Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Schematic illustration of the Id2-DBM-δHLH construct. In this construct, the HLH domain encoded by amino acid residues from 35 to 76 was deleted from Id2, and the key arginine and leucine residues within the D-box motif (RxxLxxxN) at residues 100 to 107 were changed to glycine and valine. (B) Western blot analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total proteins were extracted from cells transfected with the indicated plasmids, and then underwent SDS-PAGE electrophoresis and immunoblotting analysis using the indicated antibodies. β-actin was used as the loading control. (C) RT-PCR analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total RNA was extracted from cells transfected with the indicated plasmids and used for RT-PCR analysis of the mRNA expression of Id2 and its mutants. β-actin was the loading control. The results shown in B and C are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654660&req=5

Figure 1: (A) Schematic illustration of the Id2-DBM-δHLH construct. In this construct, the HLH domain encoded by amino acid residues from 35 to 76 was deleted from Id2, and the key arginine and leucine residues within the D-box motif (RxxLxxxN) at residues 100 to 107 were changed to glycine and valine. (B) Western blot analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total proteins were extracted from cells transfected with the indicated plasmids, and then underwent SDS-PAGE electrophoresis and immunoblotting analysis using the indicated antibodies. β-actin was used as the loading control. (C) RT-PCR analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total RNA was extracted from cells transfected with the indicated plasmids and used for RT-PCR analysis of the mRNA expression of Id2 and its mutants. β-actin was the loading control. The results shown in B and C are representative of three independent experiments.

Mentions: To investigate the effects of overexpression of Id2 and its mutants on the phenotypic traits of MCF-7 and SKOV-3 cells, the expression plasmids for Id2, Id2-DBM, and Id2-DBM-δHLH (schematic structure illustrated in Figure 1A) or the empty control vector were stably transfected into cells. After 10- to 14-day G418 screening, the drug-resistant clones appeared and were mixed for amplified culture. All of the parental cells were killed by G418 within this period. On western blot analysis, endogenous Id2 protein was not detected in MCF-7 cells and weakly detected in SKOV-3 cells, but ectopic transfection of Id2 markedly increased the expression of Id2 protein in both cell lines (Figure 1B). Ectopic transfection of D-box mutant Id2 further increased Id2 protein expression 2 to 3-fold that of transfection with the wild type Id2. A band corresponding to HLH-deleted Id2-DBM was detected using a rabbit anti-Id2 monoclonal antibody. Next, we further measured the mRNA levels of Id2 and its mutants in transfected cells by RT-PCR (Figure 1C) and found the mRNA expression of Id2 are consistent with protein levels in empty vector- and wild-type Id2-transfected cells. However, the mRNA level with Id2-DBM transfection did not differ from that with wild-type Id2 transfection in either MCF-7 or SKOV-3 cells. Id2-DBM-δHLH mRNA could be detected in cells, which confirms the effective expression of the HLH-deleted Id2. Collectively, these data indicated that the ectopic Id2 and its mutants can effectively be expressed in MCF-7 and SKOV-3 cells and with the D-box mutation transfection, Id2 protein could accumulate to a much higher level than with the wild type.


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)

(A) Schematic illustration of the Id2-DBM-δHLH construct. In this construct, the HLH domain encoded by amino acid residues from 35 to 76 was deleted from Id2, and the key arginine and leucine residues within the D-box motif (RxxLxxxN) at residues 100 to 107 were changed to glycine and valine. (B) Western blot analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total proteins were extracted from cells transfected with the indicated plasmids, and then underwent SDS-PAGE electrophoresis and immunoblotting analysis using the indicated antibodies. β-actin was used as the loading control. (C) RT-PCR analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total RNA was extracted from cells transfected with the indicated plasmids and used for RT-PCR analysis of the mRNA expression of Id2 and its mutants. β-actin was the loading control. The results shown in B and C are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: (A) Schematic illustration of the Id2-DBM-δHLH construct. In this construct, the HLH domain encoded by amino acid residues from 35 to 76 was deleted from Id2, and the key arginine and leucine residues within the D-box motif (RxxLxxxN) at residues 100 to 107 were changed to glycine and valine. (B) Western blot analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total proteins were extracted from cells transfected with the indicated plasmids, and then underwent SDS-PAGE electrophoresis and immunoblotting analysis using the indicated antibodies. β-actin was used as the loading control. (C) RT-PCR analysis of Id2 and its mutants in MCF-7 and SKOV-3 cells. Total RNA was extracted from cells transfected with the indicated plasmids and used for RT-PCR analysis of the mRNA expression of Id2 and its mutants. β-actin was the loading control. The results shown in B and C are representative of three independent experiments.
Mentions: To investigate the effects of overexpression of Id2 and its mutants on the phenotypic traits of MCF-7 and SKOV-3 cells, the expression plasmids for Id2, Id2-DBM, and Id2-DBM-δHLH (schematic structure illustrated in Figure 1A) or the empty control vector were stably transfected into cells. After 10- to 14-day G418 screening, the drug-resistant clones appeared and were mixed for amplified culture. All of the parental cells were killed by G418 within this period. On western blot analysis, endogenous Id2 protein was not detected in MCF-7 cells and weakly detected in SKOV-3 cells, but ectopic transfection of Id2 markedly increased the expression of Id2 protein in both cell lines (Figure 1B). Ectopic transfection of D-box mutant Id2 further increased Id2 protein expression 2 to 3-fold that of transfection with the wild type Id2. A band corresponding to HLH-deleted Id2-DBM was detected using a rabbit anti-Id2 monoclonal antibody. Next, we further measured the mRNA levels of Id2 and its mutants in transfected cells by RT-PCR (Figure 1C) and found the mRNA expression of Id2 are consistent with protein levels in empty vector- and wild-type Id2-transfected cells. However, the mRNA level with Id2-DBM transfection did not differ from that with wild-type Id2 transfection in either MCF-7 or SKOV-3 cells. Id2-DBM-δHLH mRNA could be detected in cells, which confirms the effective expression of the HLH-deleted Id2. Collectively, these data indicated that the ectopic Id2 and its mutants can effectively be expressed in MCF-7 and SKOV-3 cells and with the D-box mutation transfection, Id2 protein could accumulate to a much higher level than with the wild type.

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