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Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

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Effect of overexpression of Id2 or its mutants on the migratory capacity of MCF-7 and SKOV-3 cells. Migratory behaviors of Id2 or its mutant transfectants were determined by scratch-wound assay (top panel) and quantified as described in "Methods" (bottom panel). The scratch area at 0 h was arbitrarily assigned as 100%. (*P < 0.05; # P <0.01; N P > 0.05).
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Figure 5: Effect of overexpression of Id2 or its mutants on the migratory capacity of MCF-7 and SKOV-3 cells. Migratory behaviors of Id2 or its mutant transfectants were determined by scratch-wound assay (top panel) and quantified as described in "Methods" (bottom panel). The scratch area at 0 h was arbitrarily assigned as 100%. (*P < 0.05; # P <0.01; N P > 0.05).

Mentions: To examine whether the invasion potential of MCF-7 and SKOV-3 cells promoted by Id2 and its mutants was associated by their increase in cell motility, we analyzed the effects of Id2 and its mutants on the migration capability of cells by observing the effects of scratch wounding confluent monolayers of cells transfected with Id2 or its mutants. Overexpression of the wild-type Id2 or its mutants markedly promoted the flattening and spreading of both cell lines along the edges of the wound as compared with the control (Figure 5).


Id2 promotes the invasive growth of MCF-7 and SKOV-3 cells by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Meng Y, Gu C, Wu Z, Zhao Y, Si Y, Fu X, Han W - BMC Cancer (2009)

Effect of overexpression of Id2 or its mutants on the migratory capacity of MCF-7 and SKOV-3 cells. Migratory behaviors of Id2 or its mutant transfectants were determined by scratch-wound assay (top panel) and quantified as described in "Methods" (bottom panel). The scratch area at 0 h was arbitrarily assigned as 100%. (*P < 0.05; # P <0.01; N P > 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654660&req=5

Figure 5: Effect of overexpression of Id2 or its mutants on the migratory capacity of MCF-7 and SKOV-3 cells. Migratory behaviors of Id2 or its mutant transfectants were determined by scratch-wound assay (top panel) and quantified as described in "Methods" (bottom panel). The scratch area at 0 h was arbitrarily assigned as 100%. (*P < 0.05; # P <0.01; N P > 0.05).
Mentions: To examine whether the invasion potential of MCF-7 and SKOV-3 cells promoted by Id2 and its mutants was associated by their increase in cell motility, we analyzed the effects of Id2 and its mutants on the migration capability of cells by observing the effects of scratch wounding confluent monolayers of cells transfected with Id2 or its mutants. Overexpression of the wild-type Id2 or its mutants markedly promoted the flattening and spreading of both cell lines along the edges of the wound as compared with the control (Figure 5).

Bottom Line: The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells.Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2.E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, 28 Fu Xing Road, Beijing, 100853, PR China. mengyg6512@vipsina.com

ABSTRACT

Background: Inhibitor of differentiation 2 (Id2) is a critical factor for cell proliferation and differentiation in normal vertebrate development. Most of the biological function of Id2 has been ascribed to its helix-loop-helix motif. Overexpression of Id2 is frequently observed in various human tumors, but its role for invasion potential in tumor cells is dispute. We aimed to reveal the role of Id2 in invasion potential in poorly invasive and estrogen receptor alpha (ERalpha)-positive MCF-7 and SKOV-3 cancer cells.

Methods: MCF-7 and SKOV-3 cells were stably transfected with the wild-type, degradation-resistant full-length or helix-loop-helix (HLH)-deleted Id2, respectively. Protein levels of Id2 and its mutants and E-cadherin were determined by western blot analysis and mRNA levels of Id2 and its mutants were determined by RT-PCR. The effects of Id2 and its mutants on cell proliferation were determined by [3H]-thymidine incorporation assay and the 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method. The in vitro invasion potential of cells was evaluated by Transwell assay. Cell motility was assessed by scratch wound assay. The promoter activity of E-cadherin was determined by cotransfection and luciferase assays.

Results: Ectopic transfection of the wild-type Id2 markedly increased the protein and mRNA expression of Id2 in MCF-7 and SKOV-3 cells; the protein level but not mRNA level was further increased by transfection with the degradation-resistant Id2 form. The ectopic expression of Id2 or its mutants did not alter proliferation of either MCF-7 or SKOV-3 cells. Transfection of the wild-type Id2 significantly induced the invasion potential and migratory capacity of cells, which was further augmented by transfection with the degradation-resistant full-length or HLH-deleted Id2. E-cadherin protein expression and transactivation of the proximal E-cadherin promoter were markedly suppressed by the degradation-resistant full-length or HLH-deleted Id2 but not wild-type Id2. Ectopic expression of E-cadherin in MCF-7 and SKOV-3 cells only partially blunted the invasion potential induced by the degradation-resistant HLH-deleted Id2.

Conclusion: Overexpression of Id2 in ERalpha-positive epithelial tumor cells indeed increases the cells' invasive potential through a novel mechanism independent of dimerization to basic helix-loop-helix factors. E-cadherin contributes only in part to Id2-induced cell invasion when Id2 is accumulated to a higher level in some specific cell types.

Show MeSH
Related in: MedlinePlus