Ten-eleven translocation 1 dysfunction reduces 5-hydroxymethylcytosine expression levels in gastric cancer cells

Abstract

A sixth base, 5-hydroxymethylcytosine (5hmC), is formed by the oxidation of 5-methylcytosine (5mC) via the catalysis of the ten-eleven translocation (TET) protein family in cells. Expression levels of 5hmC are frequently depleted during carcinogenesis. However, the detailed mechanisms underlying the depletion of 5hmC expression in gastric cancer cells remains unclear, and further research is required. The present study examined the expression levels of 5mC and 5hmC and the expression levels of TET1 and TET2 in gastric cancer tissues using immunohistochemistry. The results revealed that 5hmC expression levels were markedly lower in gastric cancer tissues compared with corresponding adjacent normal tissues. Furthermore, a decrease in 5hmC expression levels was associated with a decrease in TET1 protein expression levels in gastric cancer tissues. The ectopic expression level of TET1 may increase the 5hmC expression level in gastric cancer cells. In addition, the results revealed that TET1 protein expression was markedly different in regards to subcellular localization, and mislocalization was significantly associated with the depletion of 5hmC expression levels in gastric cancer. Together, the results of the present study indicated that TET1 dysfunction reduces 5hmC expression levels, and this phenomenon may serve a crucial role in gastric cancer progression.

Keywords: DNA methylation; demethylation; gastric cancer; hydroxylmethylation.

Figure 1.

Expression levels of 5mC, 5hmC, TET1 and TET2 were analyzed using IHC in gastric cancer tissue arrays. A representative case is presented in this figure. (A) The IHC assay analyzed the 5mC and 5hmC expression levels in a patient with gastric cancer (magnification, ×100), and (B) the relative 5mC and 5hmC expression levels were scored between gastric cancer and adjacent normal tissues from 58 patients. (C) The IHC assay revealed the expression levels of TET1 and TET2 in a patient with gastric cancer (magnification, ×100). (D) The relative expression levels of TET1 and TET2 were scored between the gastric cancer tissues of 58 patients and their corresponding adjacent normal tissues. IHC, immunohistochemistry; TET, ten-eleven translocation; 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine.

Figure 2.

Expression levels of TET1 and TET2 in gastric cancer. (A) Expression levels of TET1 and TET2 were analyzed in 311 gastric cancer and 57 adjacent normal tissues from the Gene Expression Across Normal and Tumor Tissue database. (B) Expression levels of TET1 protein were decreased in gastric cancer tissues obtained from 16 patients with gastric cancer compared with the normal adjacent tissues. (C) TET1 overexpression increased the global 5hmC expression levels in the genomic DNA of AGS cells. The loading control was visualized using methylene blue staining. N, normal; T, tumor; TET, ten-eleven translocation; 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine.

Figure 3.

Various localizations of TET1 protein are associated with 5hmC status in human gastric cancer. (A) TET1 protein was exclusively expressed in the cytoplasm (left panel), in the cytoplasm and nucleus (middle panel) and predominantly in the nucleus (right panel) (magnification, ×100). (B) The results of immunohistochemistry revealed that nuclear exclusion of TET1 protein was associated with 5hmC depletion in gastric cancer tissues (magnification, ×100). (C) Nucleic TET1 protein expression was significantly associated with high 5hmC expression level in gastric cancer. (D) A schematic display of the structure of TET1 gene. An NES signal was identified in TET1 (aa877-889) using the ENS prediction tool (http://www.cbs.dtu.dk/services/NetNES/). TET, ten-eleven translocation; 5hmC, 5-hydroxymethylcytosine; NES, nuclear export signal; NLS, nuclear localization signals; NN, Neural Network; HMM, Hidden Markov Model.

Figure 4.

TET1 is involved in active DNA demethylation pathway in AGS cells. (A) Schematic diagrams of TET1 expression constructs for TET1-full length and TET1-delNLS. AGS cells were co-transfected with methylated GFP or unmethylated GFP, and TET1-FL or TET1-delNLS. Following transfection for 24 h, the GFP expression was detected by (B) fluorescence microscopy (magnification, ×100) and (C) western blotting. (D) The intensity of GFP was quantified using ImageJ software and was represented graphically. **P<0.01. Actin was used as an internal control. TET, ten-eleven translocation; GFP, green fluorescent protein; NES, nuclear export signal; NLS, nuclear localization signals; TET1-FL, full length of TET1 genes; TET1-delNLS, NLS domain of TET1 was deleted.