Differential DNA Methylation Profile of Key Genes in Malignant Prostate Epithelial Cells Transformed by Inorganic Arsenic or Cadmium

Katherine E. Pelch, Erik J. Tokar, B. Alex Merrick, Michael P. Waalkes.
Toxicol Appl Pharmacol. (2015) DOI: https://doi.org/10.1016/j.taap.2015.04.011 PMID: 25922126

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Abstract

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status.

Figures

Figure 1. S100P methylation

Differential methylation pattern of S100P in transformed cell lines. A) Genomic locus showing location of CpGs (red lines) relative to the transcriptional start site (arrow). Genomic DNA was bisulfite converted and PCR amplified (gray bars). B) Representative agarose gels from COBRA analysis. PCR amplified bisulfite converted DNA was enzymatically digested with BstUI, Hpy99I, HpyCH4IV, or TaqαI as indicated. Triplicate samples of each cell type and universally methylated (+ Con) or unmethylated (− Con) DNA were separated on each gel. C) Bisulfite sequencing results. Each box represents a CpG site where red is a methylated CpG site and blue is an umethylated CpG site. White boxes represent locations where a base call could not be accurately made. Each row is a different clone that was sequenced. Summary data for each cell type is also displayed. D) Relative gene expression in untreated cells (open bars) or cells treated with 0.5 μM 5-aza-dC (gray bars). Data is geometric mean of the fold changes + 95% confidence interval. Data were analyzed by two way ANOVA and all multiple comparisons post-hoc analysis. *p < 0.05 for comparisons of treated and untreated cells of the same type and † p < 0.05 for comparison of untreated transformants to untreated controls.

Figure 1 (2 MB)
Figure 1B supporting data: COBRA analysis (58 MB)
Figure 1C supporting data: Bisulfite sequencing data (14 MB)
Figure 1D supporting data: Gene expression in untreated and 5-aza-dC treated cells (565 KB)

Figure 2. Hyal1 methylation

Differential methylation pattern of HYAL1 in transformed cell lines. See Fig. 1 legend for description of panels A–D.

Figure 2 (748 KB)
Figure 2B supporting data: COBRA analysis (34 MB)
Figure 2C supporting data: Bisulfite sequencing data (33 MB)
Figure 2D supporting data: Gene expression in untreated and 5-aza-dC treated cells (536 KB)

Figure 3. Ntm methylation

Differential methylation pattern of NTM in transformed cell lines. See Fig. 1 legend for description of panels A–D. ND indicates not detected.

Figure 3 (2 MB)
Figure 3B supporting data: COBRA analysis (137 MB)
Figure 3C supporting data: Bisulfite sequencing data (15 MB)
Figure 3D supporting data: Gene expression in untreated and 5-aza-dC treated cells (1 MB)

Figure 4. Nes methylation

Differential methylation pattern of NES in transformed cell lines. See Fig. 1 legend for description of panels A–D.

Figure 4 (1 MB)
Figure 4B supporting data: COBRA analysis (90 MB)
Figure 4C supporting data: Bisulfite sequencing data (6 MB)
Figure 4D supporting data: Gene expression in untreated and 5-aza-dC treated cells (518 KB)

Figure 5. Aldh1a1 methylation

Methylation pattern of ALDH1A1 in transformed cell lines. See Fig. 1 legend for description of panels A–D.

Figure 5 (2 MB)
Figure 5B supporting data: COBRA analysis (19 MB)
Figure 5C supporting data: Bisulfite sequencing data (8 MB)
Figure 5D supporting data: Gene expression in untreated and 5-aza-dC treated cells (555 KB)

Tables

Table 2. Gene Expression

Relative gene expression in transformed cells. Fold change relative to control RWPE-1 cell equal to 1.0. *p < 0.05 by one-way ANOVA of log transformed fold change values followed by comparison to RWPE-1 control cells by Dunnett's multiple comparisons post-hoc test. (95% CI) aNTM detected at a cycle threshold of 27 in control RWPE-1 cells and undetected in CTPE or CAsE-PE cells.

Table 2 (91 KB)
Table 2 supporting data: cDNA generation (38 KB)
Table 2 supporting data: Data analysis (530 KB)
Table 2 supporting data: Raw data (3 MB)

Table 3. Percent Methylation

Percentage (%) of methylated CpGs across the entire amplicon. The percentage of methylated CpGs in each clone was compared by Kruskal–Wallis test and *p < 0.05 relative to RWPE-1 by Dunn's post-hoc analysis.

Table 3 (95 KB)
Table 3 supporting data: Analysis of percentage methylated CpG (47 KB)

Supplemental Materials

Supplmental Figure 1

Relative miRNA expression in untreated cells (open bars) or cells treated with 0.5 µM 5-aza-dC (gray bars). Data is geometric mean of the fold changes + 95% confidence interval. Data were analyzed by two way ANOVA and all multiple comparisons post-hoc analysis.