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Trichostatin A causes p53 to switch oxidative-damaged colorectal cancer cells from cell cycle arrest into apoptosis.

Habold C, Poehlmann A, Bajbouj K, Hartig R, Korkmaz KS, Roessner A, Schneider-Stock R - J. Cell. Mol. Med. (2008)

Bottom Line: H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death.In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells.For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response.

View Article: PubMed Central - PubMed

Affiliation: University of Magdeburg, Institute of Pathology, Magdeburg, Germany.

ABSTRACT
Many studies aim at improving therapeutic efficacy by combining strategies with oxidative stress-inducing drugs and histone deacetylase (HDAC) inhibitors in colorectal cancer. As p53 and p21(WAF1) are essential in oxidative stress-induced DNA damage, we investigated epigenetic regulation of p21(WAF1) promoter. Firstly, HCT116 p53(+)/(+) and p53(-)/(-) colorectal cancer cells were treated with H(2)O(2) for 6 hrs and 24 hrs (early/late response). Chromatin immunoprecipitation revealed transcriptional transactivation of p21(WAF1) in HCT116 p53(+)/(+) cells as shown by increased binding of p53 and acetylated H4 around two p21(WAF1) promoter sites, the responsible element (RE) and the Sp1 site, while both proteins bound preferentially on the RE. Interestingly, H3 was not involved, suggesting H4-specific transactivation of the p21(WAF1) promoter. H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death. To investigate whether a HDAC inhibitor strengthens G(2)/M arrest, we pretreated cells with Trichostatin A (TSA). In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells. For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response. Reduced p53-triggered transactivation of p21(WAF1) in late response allows cells to re-enter cell cycle, and TSA causes p53 to simultaneously induce apoptosis.

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Effects of H2O2-treatment (30 mM, 3 min) on cell cycle profiles and cell viability of HCT116 p53−/− cells after 6 hrs and 24 hrs, with and without TSA (200 ng/ml, 6 hrs) pre-treatment. (A) H2O2 induces G2/M arrest in HCT116 p53−/− cells after 24 hrs while TSA induces a slight G2/M stop.(B) Annexin-V measurement shows that neither H2O2 nor TSA induce a significant apoptosis. (C) Western Blotting of caspase 3 confirms the observed Annexin-V results. (D) Time-dependent effects (6 hrs and 24 hrs) of TSA, H2O2, and their combined treatment on cell cycle progression and apoptosis induction. Above the line, data of FACS analysis (in%) are shown and below the line apoptotic cells (in%) measured in the Annexin-V-Assay are given.
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fig04: Effects of H2O2-treatment (30 mM, 3 min) on cell cycle profiles and cell viability of HCT116 p53−/− cells after 6 hrs and 24 hrs, with and without TSA (200 ng/ml, 6 hrs) pre-treatment. (A) H2O2 induces G2/M arrest in HCT116 p53−/− cells after 24 hrs while TSA induces a slight G2/M stop.(B) Annexin-V measurement shows that neither H2O2 nor TSA induce a significant apoptosis. (C) Western Blotting of caspase 3 confirms the observed Annexin-V results. (D) Time-dependent effects (6 hrs and 24 hrs) of TSA, H2O2, and their combined treatment on cell cycle progression and apoptosis induction. Above the line, data of FACS analysis (in%) are shown and below the line apoptotic cells (in%) measured in the Annexin-V-Assay are given.

Mentions: Because colorectal cancer therapy often fails due to drug resistance of colon cancer cells mostly due to p53 mutations, we investigated the effectiveness of our developed pro-apoptotic combination strategy for HCT116 p53−/− cells. In contrast to the p53+/+ cells, we observed no significant increase in the Pre-G1 cell population (Fig. 4A and D), reflecting the fact that apoptosis was not efficiently induced. Additionally, this result was confirmed by Annexin-V measurements and caspase 3 western blotting (Fig. 4B and C), suggesting that HCT116 p53−/− cells lost their apoptotic competence if p53 is lost. However, we observed H2O2-induced G2/M arrest, which could even be reinforced after TSA pre-treatment and was associated with significantly increased p21WAF1 expression (Figs. 4 and 5B). Nevertheless, ChIP experiments and RT-PCR gave no evidence of a tran-scriptionally caused up-regulation of p21WAF1 protein after TSA treatment alone (9-fold) at an early time point (Figs. 5A, E and F), suggesting posttranscriptional processes for p21WAF1 regulation. By contrast, the p21WAF1 up-regulation after H2O2 treatment or in combination with TSA at a later time point seems to be regulated transcriptionally (Fig. 5A), but was obviously not caused by HDAC1 release or acetylated H4 recruitment at the p21WAF1 promoter (Fig. 5E and F). Furthermore, HDAC1 activity was enhanced following single H2O2 treatment after 6 hrs and 24 hrs in both HCT cell lines (Figs. 3C and 5C). However, only in p53-deficient cells this rise in activity was accompanied by an increase in the HDAC1 protein amounts (Figs. 5B, C, 3B and C). The higher HDAC1 binding on the Sp1 site (Fig. 5F), compared to the p53+/+ cells (Fig. 3F), supports our idea that p53 actively displaces HDAC1 from the p21WAF1 promoter in H2O2-damaged, TSA pre-treated colorectal cancer cells.


Trichostatin A causes p53 to switch oxidative-damaged colorectal cancer cells from cell cycle arrest into apoptosis.

Habold C, Poehlmann A, Bajbouj K, Hartig R, Korkmaz KS, Roessner A, Schneider-Stock R - J. Cell. Mol. Med. (2008)

Effects of H2O2-treatment (30 mM, 3 min) on cell cycle profiles and cell viability of HCT116 p53−/− cells after 6 hrs and 24 hrs, with and without TSA (200 ng/ml, 6 hrs) pre-treatment. (A) H2O2 induces G2/M arrest in HCT116 p53−/− cells after 24 hrs while TSA induces a slight G2/M stop.(B) Annexin-V measurement shows that neither H2O2 nor TSA induce a significant apoptosis. (C) Western Blotting of caspase 3 confirms the observed Annexin-V results. (D) Time-dependent effects (6 hrs and 24 hrs) of TSA, H2O2, and their combined treatment on cell cycle progression and apoptosis induction. Above the line, data of FACS analysis (in%) are shown and below the line apoptotic cells (in%) measured in the Annexin-V-Assay are given.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822547&req=5

fig04: Effects of H2O2-treatment (30 mM, 3 min) on cell cycle profiles and cell viability of HCT116 p53−/− cells after 6 hrs and 24 hrs, with and without TSA (200 ng/ml, 6 hrs) pre-treatment. (A) H2O2 induces G2/M arrest in HCT116 p53−/− cells after 24 hrs while TSA induces a slight G2/M stop.(B) Annexin-V measurement shows that neither H2O2 nor TSA induce a significant apoptosis. (C) Western Blotting of caspase 3 confirms the observed Annexin-V results. (D) Time-dependent effects (6 hrs and 24 hrs) of TSA, H2O2, and their combined treatment on cell cycle progression and apoptosis induction. Above the line, data of FACS analysis (in%) are shown and below the line apoptotic cells (in%) measured in the Annexin-V-Assay are given.
Mentions: Because colorectal cancer therapy often fails due to drug resistance of colon cancer cells mostly due to p53 mutations, we investigated the effectiveness of our developed pro-apoptotic combination strategy for HCT116 p53−/− cells. In contrast to the p53+/+ cells, we observed no significant increase in the Pre-G1 cell population (Fig. 4A and D), reflecting the fact that apoptosis was not efficiently induced. Additionally, this result was confirmed by Annexin-V measurements and caspase 3 western blotting (Fig. 4B and C), suggesting that HCT116 p53−/− cells lost their apoptotic competence if p53 is lost. However, we observed H2O2-induced G2/M arrest, which could even be reinforced after TSA pre-treatment and was associated with significantly increased p21WAF1 expression (Figs. 4 and 5B). Nevertheless, ChIP experiments and RT-PCR gave no evidence of a tran-scriptionally caused up-regulation of p21WAF1 protein after TSA treatment alone (9-fold) at an early time point (Figs. 5A, E and F), suggesting posttranscriptional processes for p21WAF1 regulation. By contrast, the p21WAF1 up-regulation after H2O2 treatment or in combination with TSA at a later time point seems to be regulated transcriptionally (Fig. 5A), but was obviously not caused by HDAC1 release or acetylated H4 recruitment at the p21WAF1 promoter (Fig. 5E and F). Furthermore, HDAC1 activity was enhanced following single H2O2 treatment after 6 hrs and 24 hrs in both HCT cell lines (Figs. 3C and 5C). However, only in p53-deficient cells this rise in activity was accompanied by an increase in the HDAC1 protein amounts (Figs. 5B, C, 3B and C). The higher HDAC1 binding on the Sp1 site (Fig. 5F), compared to the p53+/+ cells (Fig. 3F), supports our idea that p53 actively displaces HDAC1 from the p21WAF1 promoter in H2O2-damaged, TSA pre-treated colorectal cancer cells.

Bottom Line: H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death.In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells.For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response.

View Article: PubMed Central - PubMed

Affiliation: University of Magdeburg, Institute of Pathology, Magdeburg, Germany.

ABSTRACT
Many studies aim at improving therapeutic efficacy by combining strategies with oxidative stress-inducing drugs and histone deacetylase (HDAC) inhibitors in colorectal cancer. As p53 and p21(WAF1) are essential in oxidative stress-induced DNA damage, we investigated epigenetic regulation of p21(WAF1) promoter. Firstly, HCT116 p53(+)/(+) and p53(-)/(-) colorectal cancer cells were treated with H(2)O(2) for 6 hrs and 24 hrs (early/late response). Chromatin immunoprecipitation revealed transcriptional transactivation of p21(WAF1) in HCT116 p53(+)/(+) cells as shown by increased binding of p53 and acetylated H4 around two p21(WAF1) promoter sites, the responsible element (RE) and the Sp1 site, while both proteins bound preferentially on the RE. Interestingly, H3 was not involved, suggesting H4-specific transactivation of the p21(WAF1) promoter. H(2)O(2) addition resulted in G(2)/M arrest of both HCT116 cell lines without significant cell death. To investigate whether a HDAC inhibitor strengthens G(2)/M arrest, we pretreated cells with Trichostatin A (TSA). In HCT116 p53(+)/(+) cells, we found (i) remarkably increased acetylated H4 around both p21(WAF1) promoter regions, especially at the Sp1 site; (ii) increased acetylation of p53 at lysines 320 and 382;(iii) displacement of HDAC1 from the Sp1 site, thus inhibiting its repression effect and increasing p53 binding.p53 seems to trigger H4-acetylation around the p21(WAF1) promoter because there was nearly no H4 acetylation in HCT116 p53(-)/(-) cells. For the first time we show that there is a time-dependent TSA mode of action with increased p53-dependent histone H4 acetylation at the p21(WAF1) promoter in early response, and decreased acetylation in late response. Reduced p53-triggered transactivation of p21(WAF1) in late response allows cells to re-enter cell cycle, and TSA causes p53 to simultaneously induce apoptosis.

Show MeSH
Related in: MedlinePlus