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Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-beta induced epithelial to mesenchymal transition.

Hill JJ, Tremblay TL, Cantin C, O'Connor-McCourt M, Kelly JF, Lenferink AE - Proteome Sci (2009)

Bottom Line: Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).Interestingly, despite the fact that TGF-beta induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap.These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. Jennifer.Hill@nrc-cnrc.gc.ca

ABSTRACT

Background: TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-beta can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-beta in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.

Results: Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).

Conclusion: Interestingly, despite the fact that TGF-beta induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.

No MeSH data available.


Related in: MedlinePlus

Protein expression changes associated with TGF-β mediated EMT in BRI-JM01 cells, identified by 2DE. 2DE studies identified 5 proteins that changed in expression after 24 hours of TGF-β treatment. Protein ratios (TGF-β/CTL) calculated from the 2DE data is shown in the left-hand column. All protein changes were subsequently verified by western blotting of BRI-JM01 cell lysates from 2 biological repeats, as shown. β-actin was used as a loading control (LC).
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Figure 2: Protein expression changes associated with TGF-β mediated EMT in BRI-JM01 cells, identified by 2DE. 2DE studies identified 5 proteins that changed in expression after 24 hours of TGF-β treatment. Protein ratios (TGF-β/CTL) calculated from the 2DE data is shown in the left-hand column. All protein changes were subsequently verified by western blotting of BRI-JM01 cell lysates from 2 biological repeats, as shown. β-actin was used as a loading control (LC).

Mentions: To identify EMT-related changes in protein expression in BRI-JM01 cells, our preliminary experiments utilized a traditional proteomics workflow: two dimensional gel electrophoresis followed by identification of differentially expressed protein spots by ESI-LC-MS/MS. Initial experiments using standard 2DE to separate BRI-JM01 whole cell lysates produced very complex 2-dimensional spot patterns that did not allow us to identify any consistent spot intensity changes after 24 hours of TGF-β treatment. Thus, we performed a phase separation in Triton X-114 to enrich for hydrophobic proteins and added ASB-14, a zwitterionic detergent, to the IEF buffer. We found that ASB-14 helped to solubilize high molecular weight transmembrane proteins and small membrane-associated proteins, such as the lipid-modified monomeric G-proteins (data not shown). Examples of the 2D-gels analyzed in these experiments are shown in Supplementary Figure 1 (see Additional File 1). Even after hydrophobic protein enrichment, only five proteins showed a consistent change in spot intensity after analysis of gels spanning two pH ranges (3–6 and 5–8) from three biological repetitions. All five differentially expressed proteins were up-regulated upon TGF-β treatment and included Integrin α2, Integrin α5, activated leukocyte cell adhesion molecule (ALCAM), Tropomodulin-3, and 14-3-3σ. All of the changes identified in this 2DE study were subsequently validated by western blotting of BRI-JM01 whole cell lysates, as shown in Figure 2. Of the five proteins identified, three are cell surface glycoproteins, suggesting that this subset of proteins may undergo more changes in protein expression than soluble, cytoplasmic proteins.


Glycoproteomic analysis of two mouse mammary cell lines during transforming growth factor (TGF)-beta induced epithelial to mesenchymal transition.

Hill JJ, Tremblay TL, Cantin C, O'Connor-McCourt M, Kelly JF, Lenferink AE - Proteome Sci (2009)

Protein expression changes associated with TGF-β mediated EMT in BRI-JM01 cells, identified by 2DE. 2DE studies identified 5 proteins that changed in expression after 24 hours of TGF-β treatment. Protein ratios (TGF-β/CTL) calculated from the 2DE data is shown in the left-hand column. All protein changes were subsequently verified by western blotting of BRI-JM01 cell lysates from 2 biological repeats, as shown. β-actin was used as a loading control (LC).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651118&req=5

Figure 2: Protein expression changes associated with TGF-β mediated EMT in BRI-JM01 cells, identified by 2DE. 2DE studies identified 5 proteins that changed in expression after 24 hours of TGF-β treatment. Protein ratios (TGF-β/CTL) calculated from the 2DE data is shown in the left-hand column. All protein changes were subsequently verified by western blotting of BRI-JM01 cell lysates from 2 biological repeats, as shown. β-actin was used as a loading control (LC).
Mentions: To identify EMT-related changes in protein expression in BRI-JM01 cells, our preliminary experiments utilized a traditional proteomics workflow: two dimensional gel electrophoresis followed by identification of differentially expressed protein spots by ESI-LC-MS/MS. Initial experiments using standard 2DE to separate BRI-JM01 whole cell lysates produced very complex 2-dimensional spot patterns that did not allow us to identify any consistent spot intensity changes after 24 hours of TGF-β treatment. Thus, we performed a phase separation in Triton X-114 to enrich for hydrophobic proteins and added ASB-14, a zwitterionic detergent, to the IEF buffer. We found that ASB-14 helped to solubilize high molecular weight transmembrane proteins and small membrane-associated proteins, such as the lipid-modified monomeric G-proteins (data not shown). Examples of the 2D-gels analyzed in these experiments are shown in Supplementary Figure 1 (see Additional File 1). Even after hydrophobic protein enrichment, only five proteins showed a consistent change in spot intensity after analysis of gels spanning two pH ranges (3–6 and 5–8) from three biological repetitions. All five differentially expressed proteins were up-regulated upon TGF-β treatment and included Integrin α2, Integrin α5, activated leukocyte cell adhesion molecule (ALCAM), Tropomodulin-3, and 14-3-3σ. All of the changes identified in this 2DE study were subsequently validated by western blotting of BRI-JM01 whole cell lysates, as shown in Figure 2. Of the five proteins identified, three are cell surface glycoproteins, suggesting that this subset of proteins may undergo more changes in protein expression than soluble, cytoplasmic proteins.

Bottom Line: Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).Interestingly, despite the fact that TGF-beta induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap.These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, ON, Canada. Jennifer.Hill@nrc-cnrc.gc.ca

ABSTRACT

Background: TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. However, later in tumor development TGF-beta can become tumor promoting through mechanisms including the induction of epithelial-to-mesenchymal transition (EMT), a process that is thought to contribute to tumor progression, invasion and metastasis. To identify EMT-related breast cancer therapeutic targets and biomarkers, we have used two proteomic approaches to find proteins that change in abundance upon the induction of EMT by TGF-beta in two mouse mammary epithelial cell lines, NMuMG and BRI-JM01.

Results: Preliminary experiments based on two-dimensional electrophoresis of a hydrophobic cell fraction identified only 5 differentially expressed proteins from BRI-JM01 cells. Since 3 of these proteins were glycoproteins, we next used the lectin, wheat germ agglutinin (WGA), to enrich for glycoproteins, followed by relative quantification of tryptic peptides using a label-free LC-MS based method. Using these approaches, we identified several proteins that are modulated during the EMT process, including cell adhesion molecules (several members of the Integrin family, Fibronectin, Activated leukocyte cell adhesion molecule, and Neural cell adhesion molecule 1) and regulators of cellular signaling (Tumor-associated calcium signal transducer 2, Basigin).

Conclusion: Interestingly, despite the fact that TGF-beta induces similar EMT phenotypes in NMuMG and BRI-JM01 cells, the proteomic results for the two cell lines showed only minimal overlap. These differences likely result in part from the conservative cut-off values used to define differentially-expressed proteins in these experiments. Alternatively, it is possible that the two cell lines may use different mechanisms to achieve an EMT transition.

No MeSH data available.


Related in: MedlinePlus