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Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth.

Zhang X, Xu LH, Yu Q - Mol. Cancer (2010)

Bottom Line: Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood.Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates.We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai Institute of Materia Medica, China Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. xingzhangg@hotmail.com

ABSTRACT

Background: Apoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells.

Results: We observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar.

Conclusions: The data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

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Detachment from ECM induced apoptosis in normal epithelial MDCK and HBE cells. A, DNA laddering analysis of cell death. Normal cells and tumor cells from regular cell culture dishes (1 × 107 ) were plated into 100-mm polyHEMA-coated dishes for 24 h and harvested. Cytosolic DNA was extracted and analyzed by agarose gel electrophoresis. B and C Annexin V staining analysis of cell death. 5 × 105 MDCK, HBE, and H460 cells from regular cell culture dishes were plated into 60-mm polyHEMA-coated dishes for 24 h, harvested, fixed, and assessed by Annexin V/propidium iodide (AV/PI) DNA staining and flow cytometry. B, Percent of early apoptotic cells determined by flow cytometry using AV/PI binding. Quadrant D1: PI/AV +/-, necrotic; D2: PI/AV +/+, late apoptotic/necrotic; D3: PI/AV -/-, live cells; D4: PI/AV -/+, early apoptotic cells. C, Histograms of flow cytometry data. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. D, Immunoblotting analysis of cleaved caspase-3. After the indicated time periods cells were lysed and total protein was extracted separated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. "***": P < 0.001.
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Figure 2: Detachment from ECM induced apoptosis in normal epithelial MDCK and HBE cells. A, DNA laddering analysis of cell death. Normal cells and tumor cells from regular cell culture dishes (1 × 107 ) were plated into 100-mm polyHEMA-coated dishes for 24 h and harvested. Cytosolic DNA was extracted and analyzed by agarose gel electrophoresis. B and C Annexin V staining analysis of cell death. 5 × 105 MDCK, HBE, and H460 cells from regular cell culture dishes were plated into 60-mm polyHEMA-coated dishes for 24 h, harvested, fixed, and assessed by Annexin V/propidium iodide (AV/PI) DNA staining and flow cytometry. B, Percent of early apoptotic cells determined by flow cytometry using AV/PI binding. Quadrant D1: PI/AV +/-, necrotic; D2: PI/AV +/+, late apoptotic/necrotic; D3: PI/AV -/-, live cells; D4: PI/AV -/+, early apoptotic cells. C, Histograms of flow cytometry data. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. D, Immunoblotting analysis of cleaved caspase-3. After the indicated time periods cells were lysed and total protein was extracted separated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. "***": P < 0.001.

Mentions: However, we did not observe a correlation between cell aggregation and survival in the case of tumor cells in suspension. The H460 tumor cells, which formed large and tight aggregates in suspension, underwent partial anoikis in suspension (Fig. 2A-C). On the other hand, the SK-LU-1 tumor cells, which did not form large aggregates and grew slowly in suspension (Fig. 1C), were completely resistant to anoikis (Fig. 2A). While aggregation of H460 cells provided adequate signals for cell growth (Fig. 1C), it was not sufficient to protect the cells from undergoing anoikis (Fig. 2A). These data suggest that the signals for tumor cell anchorage-independent survival and growth were distinct.


Cell aggregation induces phosphorylation of PECAM-1 and Pyk2 and promotes tumor cell anchorage-independent growth.

Zhang X, Xu LH, Yu Q - Mol. Cancer (2010)

Detachment from ECM induced apoptosis in normal epithelial MDCK and HBE cells. A, DNA laddering analysis of cell death. Normal cells and tumor cells from regular cell culture dishes (1 × 107 ) were plated into 100-mm polyHEMA-coated dishes for 24 h and harvested. Cytosolic DNA was extracted and analyzed by agarose gel electrophoresis. B and C Annexin V staining analysis of cell death. 5 × 105 MDCK, HBE, and H460 cells from regular cell culture dishes were plated into 60-mm polyHEMA-coated dishes for 24 h, harvested, fixed, and assessed by Annexin V/propidium iodide (AV/PI) DNA staining and flow cytometry. B, Percent of early apoptotic cells determined by flow cytometry using AV/PI binding. Quadrant D1: PI/AV +/-, necrotic; D2: PI/AV +/+, late apoptotic/necrotic; D3: PI/AV -/-, live cells; D4: PI/AV -/+, early apoptotic cells. C, Histograms of flow cytometry data. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. D, Immunoblotting analysis of cleaved caspase-3. After the indicated time periods cells were lysed and total protein was extracted separated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. "***": P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Detachment from ECM induced apoptosis in normal epithelial MDCK and HBE cells. A, DNA laddering analysis of cell death. Normal cells and tumor cells from regular cell culture dishes (1 × 107 ) were plated into 100-mm polyHEMA-coated dishes for 24 h and harvested. Cytosolic DNA was extracted and analyzed by agarose gel electrophoresis. B and C Annexin V staining analysis of cell death. 5 × 105 MDCK, HBE, and H460 cells from regular cell culture dishes were plated into 60-mm polyHEMA-coated dishes for 24 h, harvested, fixed, and assessed by Annexin V/propidium iodide (AV/PI) DNA staining and flow cytometry. B, Percent of early apoptotic cells determined by flow cytometry using AV/PI binding. Quadrant D1: PI/AV +/-, necrotic; D2: PI/AV +/+, late apoptotic/necrotic; D3: PI/AV -/-, live cells; D4: PI/AV -/+, early apoptotic cells. C, Histograms of flow cytometry data. Data from at least three separate experiments were analyzed using Student's t test. "***": P < 0.001. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. D, Immunoblotting analysis of cleaved caspase-3. After the indicated time periods cells were lysed and total protein was extracted separated by SDS-PAGE, and analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control. A: Attached culture; S: Suspension culture in polyHEMA-coated dishes. "***": P < 0.001.
Mentions: However, we did not observe a correlation between cell aggregation and survival in the case of tumor cells in suspension. The H460 tumor cells, which formed large and tight aggregates in suspension, underwent partial anoikis in suspension (Fig. 2A-C). On the other hand, the SK-LU-1 tumor cells, which did not form large aggregates and grew slowly in suspension (Fig. 1C), were completely resistant to anoikis (Fig. 2A). While aggregation of H460 cells provided adequate signals for cell growth (Fig. 1C), it was not sufficient to protect the cells from undergoing anoikis (Fig. 2A). These data suggest that the signals for tumor cell anchorage-independent survival and growth were distinct.

Bottom Line: Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood.Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates.We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai Institute of Materia Medica, China Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China. xingzhangg@hotmail.com

ABSTRACT

Background: Apoptosis caused by inadequate or inappropriate cell-matrix interactions is defined as anoikis. Although transformed cells are known to be anoikis-resistant, the underlying mechanisms have not been well understood. We investigated the mechanisms of anoikis resistance of tumor cells.

Results: We observed that cell aggregation in suspension promoted cell survival and proliferation. We demonstrated a correlation between tumor cell aggregation in suspension and cell growth in soft agar. Analysis of tyrosine kinase-mediated cell survival and growth signaling pathways revealed increased levels of tyrosine-phosphorylation of PECAM-1 and Pyk2 in cell aggregates. We also showed that PECAM-1 and Pyk2 physically interact with each other, and that PECAM-1 carrying a deletion of exons 11-16 could no longer bind to Pyk2. Furthermore, RNA interference-mediated reduction of Pyk2 and PECAM-1 protein levels reduced cell aggregation and inhibited the growth of tumor cells in soft agar.

Conclusions: The data demonstrated that Pyk2 and PECAM-1 were critical mediators of both anchorage-independent growth and anoikis resistance in tumor cells.

Show MeSH
Related in: MedlinePlus