Limits...
Inhibition of Id proteins by a peptide aptamer induces cell-cycle arrest and apoptosis in ovarian cancer cells.

Mern DS, Hasskarl J, Burwinkel B - Br. J. Cancer (2010)

Bottom Line: Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.These effects were counteracted by ectopically overexpressed Id1 and Id3.Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz-University Group Molecular Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany. d.mern@dkfz-heidelberg.de

ABSTRACT

Background: Inhibitors of DNA-binding proteins (Id1-4), lacking the basic DNA-binding domain, function as dominant inhibitors of cell-cycle regulators. Overexpression of Id proteins promotes cancer cell proliferation and resistance against apoptosis. Level of Id protein expression, especially of Id1, correlates with poor differentiation, enhanced malignant potential and more aggressive clinical behaviour of ovarian tumours. Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.

Methods: A peptide aptamer, Id1/3-PA7, targeting Id1 and Id3, was isolated from a randomised combinatorial expression library using yeast and mammalian two-hybrid systems. Id1/3-PA7 was fused, expressed and purified with a cell-penetrating protein transduction domain.

Results: Intracellular-delivered Id1/3-PA7 colocalised to Id1 and Id3. It induced cell-cycle arrest and apoptosis in ovarian cancer cells ES-2 and PA-1. It activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitor (CDKN2A) in a dose-dependent manner that is paralleled by the cleavage of poly-ADP ribose polymerase. These effects were counteracted by ectopically overexpressed Id1 and Id3.

Conclusion: Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.

Show MeSH

Related in: MedlinePlus

Colocalisation of the intracellular-delivered peptide aptamer Id1/3-PA7 to Id1 and Id3. (A, B) His-tag coimmunoprecipitation of Id1/3-PA7 with Id1 and Id3 in ovarian cancer cells ES-2 and PA-1. (C, D) His-tag coimmunoprecipitation of TrxA as negative control. (E–H) Coimmunofluorescence staining of Id1/3-PA7 with Id1 and Id3 in ES-2 and PA-1 cells. DAPI, 4,6-diamidino-2-phenylindole.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2967066&req=5

fig1: Colocalisation of the intracellular-delivered peptide aptamer Id1/3-PA7 to Id1 and Id3. (A, B) His-tag coimmunoprecipitation of Id1/3-PA7 with Id1 and Id3 in ovarian cancer cells ES-2 and PA-1. (C, D) His-tag coimmunoprecipitation of TrxA as negative control. (E–H) Coimmunofluorescence staining of Id1/3-PA7 with Id1 and Id3 in ES-2 and PA-1 cells. DAPI, 4,6-diamidino-2-phenylindole.

Mentions: Using yeast and mammalian two-hybrid systems, we isolated from the randomised expression library a constrained peptide aptamer Id1/3-PA7 that interacts specifically with Id1 and Id3 (Mern et al, 2010). Here, we used Id1/3-PA7 to analyse its functional interference in Id1- and Id3-overexpressing ovarian cancer cells ES-2 and PA-1. For its delivery into ovarian cancer cells, Id1/3-PA7 was fused with cell-penetrating PTD (Elliott and O'Hare, 1997; Phelan et al, 1998; Narita et al, 2001), expressed in bacteria and purified under native conditions. Ovarian cancer cells ES-2 and PA-1 were treated with Id1/3-PA7 (5 μg per 106 cells). At 1.5 h after treatment, the internalisation of the peptide aptamer into cells and its colocalisation with Id1 and Id3 were verified by His-tag coimmunoprecipitation (Figure 1A and B). Cells treated with TrxA (5 μg per 106 cells) were used as negative control in coimmunoprecipitation (Figure 1C and D). In addition, the colocalisation was verified by coimmunofluorescence staining (Figure 1E–H).


Inhibition of Id proteins by a peptide aptamer induces cell-cycle arrest and apoptosis in ovarian cancer cells.

Mern DS, Hasskarl J, Burwinkel B - Br. J. Cancer (2010)

Colocalisation of the intracellular-delivered peptide aptamer Id1/3-PA7 to Id1 and Id3. (A, B) His-tag coimmunoprecipitation of Id1/3-PA7 with Id1 and Id3 in ovarian cancer cells ES-2 and PA-1. (C, D) His-tag coimmunoprecipitation of TrxA as negative control. (E–H) Coimmunofluorescence staining of Id1/3-PA7 with Id1 and Id3 in ES-2 and PA-1 cells. DAPI, 4,6-diamidino-2-phenylindole.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2967066&req=5

fig1: Colocalisation of the intracellular-delivered peptide aptamer Id1/3-PA7 to Id1 and Id3. (A, B) His-tag coimmunoprecipitation of Id1/3-PA7 with Id1 and Id3 in ovarian cancer cells ES-2 and PA-1. (C, D) His-tag coimmunoprecipitation of TrxA as negative control. (E–H) Coimmunofluorescence staining of Id1/3-PA7 with Id1 and Id3 in ES-2 and PA-1 cells. DAPI, 4,6-diamidino-2-phenylindole.
Mentions: Using yeast and mammalian two-hybrid systems, we isolated from the randomised expression library a constrained peptide aptamer Id1/3-PA7 that interacts specifically with Id1 and Id3 (Mern et al, 2010). Here, we used Id1/3-PA7 to analyse its functional interference in Id1- and Id3-overexpressing ovarian cancer cells ES-2 and PA-1. For its delivery into ovarian cancer cells, Id1/3-PA7 was fused with cell-penetrating PTD (Elliott and O'Hare, 1997; Phelan et al, 1998; Narita et al, 2001), expressed in bacteria and purified under native conditions. Ovarian cancer cells ES-2 and PA-1 were treated with Id1/3-PA7 (5 μg per 106 cells). At 1.5 h after treatment, the internalisation of the peptide aptamer into cells and its colocalisation with Id1 and Id3 were verified by His-tag coimmunoprecipitation (Figure 1A and B). Cells treated with TrxA (5 μg per 106 cells) were used as negative control in coimmunoprecipitation (Figure 1C and D). In addition, the colocalisation was verified by coimmunofluorescence staining (Figure 1E–H).

Bottom Line: Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.These effects were counteracted by ectopically overexpressed Id1 and Id3.Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz-University Group Molecular Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany. d.mern@dkfz-heidelberg.de

ABSTRACT

Background: Inhibitors of DNA-binding proteins (Id1-4), lacking the basic DNA-binding domain, function as dominant inhibitors of cell-cycle regulators. Overexpression of Id proteins promotes cancer cell proliferation and resistance against apoptosis. Level of Id protein expression, especially of Id1, correlates with poor differentiation, enhanced malignant potential and more aggressive clinical behaviour of ovarian tumours. Although overexpression of Ids has been found and shown to correlate with poor clinical outcome, their inhibition at protein level has never been studied.

Methods: A peptide aptamer, Id1/3-PA7, targeting Id1 and Id3, was isolated from a randomised combinatorial expression library using yeast and mammalian two-hybrid systems. Id1/3-PA7 was fused, expressed and purified with a cell-penetrating protein transduction domain.

Results: Intracellular-delivered Id1/3-PA7 colocalised to Id1 and Id3. It induced cell-cycle arrest and apoptosis in ovarian cancer cells ES-2 and PA-1. It activated the E-box promoter and increased the expression level of cyclin-dependent kinase inhibitor (CDKN2A) in a dose-dependent manner that is paralleled by the cleavage of poly-ADP ribose polymerase. These effects were counteracted by ectopically overexpressed Id1 and Id3.

Conclusion: Id1/3-PA7 could represent an exogenous anti-tumour agent that can significantly trigger cell-cycle arrest and apoptosis in ovarian cancer.

Show MeSH
Related in: MedlinePlus