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Selective inhibition of p300 HAT blocks cell cycle progression, induces cellular senescence, and inhibits the DNA damage response in melanoma cells.

Yan G, Eller MS, Elm C, Larocca CA, Ryu B, Panova IP, Dancy BM, Bowers EM, Meyers D, Lareau L, Cole PA, Taverna SD, Alani RM - J. Invest. Dermatol. (2013)

Bottom Line: The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes.Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms.In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to have critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

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Effect of p300 HAT inhibition on cancer cell proliferation. (a) 3H-thymidine uptake in melanoma cells treated for 24 h with 10 μM C646, 10 μM C37, or 0.1% DMSO. Boxed: B-raf wildtype; unboxed: B-raf(V600E). The data are normalized to DMSO. N=3; error bars: standard deviations (SD). (b) Cell cycle profiles of WM35, WM983B and 1205Lu after 24 h treatment. Right panel: %S phase normalized to DMSO. N=3. P-values were calculated from Student's t-test. Representative histograms are shown in Figure S3. (c) Data from the one-dose NCI-60 screen. Bars indicate ‘Growth Percent (of each cell line) – Mean Growth Percent (of all cell lines)’; smaller numbers correspond to greater growth inhibition. See Figure S1, S2 for details.
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Figure 1: Effect of p300 HAT inhibition on cancer cell proliferation. (a) 3H-thymidine uptake in melanoma cells treated for 24 h with 10 μM C646, 10 μM C37, or 0.1% DMSO. Boxed: B-raf wildtype; unboxed: B-raf(V600E). The data are normalized to DMSO. N=3; error bars: standard deviations (SD). (b) Cell cycle profiles of WM35, WM983B and 1205Lu after 24 h treatment. Right panel: %S phase normalized to DMSO. N=3. P-values were calculated from Student's t-test. Representative histograms are shown in Figure S3. (c) Data from the one-dose NCI-60 screen. Bars indicate ‘Growth Percent (of each cell line) – Mean Growth Percent (of all cell lines)’; smaller numbers correspond to greater growth inhibition. See Figure S1, S2 for details.

Mentions: Previous data from our group and others have suggested that p300 HAT is important for tumor cell growth (Bowers et al, 2010). To evaluate the specific functional significance of p300 HAT in human melanomas, we explored the effect of p300 HAT blockade on melanoma cell proliferation using the selective inhibitor C646 in a 3H-thymidine incorporation assay. 3H-thymidine assay results were further validated using an XTT assay (Figure S3), recognizing the limitations associated with tetrazolium salt-based cell viability assays (Scudiero et al, 1988, Wang, Henning and Heber, 2010). Ten cell lines representing radial, vertical and metastatic phases of melanoma progression were evaluated with the majority demonstrating significant growth inhibition following treatment with C646 (Figure 1a) versus the non-functional control compound, C37 (Bowers et al, 2010). Furthermore, the degree of growth inhibition by C646 was not associated with tumor stage or B-raf status. Of note, previous studies from our group failed to demonstrate significant differences in EP300 transcript levels between our melanoma lines and melanocytes (Ryu et al, 2007).


Selective inhibition of p300 HAT blocks cell cycle progression, induces cellular senescence, and inhibits the DNA damage response in melanoma cells.

Yan G, Eller MS, Elm C, Larocca CA, Ryu B, Panova IP, Dancy BM, Bowers EM, Meyers D, Lareau L, Cole PA, Taverna SD, Alani RM - J. Invest. Dermatol. (2013)

Effect of p300 HAT inhibition on cancer cell proliferation. (a) 3H-thymidine uptake in melanoma cells treated for 24 h with 10 μM C646, 10 μM C37, or 0.1% DMSO. Boxed: B-raf wildtype; unboxed: B-raf(V600E). The data are normalized to DMSO. N=3; error bars: standard deviations (SD). (b) Cell cycle profiles of WM35, WM983B and 1205Lu after 24 h treatment. Right panel: %S phase normalized to DMSO. N=3. P-values were calculated from Student's t-test. Representative histograms are shown in Figure S3. (c) Data from the one-dose NCI-60 screen. Bars indicate ‘Growth Percent (of each cell line) – Mean Growth Percent (of all cell lines)’; smaller numbers correspond to greater growth inhibition. See Figure S1, S2 for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380234&req=5

Figure 1: Effect of p300 HAT inhibition on cancer cell proliferation. (a) 3H-thymidine uptake in melanoma cells treated for 24 h with 10 μM C646, 10 μM C37, or 0.1% DMSO. Boxed: B-raf wildtype; unboxed: B-raf(V600E). The data are normalized to DMSO. N=3; error bars: standard deviations (SD). (b) Cell cycle profiles of WM35, WM983B and 1205Lu after 24 h treatment. Right panel: %S phase normalized to DMSO. N=3. P-values were calculated from Student's t-test. Representative histograms are shown in Figure S3. (c) Data from the one-dose NCI-60 screen. Bars indicate ‘Growth Percent (of each cell line) – Mean Growth Percent (of all cell lines)’; smaller numbers correspond to greater growth inhibition. See Figure S1, S2 for details.
Mentions: Previous data from our group and others have suggested that p300 HAT is important for tumor cell growth (Bowers et al, 2010). To evaluate the specific functional significance of p300 HAT in human melanomas, we explored the effect of p300 HAT blockade on melanoma cell proliferation using the selective inhibitor C646 in a 3H-thymidine incorporation assay. 3H-thymidine assay results were further validated using an XTT assay (Figure S3), recognizing the limitations associated with tetrazolium salt-based cell viability assays (Scudiero et al, 1988, Wang, Henning and Heber, 2010). Ten cell lines representing radial, vertical and metastatic phases of melanoma progression were evaluated with the majority demonstrating significant growth inhibition following treatment with C646 (Figure 1a) versus the non-functional control compound, C37 (Bowers et al, 2010). Furthermore, the degree of growth inhibition by C646 was not associated with tumor stage or B-raf status. Of note, previous studies from our group failed to demonstrate significant differences in EP300 transcript levels between our melanoma lines and melanocytes (Ryu et al, 2007).

Bottom Line: The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes.Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms.In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to have critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity and scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly, and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. In addition, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells after combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

Show MeSH
Related in: MedlinePlus