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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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Expression of selected growth regulatory genes in C646 sensitive and insensitive cell lines. (a) Western blot analyses on WM35, 1205Lu and WM983B cells. Lanes 1-4 represent 24 h of treatment with 0.2% DMSO, 10 μM C646, 20 μM C646 and 20 μM C37, respectively. (b) qPCR analysis of CCNA2 transcripts in WM35 and 1205Lu cells after 24 h treatment. N≥3. Please note that the Western blot antibody detects both CCNA1 and CCNA2, while the qPCR only detects CCNA2. (c) Relative expression of CCNA2 after siRNA transfection, as determined by qRT-PCR. (d) Relative cell proliferation measured by the MTT assay 96 h after plating the siRNA-transfected cells.
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Figure 3: Expression of selected growth regulatory genes in C646 sensitive and insensitive cell lines. (a) Western blot analyses on WM35, 1205Lu and WM983B cells. Lanes 1-4 represent 24 h of treatment with 0.2% DMSO, 10 μM C646, 20 μM C646 and 20 μM C37, respectively. (b) qPCR analysis of CCNA2 transcripts in WM35 and 1205Lu cells after 24 h treatment. N≥3. Please note that the Western blot antibody detects both CCNA1 and CCNA2, while the qPCR only detects CCNA2. (c) Relative expression of CCNA2 after siRNA transfection, as determined by qRT-PCR. (d) Relative cell proliferation measured by the MTT assay 96 h after plating the siRNA-transfected cells.

Mentions: Given the results from our microarray study, we sought to further evaluate the effect of p300 HAT inhibition on cell cycle regulatory protein expression in the melanoma cell lines. We found that cyclins A2 (CCNA2) and E2 (CCNE2) were among the most downregulated genes after 24 h of C646 treatment of WM35 cells (Table 1). Remarkably, we also observed a dose-dependent decrease in the protein levels of cyclins A and E in these cells (Figure 3a). Furthermore, this decrease was accompanied by an increased expression of p53 and its downstream effector p21, a known inhibitor of cyclin-dependent kinases (Figure 3a). No changes were noted in the level of cyclin D1, which is known to be overexpressed rather than repressed in senescent cells (Burton et al, 2007, Han et al, 1999, Meyyappan et al, 1998), nor were there any changes in the levels of B-raf, Bcl-2, PARP, or PCNA (Figure S6). Interestingly, in WM983B cells, in which the TP53 gene is mutated (Weiss et al, 1994), C646 continued to induce cell cycle arrest (Figure 1b) characterized by cyclin A repression and p21 activation (Figure 3a), suggesting a p53-independent mechanism for growth arrest in these cells. In contrast to the responsive cells, the C646-resistant cell line 1205Lu showed minimal effects on cyclin A expression following C646 treatment; however, p53 and p21 were both up-regulated to a similar extent as seen in the WM35 cells (Figure 3a), suggesting the existence of a bypass mechanism preventing p21-associated growth arrest. Notably, the mRNA level of CCNA2 decreased dose-dependently in C646-treated WM35 cells; however, such effects were significantly lessened in 1205Lu cells (Figure 3b), suggesting that the extent of cyclin A attenuation in these cells may not be sufficient for cell cycle arrest. The involvement of cyclin A in melanoma cell cycle regulation was further demonstrated with the siRNA knockdown of CCNA2 mRNA, which also resulted in decreased proliferation of the C646-responsive WM35 cells (Figure 3c, d).


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)

Expression of selected growth regulatory genes in C646 sensitive and insensitive cell lines. (a) Western blot analyses on WM35, 1205Lu and WM983B cells. Lanes 1-4 represent 24 h of treatment with 0.2% DMSO, 10 μM C646, 20 μM C646 and 20 μM C37, respectively. (b) qPCR analysis of CCNA2 transcripts in WM35 and 1205Lu cells after 24 h treatment. N≥3. Please note that the Western blot antibody detects both CCNA1 and CCNA2, while the qPCR only detects CCNA2. (c) Relative expression of CCNA2 after siRNA transfection, as determined by qRT-PCR. (d) Relative cell proliferation measured by the MTT assay 96 h after plating the siRNA-transfected cells.
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Figure 3: Expression of selected growth regulatory genes in C646 sensitive and insensitive cell lines. (a) Western blot analyses on WM35, 1205Lu and WM983B cells. Lanes 1-4 represent 24 h of treatment with 0.2% DMSO, 10 μM C646, 20 μM C646 and 20 μM C37, respectively. (b) qPCR analysis of CCNA2 transcripts in WM35 and 1205Lu cells after 24 h treatment. N≥3. Please note that the Western blot antibody detects both CCNA1 and CCNA2, while the qPCR only detects CCNA2. (c) Relative expression of CCNA2 after siRNA transfection, as determined by qRT-PCR. (d) Relative cell proliferation measured by the MTT assay 96 h after plating the siRNA-transfected cells.
Mentions: Given the results from our microarray study, we sought to further evaluate the effect of p300 HAT inhibition on cell cycle regulatory protein expression in the melanoma cell lines. We found that cyclins A2 (CCNA2) and E2 (CCNE2) were among the most downregulated genes after 24 h of C646 treatment of WM35 cells (Table 1). Remarkably, we also observed a dose-dependent decrease in the protein levels of cyclins A and E in these cells (Figure 3a). Furthermore, this decrease was accompanied by an increased expression of p53 and its downstream effector p21, a known inhibitor of cyclin-dependent kinases (Figure 3a). No changes were noted in the level of cyclin D1, which is known to be overexpressed rather than repressed in senescent cells (Burton et al, 2007, Han et al, 1999, Meyyappan et al, 1998), nor were there any changes in the levels of B-raf, Bcl-2, PARP, or PCNA (Figure S6). Interestingly, in WM983B cells, in which the TP53 gene is mutated (Weiss et al, 1994), C646 continued to induce cell cycle arrest (Figure 1b) characterized by cyclin A repression and p21 activation (Figure 3a), suggesting a p53-independent mechanism for growth arrest in these cells. In contrast to the responsive cells, the C646-resistant cell line 1205Lu showed minimal effects on cyclin A expression following C646 treatment; however, p53 and p21 were both up-regulated to a similar extent as seen in the WM35 cells (Figure 3a), suggesting the existence of a bypass mechanism preventing p21-associated growth arrest. Notably, the mRNA level of CCNA2 decreased dose-dependently in C646-treated WM35 cells; however, such effects were significantly lessened in 1205Lu cells (Figure 3b), suggesting that the extent of cyclin A attenuation in these cells may not be sufficient for cell cycle arrest. The involvement of cyclin A in melanoma cell cycle regulation was further demonstrated with the siRNA knockdown of CCNA2 mRNA, which also resulted in decreased proliferation of the C646-responsive WM35 cells (Figure 3c, d).

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