Expression level and DNA methylation status of glutathione-S-transferase genes in normal murine prostate and TRAMP tumors

Abstract

Background: Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors.

Methods: Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured.

Results: Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells.

Conclusions: Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.

Fig. 1

Gst mRNA expression in normal prostates from WT mice and TRAMP tumors. qRT-PCR of Gst genes was performed as described in Materials and Methods using the samples described in Table 1. Gst mRNA copy number is plotted relative to 18s rRNA expression. (A) Gst expression in normal murine prostate (N = 15). Gst family members analyzed are shown on the x-axis. (B) GstA4 (C) GstK1 (D) GstM1 (E) GstO1 and (F) GstP1 mRNA expression in normal prostate (N), primary prostate tumor (P), and metastases (M) (N = 15/group). The metastases group includes lymph node, kidney, and liver metastases (5 each). Error bars = Standard deviation (SD). The results of unpaired two-tailed T-test comparisons are shown for the indicated groups. In all cases, differences in Gst mRNA expression between Normal Prostate and Metastatic tumors were not significant.

Fig. 2

Western blot analysis of GstM1 and GstP1 protein expression in normal prostates from WT mice and TRAMP tumors. Cytosolic GstM1 and GstP1 protein levels were measured as described in Materials and Methods using the samples described in Table 1; representative Western blots are shown. (A) GstM1 Western blots. (B) GstP1 Western blots. Data labels: Normal prostates (N), primary tumors (P), kidney metastases (KM), liver metastases (LM), and lymph node metastases (LNM). For both panels, Ponceau S total protein staining served as a loading control.

Fig. 3

5’ end of murine Gst genes, indicating position of CpG islands and primer sites used for DNA methylation analyses. (A) GstA4, (B) GstK1, (C) GstM1, (D) GstO1, and (E) GstP1. For each diagram, the predicted transcriptional start sites from the UC Santa Cruz Genome Browser are shown with bent right arrows, and exons are shown with black filled bars. Hash marks indicate CpG sites. Gray filled bars show 5’ CpG islands; CpG island characteristics as determined using CpG island searcher (http://www.uscnorris.com/cpgislands2/cpg.aspx) are shown beneath the gray bars. The approximate position and 5’ nucleotide coordinates of primers used for MAQMA methylation analysis are shown by inward facing arrows.

Fig. 4

Gst gene methylation in normal murine prostate and inverse association with Gst mRNA expression. (A) DNA methylation of the 5’ regions of Gst genes diagrammed in Fig. 4 were determined by MassARRAY Quantitative DNA Methylation Analyses (MAQMA) as described in the Materials and Methods. Results plotted are the average methylation value of all CpG sites over the entire sequenced region, and all data are averaged over 5 normal prostate samples. Error bars = SD. (B) Gst methylation values plotted against Gst mRNA expression values shown in Fig. 1A. Non-linear regression (one-phase decay) correlation coefficient R² value was caculated using GraphPad Prism and is shown. (C) Gst methylation values plotted against Gst mRNA expression values shown in Fig. 1A, after removal of GstA4 data. Correlation coefficient R² value was calculated as described in panel C. Symbols : square = GstM1; circle = GstA4; inverted triangle = GstP1; diamond = GstO1; triangle = GstK1.

Fig. 5

Individual CpG site DNA methylation of Gst genes in normal prostates from WT mice and TRAMP tumors. (A) GstA4, (B) GstK1, (C) GstM1, (D) GstO1, and (E) GstP1. DNA methylation of the 5’ regions of Gst genes diagrammed in Fig. 3 were determined by MassARRAY Quantitative DNA Methylation Analyses (MAQMA) as described in the Materials and Methods. Results plotted are the average methylation value of each CpG site (or clusters of CpG sites) within each sequenced region, and data are averaged for normal prostate (N) (N=5), primary tumor (P) (N=15), and metastatic tumors (M) (N = 15). Infrequently, CpG sites failed MAQMA analysis; in these instances no data are shown (absence of bars on the graph). Right arrows below X-axes indicate the approximate position of the predicted transcriptional start site for each gene. Error bars = SD.

Fig. 6

Averaged Gst gene methylation in normal prostates from WT mice and TRAMP tumors. (A) GstA4, (B) GstK1, (C) GstM1, (D) GstO1, and (E) GstP1. DNA methylation of the 5’ regions of Gst genes diagrammed in Fig. 3 were determined by MassARRAY Quantitative DNA Methylation Analyses (MAQMA) as described in the Materials and Methods. Results plotted are the average methylation value of all CpG sites over the entire sequenced region, and all data are averaged for normal prostates (N) (N=5), primary tumors (P) (N=15), and metastatic tumors (M) (N = 15). The methylation data for the individual sites comprising each region are shown in Fig. 5. Error bars = SD. In all cases, the differences between groups were not significant (data not shown).

Fig. 7

Effect of decitabine and TSA treatment on Gst gene expression in TRAMP-C2 cells. TRAMP-C2 cells were treated with 1.0 μM decitabine (DAC) and/or 600 nM trichostatin A (TSA) as described in the Materials and Methods, and cells were harvest at five days post-DAC treatment and/or one day post TSA treatment. The vehicle control consisted of treatment of TRAMP-C2 cells with PBS and DMSO for five days and one day, respectively. Gst mRNA expression was measured by qRT-PCR as described in the Materials and Methods. (A) GstA4, (B) GstK1, (C) GstM1, (D) GstO1, and (E) GstP1. Error bars = SD. Students T-test (unpaired, one-tailed) was performed to test for significant differences in Gst gene expression between control cells and cells treated with DAC and TSA. Results (P-values) are shown on the figure.