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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

Effective enrichment of glycoproteins by WGA lectin affinity workflow. Immobilized wheat germ agglutinin was used to enrich glycosylated proteins from BRI-JM01 and NMuMG cells with or without the induction of EMT by TGF-β. Proteins from control and TGF-β treated BRI-JM01 (A) and NMuMG (B) cells were fractionated on a WGA affinity column. To determine the efficiency of glycoprotein enrichment by WGA, proteins from the unfractionated cellular lysate (input, 'IN'), the WGA flow-through ('FT') fraction, and WGA elution ('EL') fraction were separated by SDS-PAGE and visualized by both a glycoprotein-specific stain and Sypro Ruby, a total protein stain.
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Figure 4: Effective enrichment of glycoproteins by WGA lectin affinity workflow. Immobilized wheat germ agglutinin was used to enrich glycosylated proteins from BRI-JM01 and NMuMG cells with or without the induction of EMT by TGF-β. Proteins from control and TGF-β treated BRI-JM01 (A) and NMuMG (B) cells were fractionated on a WGA affinity column. To determine the efficiency of glycoprotein enrichment by WGA, proteins from the unfractionated cellular lysate (input, 'IN'), the WGA flow-through ('FT') fraction, and WGA elution ('EL') fraction were separated by SDS-PAGE and visualized by both a glycoprotein-specific stain and Sypro Ruby, a total protein stain.

Mentions: To confirm the efficient enrichment of glycoproteins from cell lysates of BRI-JM01 and NMuMG cells, we visualized proteins in the unfractionated cell lysate (input), WGA flow-through, and WGA elution fraction using a glycosylation-specific staining procedure (Figure 4). As expected, glycoproteins were depleted in the flow-through fractions and enriched in the elution fractions. Subsequent staining of the same gel with Sypro Ruby to visualize the total protein content of each fraction showed that each lane contained a similar amount of total protein. The overall protein banding pattern in the WGA eluant fraction was distinct from the banding pattern in the input and flow-through, confirming that a unique subpopulation of proteins is enriched by WGA. Interestingly, the majority of proteins appear to be unchanged in abundance after treatment with TGF-β.


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)

Effective enrichment of glycoproteins by WGA lectin affinity workflow. Immobilized wheat germ agglutinin was used to enrich glycosylated proteins from BRI-JM01 and NMuMG cells with or without the induction of EMT by TGF-β. Proteins from control and TGF-β treated BRI-JM01 (A) and NMuMG (B) cells were fractionated on a WGA affinity column. To determine the efficiency of glycoprotein enrichment by WGA, proteins from the unfractionated cellular lysate (input, 'IN'), the WGA flow-through ('FT') fraction, and WGA elution ('EL') fraction were separated by SDS-PAGE and visualized by both a glycoprotein-specific stain and Sypro Ruby, a total protein stain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effective enrichment of glycoproteins by WGA lectin affinity workflow. Immobilized wheat germ agglutinin was used to enrich glycosylated proteins from BRI-JM01 and NMuMG cells with or without the induction of EMT by TGF-β. Proteins from control and TGF-β treated BRI-JM01 (A) and NMuMG (B) cells were fractionated on a WGA affinity column. To determine the efficiency of glycoprotein enrichment by WGA, proteins from the unfractionated cellular lysate (input, 'IN'), the WGA flow-through ('FT') fraction, and WGA elution ('EL') fraction were separated by SDS-PAGE and visualized by both a glycoprotein-specific stain and Sypro Ruby, a total protein stain.
Mentions: To confirm the efficient enrichment of glycoproteins from cell lysates of BRI-JM01 and NMuMG cells, we visualized proteins in the unfractionated cell lysate (input), WGA flow-through, and WGA elution fraction using a glycosylation-specific staining procedure (Figure 4). As expected, glycoproteins were depleted in the flow-through fractions and enriched in the elution fractions. Subsequent staining of the same gel with Sypro Ruby to visualize the total protein content of each fraction showed that each lane contained a similar amount of total protein. The overall protein banding pattern in the WGA eluant fraction was distinct from the banding pattern in the input and flow-through, confirming that a unique subpopulation of proteins is enriched by WGA. Interestingly, the majority of proteins appear to be unchanged in abundance after treatment with TGF-β.

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