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. 2011 Apr 20:4:17.
doi: 10.1186/1756-8722-4-17.

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

Xiaoxin S Xu  1 , Le Wang, Judith Abrams, Gan Wang
Affiliations

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

Xiaoxin S Xu et al. J Hematol Oncol. .

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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Figures

Figure 1
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.
Figure 2
Figure 2
Detection of CREB-1 and Sp1 protein obtained from the chromatin immunoprecipitation (ChIP). A ChIP protocol was performed to pull down the CREB-1 and Sp1 proteins from the individual cell lysates using antibodies against CREB-1 and Sp1 respectively. Half of the agarose beads obtained from the ChIP study were analyzed by western blots to determine the amount of the transcription factors precipitated from individual cell lysates. The remainder of the beads was analyzed by real time PCR to determine the amount of the XPC gene promoter DNA co-precipitated with the individual transcription factors.
Figure 3
Figure 3
Immunohistochemistry (IHC) stain of XPC and HDAC4 proteins in both normal and cancerous bladder tissue specimens using bladder tumor tissue arrays. The bladder tumor tissue arrays purchased from US BioMax Inc. were stained with either XPC or HDAC4 antibodies in an immunohistochemistry (IHC) protocol. The presence of XPC or HDAC4 protein was determined by light microscopy and the image was recorded by a DP Controller software (Olympus Corp., Center Valley, PA).
Figure 4
Figure 4
Detection of expression of HDAC4, HDAC1, and HDAC2 in various bladder cancer cells. The cell lysates prepared from the HTB2, HTB3, HTB4, HTB5, HTB9, HT1197, HT1376 bladder cancer cells and GM00637 normal human fibroblast cells (30 μg total protein) were analyzed by western blots to determine the protein levels of HDAC4, HDAC1, HDAC2, and β-actin in each cell lysate. The antibodies against HDAC4 (A-4), HDAC1 (C-19), HDAC2 (H-54) and β-actin (C-2) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and used in the western blots study.
Figure 5
Figure 5
The cisplatin-induced caspase 3 activity in both untreated and VPA-treated HTB4 and HTB9 bladder cancer cells. The VPA treatment (5 mM) was done 24 hours prior to the cisplatin treatment. The cells were treated with cisplatin at the indicated concentrations for 3 hours and then cultured in the cell culture incubator for 40 hours before the cells were harvested and the caspase 3 activity was measured. The caspase 3 activity was measured as nanomole of AMC/minute/mg of protein. (* statistical difference to that of the untreated cells with p value < 0.01).
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