Evaluation of Metabolic Labeling for Comparative Proteomics in Breast Cancer Cells

Gehrmann, Marion; Hathout, Yetrib; Fenselau, Catherine

doi:10.1021/pr049906k

Cited by 50 publications

(51 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…SILAC has been used to provide both targeted (10,31) and more global quantitative analyses of cellular proteomes (20,25). We have documented the suitability of this approach using both primary SCs and immortalized S16 SCs.…”

Section: Discussionmentioning

confidence: 99%

“…However, the presence of unlabeled amino acids in FCS can compete with the isotopically labeled amino acid and lead to a mixture of labeled and unlabeled amino-acyl tRNAs that both contribute to protein synthesis. Although the use of dFCS avoids this problem (9), some cells require low molecular weight serum components for growth that are also removed by dialysis (20). Because SILAC analysis has not been performed on primary or immortalized SCs, it was necessary to assess the ability of these cells to grow in dFCS and incorporate heavy amino acids into their proteomes.…”

Section: Silac As An Analytical Approach In Primary and Immortalized Scsmentioning

confidence: 99%

See 1 more Smart Citation

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1

Alterman

Dobrowsky

2005

Journal of Lipid Research

121

104

View full text Add to dashboard Cite

Caveolae and lipid raft domains are distinct but related regions of the plasma membrane that are enriched in cholesterol, sphingomyelin, and glycosphingolipids (1, 2). Whereas lipid raft domains are present in many, if not all, cells (3), caveolae are specialized, morphologically distinct lipid raft domains that require the cell-specific expression of the protein caveolin-1 (Cav-1) (4).The integrity of lipid rafts is very dependent upon the presence of cholesterol (5). Relative to phospholipids, sphingolipids are more hydrophobic, undergo more hydrogen bonding, and tend to cluster within cell membranes (6). Moreover, the ability of cholesterol to pack tightly with saturated lipids is essential to promoting the liquid-ordered structure of lipid raft domains (6). The loss of cholesterol from caveolae by treatment with cholesterol-sequestering agents, through increased sterol oxidation, or by inhibiting its de novo synthesis leads to a more disordered state and the loss of raft-associated proteins. Indeed, because Cav-1 is a cholesterol binding protein, it is often used to monitor the loss of caveolar integrity after cholesterol displacement or depletion. Recent reports have provided evidence that ceramide production in raft model membranes and astrocytes causes a significant displacement of cholesterol from lipid raft membranes (7,8). However, whether a ceramide-induced displacement of cholesterol can lead to changes in the protein composition of caveolin-enriched membranes (CEMs) is unknown.The recent development of stable isotope labeling of cells in culture (SILAC) (9) has enabled a quantitative, hypothesis-driven assessment of changes in a single protein (10) as well as the unbiased, quantitative characterization of large proteomes (11). The basic premise of SILAC is that two populations of cells are grown in medium containing either unlabeled amino acids or an amino acid containing deuterium or 13 C substitutions. After a suffiAbbreviations: bSMase, bacterial sphingomyelinase; Cav-1, caveolin-1; CEM, caveolin-enriched membrane; dFCS, dialyzed fetal calf serum; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; M ␤ CD, methyl-␤ -cyclodextrin; SC, Schwann cell; SILAC, stable isotope labeling with amino acids in cell culture.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Silac As An Analytical Approach In Primary and Immortalized Scsmentioning

confidence: 99%

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1

Alterman

Dobrowsky

2005

Journal of Lipid Research

121

104

View full text Add to dashboard Cite

show abstract

“…For quantitative PCR approaches, the use of certain reference genes such as GAPDH can also be problematic. It has been found that GAPDH expression was increased in the Adriamycin-resistant MCF7/Adr cells [46,47]. In a metabolic marker-focused proteomic profiling analysis of 101 breast cancer tissues, Isidoro et al [48] also found that the expression of GAPDH was significantly increased in patients with poor survival.…”

Section: Concluding Remarks and Perspectivesmentioning

confidence: 99%

Use of arrays to investigate the contribution of ATP-binding cassette transporters to drug resistance in cancer chemotherapy and prediction of chemosensitivity

Zhang

2007

Cell Res

View full text Add to dashboard Cite

Multidrug resistance (MDR) is a major problem in cancer chemotherapy. One of the best known mechanisms of MDR is the elevated expression of ATP-binding cassette (ABC) transporters. While some members of human ABC transporters have been shown to cause drug resistance with elevated expression, it is not yet known whether the over-expression of other members could also contribute to drug resistance in many model cancer cell lines and clinics. The recent development of microarrays and quantitative PCR arrays for expression profiling analysis of ABC transporters has helped address these issues. In this article, various arrays with limited or full list of ABC transporter genes and their use in identifying ABC transporter genes in drug resistance and chemo-sensitivity prediction will be reviewed.

show abstract

“…Proteomics studies reported so far have mainly been performed with breast cancer cell lines using either two-dimensional gel electrophoresis (11)(12)(13)(14) or LC-MS for protein separation (15)(16)(17). However, it is known that the proteomic makeup of a cultured cell is rather different from that of a tumor cell surrounded by its native microenvironment (18).…”

Section: Molecular and Cellular Proteomics 8:1278 -1294 2009mentioning

confidence: 99%

Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer

Umar

Kang

Timmermans

et al. 2009

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ϳ5,500 pooled tumor cells (corresponding to ϳ550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n ‫؍‬ 24 and n ‫؍‬ 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 nonredundant proteins identified with >2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n ‫؍‬ 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p ‫؍‬ 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

show abstract

Evaluation of Metabolic Labeling for Comparative Proteomics in Breast Cancer Cells

Cited by 50 publications

References 24 publications

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1

Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1

Use of arrays to investigate the contribution of ATP-binding cassette transporters to drug resistance in cancer chemotherapy and prediction of chemosensitivity

Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer

Contact Info

Product

Resources

About