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

The HTB4 (T24), HTB9, HTB2, HTB3, HTB5, HT1197, and HT1376 bladder cancer cells were purchased from American Type Culture Collection (ATCC) (Rockville, MD). The GM00637 human fibroblast cells were purchased from the Coriell Institute for Medical Research (Camden, NJ). The HTB2 and HTB4 cells were cultured in a McCoy's 5A medium supplemented with 10% FBS at 37°C with 5% CO₂. The HTB9 cells were cultured in RPMI1640 medium supplemented with 1× non-essential amino acids (NEAA) and 10% FBS at 37°C with 5% CO₂. The HTB3, HTB5, HT1197, and HT1376 bladder cancer cells were cultured in minimal essential medium (MEM) supplemented with 10% FBS and 1× NEAA at 37°C with 5% CO₂. The GM00637 cells were cultured in MEM supplemented with 10% FBS, 2× essential amino acids (EAA), 2x NEAA, and 2x vitamins (Vt) at 37°C with 5% CO₂.

The oligonucleotides used in this study are listed in Table 1 and were synthesized by Retrogen, Inc. (San Diego, CA). The primers used for determining the level of XPC mRNA by real time PCR were designed to bind to the XPC mRNA sequence at exon 5 and exon 6 thus amplifying a 120 bp DNA fragment. The primers used for determining the level of XPA mRNA by real time PCR were designed to bind to the XPA mRNA at exon 3 and exon 4 in order to amplify a 110 bp DNA fragment. The primers used for detection of the immuno-precipitation XPC gene promoter sequence were designed to bind to the XPC gene 5' regulatory region sequence at the -95 to -75 region and the +80 to +50 region to amplify a 175 bp DNA fragment.

The VPA was purchased from Sigma Corp. (St. Louis, MO). The HTB4 and HTB9 cells were seeded onto 100 mm cell culture dishes at a density of 1 × 10⁶ cells/dish and incubated at 37°C overnight. The VPA was added to the cell culture medium to a final concentration of 5 mM. The cells were cultured in the VPA-containing medium for 48 hours and then used for further studies.

Total RNA was isolated from both untreated and VPA-treated HTB4 and HTB9 bladder cancer cells using an RNeasy mini isolation kit (Qiagen). A reverse transcription-based quantitative PCR (real time PCR) was then performed to determine the mRNA levels of both xpc and xpa genes from each RNA sample using a Sybr green-based DNA quantification method (Applied Biosystems, Foster City, CA). The mRNA level of the β-actin gene was also determined for each RNA sample by using the real time PCR. The reverse transcription assay was carried out using 2 μg of total RNA utilizing the protocol suggested by the manufacturer (Applied Biosystems). The PCR procedure was performed using Taq-Man Universal PCR master mix with 100 ng cDNA in a total volume of 20 μl. The PCR assays were completed using the ABI prism 7500 Fast PCR system with the following conditions: 2 min at 94°C, followed by 40 cycles of 15 seconds at 95°C, 30 seconds at 56°C, and 60 seconds at 72°C. The real time PCR data was analyzed using a comparative cycle threshold (C_t) method. Relative quantification was performed to determine gene expression between untreated and VPA-treated cells. The actin gene was used as an internal control for normalization. Relative transcriptions of the XPC and XPA mRNAs were calculated as 2^-ΔΔCt where ΔC_t was calculated by subtracting the average actin gene C_t from the average XPC or XPA gene C_t value in the same cell line. The ΔΔC_t was obtained by the ΔC_t of the VPA-treated cells subtracted from the ΔC_t of the untreated cells.

Cells were harvested and lysed in RIPA cell lysis buffer (1xPBS, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS). The cell lysates (30 μg total protein) were analyzed by SDS-PAGE using a 10% gel. The proteins were transferred to a PVDF membrane and hybridized with the indicated antibodies for detection of the desired target proteins. The same membrane was then soaked in a stripping solution (62.5 mM Tris, pH 6.8, 2% SDS, 0.7% 2-mercaptoethanol) at 50°C for 30 min and then hybridized with a β-actin antibody (Oncogene, Cambridge, MA) to determine the level of β-actin in each sample. Quantification of the western results was performed using a Kodak Image Station 440CF system and the level of the target protein in each cell lysate was expressed as a relative level to that of β-actin in the same cell lysate. The level of XPC protein in the VPA-treated cells was calculated as a percentage compared to that of the XPC protein in the untreated cells. The statistical analysis of the western data was done using GraphPad PRISM 4.0 software.

The cells were harvested and washed in 1xPBS buffer once. The cells were then resuspended into 1xPBS buffer containing 1% formaldehyde and incubated at 37°C for 15 minutes. The cells were collected and washed three times with 1xPBS buffer. The cells were then resuspended into SDS lysis buffer (1 × 10⁶ cells/200 μl) and incubated on ice for 10 minutes. The cells were sonicated in order to shear the genomic DNA to lengths of 200-1000 bp. The cell lysates were centrifuged at 4°C for 10 minutes and the supernatants were collected. For the ChIP assay, cell lysate (200 μl) was diluted at a ratio of 1:10 in the ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH8.1, 167 mM NaCl) and incubated with either Protein A-conjugated agarose beads (for Sp1) or Protein G-conjugated agarose beads (for CREB1) at 4°C for 60 minutes. The cell lysates were centrifuged at 4°C for 5 minutes to remove the agarose beads. The cell lysates were then incubated with 2 μg of CREB1 antibody (X-12 from Santa Cruz) or Sp1 antibody (H-225 from Santa Cruz) at 4°C overnight using a rotating mixer. The Protein A-conjugated agarose beads (for Sp1) or Protein G-conjugated agarose beads (for CREB1) were then added and the reactants were incubated at 4°C for 2 hours with a rotating mixer. The beads were collected and washed three time in 1xPBS buffer and three times in ChIP washing buffer (0.1%SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 150 mM NaCl). Half of the beads were analyzed by western blot to determine the amount of the CREB1 or Sp1 proteins precipitated by the ChIP protocol. The remainder of the beads were resuspended into 200 μl of DNA elution buffer (0.1M Na₂CO₃, 1% SDS, 200 mM NaCl) and incubated at 65°C for 6 hours to reverse the protein-DNA cross-links. The DNA was recovered by phenol/chloroform extraction and ethanol precipitation. The relative level of XPC gene promoter region DNA co-precipitated with the beads was determined by a quantitative PCR (qPCR) protocol using the Applied Biosystems' Fast 7500 Real Time PCR system (Applied Biosystems, Foster City, CA). The level of the XPC gene promoter region DNA co-precipitated with the CREB1 or Sp1 in the untreated cells was accounted as 100% and the level of the XPC gene promoter region DNA co-precipitated with the beads in the VPA-treated cells was calculated as a fold change relative to that of the untreated cells.

The bladder tumor tissue arrays BL208, BL2081 and BL2082 were purchased from US BioMax Inc. (Rockville, MD) and were used in the IHC staining study. The formalin-fixed paraffin-embedded (FFPE) bladder tumor tissue array slides were first deparaffinized in 100% xylenes; the slides were then hydrated through a series of graded alcohols (100%, 95%, 80%, 70%, and 30%) for 5 minutes each. The slides were washed once in H₂O for 5 minutes. The slides were then incubated in 10 mM sodium citrate buffer (pH6.0) for 15 minutes at 95°C to unmask the antigen. The bladder tumor tissue array slides were then incubated in 1% hydrogen peroxide at room temperature for 10 minutes to quench endogenous peroxidase activity. The slides were incubated in 1.5% normal blocking serum in 1xPBS for 1 hour and then incubated with the primary antibody at 1:100 dilution in 1xPBS for 30 minutes. The slides were washed in 1xPBS three times and then incubated with a biotin-conjugated secondary antibody (Santa Cruz) at room temperature for 30 minutes. The slides were then washed three times in 1xPBS and incubated with an avidin-biotin enzyme reagent (Santa Cruz) for 30 minutes. The slides were incubated in peroxidase substrate (Santa Cruz) for 1 to 10 minutes until the desired stain intensity developed. The slides were counterstained in Gill's formulation #2 hematoxylin (Santa Cruz) for 10 seconds and then washed in deionized H₂O with several H₂O changes. The slides were dehydrated through graded alcohols (30 - 100%) and xylenes and mounted with glass coverslips using a Clarion permanent mounting medium (Santa Cruz, CA). The HDAC-positive cells were determined using light microscopy. Two hundred cells were counted from each tissue specimen. A HDAC-negative tissue specimen was established if >20% of the counted cells were HDAC-positive cells and a HDAC-positive tissue specimen was established if <20% of the counted cells were HDAC-positive cells.

The caspase-3 activity was measured using a protocol described previously [39, 40]. Essentially, the cells were harvested 40 hours after the cisplatin treatment and lysed in insect cell lysis buffer (BD Biosciences). The protein concentrations of the cell lysates were determined. The caspase-3 assay was carried out in a 96-well plate using fluorogenic Ac-DEVD-AMC as a substrate (BD Biosciences). Caspase-3 activity was determined by a spectrafluorometer (Molecular Devices) for detection of free AMC released from the substrate during a 15-minute incubation period at 37°C with an excitation wavelength of 380 nm and an emission wavelength of 430-460 nm. Caspase-3 activity was measured as nanomole of AMC/min/mg protein

In order to determine the role that the HDACs may play in XPC gene silencing and bladder cancer development, we first determined the effect of HDAC inhibitor treatment on activation of XPC gene transcription in HTB4 and HTB9 bladder cancer cells. Both HTB4 and HTB9 cancer cells were treated with the HDAC inhibitor of VPA (5 mM) for 48 hours and the total RNA was isolated. Total RNA was also isolated from the untreated HTB4 and HTB9 cells. A reverse transcription-based quantitative PCR (real time PCR) was performed to determine the level of the XPC mRNA in each RNA sample (Table 2). The level of XPA mRNA was also determined for each RNA sample in the study. The XPA protein is an important NER component but the level of XPA mRNA was not affected by the DNA damaging treatment [16]. The level of β-actin mRNA was determined for each RNA sample as an internal control. The level of the XPC mRNA increased 2.8 ± 0.4 and 2.4 ± 0.3 fold in the HTB4 and HTB9 cells respectively with the VPA treatment (Table 2). In contrast, the level of the XPA mRNA was not significantly altered in these cells following the VPA treatment (Table 2). These results suggest that the HDACs indeed play an important role in XPC gene silencing for both HTB4 and HTB9 bladder cancer cells, and treatment with the VPA HDAC inhibitor causes activation of the XPC transcription in both bladder cancer cell lines.

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.

To further determine the role of HDACs in XPC gene silencing and bladder cancer development, we determined the correlation between the presence of HDACs and the occurrence of bladder cancer using bladder tumor tissue arrays with an immunohistochemistry (IHC) staining procedure (Figure 3 and Table 4). The bladder tumor tissue arrays were purchased from US BioMax, Inc. (Rockville, MD) and used in this study. Both HDAC2 and HDAC4 were chosen for this study because the work of others has revealed abnormal levels of these proteins in many types of cancer [47‐55]. The results of our IHC study indicated that the frequency of the HDAC4-positive tissue specimens was much higher in the bladder tumors than in the normal bladder tissues (Figure 3 panel and Table 4). The statistical analysis of the data further revealed a significant difference in the frequency of HDAC4-positive tissue specimens between normal and cancerous bladder tissues (p < 0.001) as well as a marginal significance between the increasing incidence of HDAC4 positivity and the increasing severity of the bladder tumors (p = 0.08) (Table 4). The frequency of the HDAC2-positive specimens, however, was similar between normal and cancerous bladder tissues (data not shown). These results suggest that over-expression of the HDAC4 is strongly correlated with the development of bladder cancer.

The results of our IHC studies revealed strong correlation between over-expression of the HDAC4 and the occurrence of bladder tumors. To validate the IHC result, we further determined expression of several HDACs, including HDAC4, HDAC1, and HDAC2, in the HTB4, HTB9, HTB2, HTB3, HTB5, HT1197 and HT1376 bladder cancer cells (Figure 4). The expression of these HDACs in the GM00637 normal human fibroblast cells was also determined in the western blotting study and used as a control. The results obtained from our western blots study indicated that the protein levels of the HDAC1 and HDAC2 were similar in all the tested cells (Figure 4 middle panels). In contrast, the expression levels of HDAC4 were greatly increased in most of the tested bladder cancer cells except the HTB4 bladder cancer cells in comparison to that of the GM00637 normal human fibroblast cells (Figure 4 top panel). This result confirmed our IHC results and suggested the important role of HDAC4 over-expression in the bladder cancer development.

Extensive studies have demonstrated the cisplatin-induced apoptosis as major mechanism in cell killing [16, 39, 56‐58]. Because of the important function of XPC protein in the cisplatin-caused apoptosis [16] and the role HDACs in XPC gene silencing, we further investigated the effect of the HDAC inhibitor VPA in cisplatin-induced apoptosis of bladder cancer cells. The HTB4 and HTB9 bladder cancer cells were treated with VPA (5 mM) for 48 hours before they were treated with cisplatin. The cells were harvested 40 hours after the cisplatin treatment and the caspase-3 activity was determined (Figure 5). The caspase-3 activity was also determined from the HBT4 and HTB9 cells that were treated with cisplatin but without the prior VPA treatment (Figure 5). The cisplatin treatment itself caused an increase in caspase-3 activity in both HTB4 and HTB9 bladder cancer cells at high concentrations (20 μM and 40 μM) but not at lower concentrations (5 μM and 10 μM) (Figure 5). When these cells were treated with VPA prior to the cisplatin treatment, however, the caspase-3 activity was significantly increased at lower concentrations as well (Figure 5). For example, when treated only with cisplatin at 10 μM, the caspase 3 activity was increased by a 1.5 and 2 fold in the HTB4 and HTB9 cells respectively; when the cells were treated with 5 mM VPA prior to the cisplatin treatment, however, the 10 μM cisplatin treatment resulted in a 7.3 and 6.6 fold increase of the caspase-3 activity in the HTB4 and HTB9 cells respectively (Figure 5). These results suggest that the prior treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor VPA sensitizes these bladder cancer cells to the anticancer drug cisplatin.