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

Id1/3-PA7 induces cycle arrest and apoptosis in ovarian cancer cells. (A–J) Quantification of cell-incorporated bromodeoxyuridine (BrdU) (fluorescein isothiocyanate (FITC) anti-BrdU) and total DNA content (7-AAD) in untreated and TrxA-treated (5 μg per 106 cells) or Id1/3-PA7-treated (5 μg per 106 cells) ES-2 cells (A, C) and PA-1 cells (D–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells showed S-phase reduction of actively cycling ES-2 cells (G) and PA-1 cells (I), increasing apoptotic cells in sub-G0/G1- and G2/M-resided ES-2 cells (H) and PA-1 cells (J). There were no significant differences between untreated and TrxA-treated cells (A–J). Bars: black, untreated; white dotted, TrxA treated; hatched, Id1/3-PA7 treated. Results are represented as mean values of three independent experiments with s.d. (P⩽0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2967066&req=5

fig4: Id1/3-PA7 induces cycle arrest and apoptosis in ovarian cancer cells. (A–J) Quantification of cell-incorporated bromodeoxyuridine (BrdU) (fluorescein isothiocyanate (FITC) anti-BrdU) and total DNA content (7-AAD) in untreated and TrxA-treated (5 μg per 106 cells) or Id1/3-PA7-treated (5 μg per 106 cells) ES-2 cells (A, C) and PA-1 cells (D–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells showed S-phase reduction of actively cycling ES-2 cells (G) and PA-1 cells (I), increasing apoptotic cells in sub-G0/G1- and G2/M-resided ES-2 cells (H) and PA-1 cells (J). There were no significant differences between untreated and TrxA-treated cells (A–J). Bars: black, untreated; white dotted, TrxA treated; hatched, Id1/3-PA7 treated. Results are represented as mean values of three independent experiments with s.d. (P⩽0.05).

Mentions: Tumour suppressor CDKN2A inhibits the cyclin-dependent kinases (Cdk4 and Cdk6) that initiate the phosphorylation of the pRb (Ruas and Peters, 1998; Sherr and Roberts, 1999). Therefore, CDKN2A has the capacity to arrest cells in the G1 phase of the cell cycle. Applying immunofluorescent staining of incorporated BrdU, an analogue of the DNA precursor thymidine, and flow cytometric analysis, we quantified individual cells that have incorporated BrdU into newly synthesised DNA as cells entering and progressing through the S (DNA synthesis) phase of the cell cycle. ES-2 and PA-1 cells were treated with TrxA or Id1/3-PA7 (5 μg per 106 cells) at 4 h intervals for 48 h. For the identification and analysis of actively cycling, as opposed to non-cycling, cell fractions, we exposed untreated and TrxA- or Id1/3-PA7-treated cells to BrdU for 6 h. The cell-cycle positions and active DNA synthetic activities of cells were determined by analysing the correlated expression of total DNA and incorporated BrdU levels as shown by the region gates applied to the 7-AAD vs BrdU dot plot (Figure 4A–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells demonstrated that the anti-proliferative and apoptotic effects of Id1/3-PA7 reduced the number of actively cycling cells in S from 93.1 to 54.8% for ES-2 cells (Figure 4G) and from 91.4 to 54.6% for PA-1 cells (Figure 4I). The number of apoptotic cells in sub-G0/G1- and in G0/G1- or G2/M-resided cells increased from 1.4 to 21.1%, from 3.2 to 13.2% and from 0.2 to 5.9%, respectively, for ES-2 cells (Figure 4H), and from 1.7 to 14,6%, from 3.9 to 16% and from 0.3 to 7.2%, respectively, for PA-1 cells (Figure 4J). There were no significant differences between untreated and TrxA-treated cells (Figure 4A–J).


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)

Id1/3-PA7 induces cycle arrest and apoptosis in ovarian cancer cells. (A–J) Quantification of cell-incorporated bromodeoxyuridine (BrdU) (fluorescein isothiocyanate (FITC) anti-BrdU) and total DNA content (7-AAD) in untreated and TrxA-treated (5 μg per 106 cells) or Id1/3-PA7-treated (5 μg per 106 cells) ES-2 cells (A, C) and PA-1 cells (D–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells showed S-phase reduction of actively cycling ES-2 cells (G) and PA-1 cells (I), increasing apoptotic cells in sub-G0/G1- and G2/M-resided ES-2 cells (H) and PA-1 cells (J). There were no significant differences between untreated and TrxA-treated cells (A–J). Bars: black, untreated; white dotted, TrxA treated; hatched, Id1/3-PA7 treated. Results are represented as mean values of three independent experiments with s.d. (P⩽0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Id1/3-PA7 induces cycle arrest and apoptosis in ovarian cancer cells. (A–J) Quantification of cell-incorporated bromodeoxyuridine (BrdU) (fluorescein isothiocyanate (FITC) anti-BrdU) and total DNA content (7-AAD) in untreated and TrxA-treated (5 μg per 106 cells) or Id1/3-PA7-treated (5 μg per 106 cells) ES-2 cells (A, C) and PA-1 cells (D–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells showed S-phase reduction of actively cycling ES-2 cells (G) and PA-1 cells (I), increasing apoptotic cells in sub-G0/G1- and G2/M-resided ES-2 cells (H) and PA-1 cells (J). There were no significant differences between untreated and TrxA-treated cells (A–J). Bars: black, untreated; white dotted, TrxA treated; hatched, Id1/3-PA7 treated. Results are represented as mean values of three independent experiments with s.d. (P⩽0.05).
Mentions: Tumour suppressor CDKN2A inhibits the cyclin-dependent kinases (Cdk4 and Cdk6) that initiate the phosphorylation of the pRb (Ruas and Peters, 1998; Sherr and Roberts, 1999). Therefore, CDKN2A has the capacity to arrest cells in the G1 phase of the cell cycle. Applying immunofluorescent staining of incorporated BrdU, an analogue of the DNA precursor thymidine, and flow cytometric analysis, we quantified individual cells that have incorporated BrdU into newly synthesised DNA as cells entering and progressing through the S (DNA synthesis) phase of the cell cycle. ES-2 and PA-1 cells were treated with TrxA or Id1/3-PA7 (5 μg per 106 cells) at 4 h intervals for 48 h. For the identification and analysis of actively cycling, as opposed to non-cycling, cell fractions, we exposed untreated and TrxA- or Id1/3-PA7-treated cells to BrdU for 6 h. The cell-cycle positions and active DNA synthetic activities of cells were determined by analysing the correlated expression of total DNA and incorporated BrdU levels as shown by the region gates applied to the 7-AAD vs BrdU dot plot (Figure 4A–F). Flow cytometric analysis of untreated cells vs Id1/3-PA7-treated cells demonstrated that the anti-proliferative and apoptotic effects of Id1/3-PA7 reduced the number of actively cycling cells in S from 93.1 to 54.8% for ES-2 cells (Figure 4G) and from 91.4 to 54.6% for PA-1 cells (Figure 4I). The number of apoptotic cells in sub-G0/G1- and in G0/G1- or G2/M-resided cells increased from 1.4 to 21.1%, from 3.2 to 13.2% and from 0.2 to 5.9%, respectively, for ES-2 cells (Figure 4H), and from 1.7 to 14,6%, from 3.9 to 16% and from 0.3 to 7.2%, respectively, for PA-1 cells (Figure 4J). There were no significant differences between untreated and TrxA-treated cells (Figure 4A–J).

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