueche-Babari, M.
Right arrow Articles by Ronen, S. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online