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Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer.

Xu XS, Wang L, Abrams J, Wang G - J Hematol Oncol (2011)

Bottom Line: Bladder cancer is strongly associated with exposure to environmental carcinogens.The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08).In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Environmental Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA.

ABSTRACT
Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors.

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Diagram of the promoter region structure of the XPC gene. The consensus sequences of transcription factors CREB-1 and Sp1 were highlighted in the box. The start codon of the XPC gene is labeled in red.
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Figure 1: Diagram of the promoter region structure of the XPC gene. The consensus sequences of transcription factors CREB-1 and Sp1 were highlighted in the box. The start codon of the XPC gene is labeled in red.

Mentions: To determine the mechanism through which the HDACs inhibit transcription of the XPC gene, we further performed a chromatin immunoprecipitation (ChIP)-based transcription factor binding study. We chose both CREB1 and Sp1 transcription factors for our ChIP study because the consensus sequences for both transcription factors are present at the 5' promoter region of the XPC gene (Figure 1) and are likely to be involved in the transcription regulation of the XPC gene. Some studies also revealed the overlapping in binding to DNA targets between the HDAC4 and the Sp1 [41-46]. The HTB4 and HTB9 cells were treated with the VPA (5 mM) for 48 hours and fixed in 1% formaldehyde. As a control, the untreated HTB4 and HTB9 cells were also harvested and fixed in 1% formaldehyde. The cells were sonicated to shear the chromosomal DNA into small fragments. A ChIP protocol was performed to pull down the CREB1 or the Sp1 transcription factor using antibodies against the individual transcription factors. Half of the beads obtained from the ChIP protocol were analyzed by western blots to determine the amount of the transcription factor pulled down by the ChIP protocol (Figure 2). The remainder of the beads was resuspended into an elution buffer (0.1M Na2CO3, 1% SDS, 200 mM NaCl) and the DNA co-precipitated with the transcription factors was recovered. The DNA was analyzed by a quantitative PCR (qPCR) protocol to determine the amount of XPC gene promoter region DNA co-precipitated with the transcription factors (Table 3). The results of our western blots revealed that similar amounts of the CREB1 and Sp1 were pulled down from both untreated and VPA-treated cells for both HTB4 and HTB9 cells, suggesting a very successful ChIP protocol (Figure 2). The results of our qPCR studies, however, indicated a very different pattern of XPC gene promoter region DNA co-precipitation following the VPA treatment. When the CREB1 antibody was used in the ChIP study, the VPA treatment resulted in a 4.6 ± 0.4 and 2.2 ± 0.4 fold increase of the co-precipitated XPC gene promoter region DNA in the HTB4 and HTB9 cells respectively (Table 3); when the Sp1 antibody was used in the ChIP study, the VPA treatment caused a 2.2 ± 0.3 and 2.0 ± 0.3 fold increase of the co-precipitated XPC gene promoter region DNA in the HTB4 and HTB9 cells respectively. These results indicate that the VPA treatment enhances the binding of the CREB1 and Sp1 transcription factors at the promoter region of the endogenous XPC gene in both HTB4 and HTB9 cells, suggesting that inhibiting the binding of CREB1 and Sp1 transcription factors to their consensus sequences plays an important role in the HDACs-mediated XPC gene silencing.


Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer.

Xu XS, Wang L, Abrams J, Wang G - J Hematol Oncol (2011)

Diagram of the promoter region structure of the XPC gene. The consensus sequences of transcription factors CREB-1 and Sp1 were highlighted in the box. The start codon of the XPC gene is labeled in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108377&req=5

Figure 1: Diagram of the promoter region structure of the XPC gene. The consensus sequences of transcription factors CREB-1 and Sp1 were highlighted in the box. The start codon of the XPC gene is labeled in red.
Mentions: To determine the mechanism through which the HDACs inhibit transcription of the XPC gene, we further performed a chromatin immunoprecipitation (ChIP)-based transcription factor binding study. We chose both CREB1 and Sp1 transcription factors for our ChIP study because the consensus sequences for both transcription factors are present at the 5' promoter region of the XPC gene (Figure 1) and are likely to be involved in the transcription regulation of the XPC gene. Some studies also revealed the overlapping in binding to DNA targets between the HDAC4 and the Sp1 [41-46]. The HTB4 and HTB9 cells were treated with the VPA (5 mM) for 48 hours and fixed in 1% formaldehyde. As a control, the untreated HTB4 and HTB9 cells were also harvested and fixed in 1% formaldehyde. The cells were sonicated to shear the chromosomal DNA into small fragments. A ChIP protocol was performed to pull down the CREB1 or the Sp1 transcription factor using antibodies against the individual transcription factors. Half of the beads obtained from the ChIP protocol were analyzed by western blots to determine the amount of the transcription factor pulled down by the ChIP protocol (Figure 2). The remainder of the beads was resuspended into an elution buffer (0.1M Na2CO3, 1% SDS, 200 mM NaCl) and the DNA co-precipitated with the transcription factors was recovered. The DNA was analyzed by a quantitative PCR (qPCR) protocol to determine the amount of XPC gene promoter region DNA co-precipitated with the transcription factors (Table 3). The results of our western blots revealed that similar amounts of the CREB1 and Sp1 were pulled down from both untreated and VPA-treated cells for both HTB4 and HTB9 cells, suggesting a very successful ChIP protocol (Figure 2). The results of our qPCR studies, however, indicated a very different pattern of XPC gene promoter region DNA co-precipitation following the VPA treatment. When the CREB1 antibody was used in the ChIP study, the VPA treatment resulted in a 4.6 ± 0.4 and 2.2 ± 0.4 fold increase of the co-precipitated XPC gene promoter region DNA in the HTB4 and HTB9 cells respectively (Table 3); when the Sp1 antibody was used in the ChIP study, the VPA treatment caused a 2.2 ± 0.3 and 2.0 ± 0.3 fold increase of the co-precipitated XPC gene promoter region DNA in the HTB4 and HTB9 cells respectively. These results indicate that the VPA treatment enhances the binding of the CREB1 and Sp1 transcription factors at the promoter region of the endogenous XPC gene in both HTB4 and HTB9 cells, suggesting that inhibiting the binding of CREB1 and Sp1 transcription factors to their consensus sequences plays an important role in the HDACs-mediated XPC gene silencing.

Bottom Line: Bladder cancer is strongly associated with exposure to environmental carcinogens.The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08).In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Environmental Health Sciences, Wayne State University, 259 Mack Avenue, Detroit, MI 48201, USA.

ABSTRACT
Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors.

Show MeSH
Related in: MedlinePlus