Frequent hypermethylation and loss of heterozygosity of the testis derived transcript gene in ovarian cancer

Abstract

Testis derived transcript (TES) is a candidate tumor suppressor gene located at the human chromosome 7q31, and its function in ovarian cancer is still unknown. Using ovarian cancer cell lines and tissue samples, we demonstrated that both loss of heterozygosity and hypermethylation of the TES gene occurred in ovarian cancer at high frequencies, and there were significant correlations between TES expression and hypermethylation or loss of heterozygosity. We also detected methylation in ovarian cancer cell line A2780 after treatment with 5-aza-2-deoxycytidine. The expression level of TES was enormously up-regulated, then caused changes to the biological behaviors of A2780 cells: cell growth properties were greatly impaired, colony formatting abilities were suppressed to very low levels, and the apoptosis rate was highly raised compared to the control group. Our findings suggest that the TES gene functions as a tumor suppressor gene and is frequently silenced by hypermethylation and loss of heterozygosity in ovarian cancers.

Figure 1

Testis derived transcript (TES) expression in normal ovary and ovarian cancer tissues. (A) Positive staining of TES protein in ovarian cancer. (B) TES expression was lost in ovarian cancer. (C) Normal ovarian tissue was stained positively.

Figure 2

Methylation analysis of testis derived transcript in ovarian cancers. C, cancer samples; M, methylated; M. Sss I, positive control; N, normal ovary tissues; U, unmethylated. (DNA modificated by CpG methyltransferase M. Sss I.)

Figure 3

Loss of heterozygosity in ovarian cancer.

Figure 4

RT‐PCR analysis of testis derived transcript with or without 5‐aza‐2‐deoxycytidine treatment in A2780 and SKOV3 cell lines.

Figure 5

Western blot of testis derived transcript (TES) in SKOV3 and A2780 cell lines before and after 5‐aza‐2‐deoxycytidine treatment. β‐Actin was used as an endogenous control.

Figure 6

Cell proliferation curve with or without 5‐aza‐2‐deoxycytidine. (A) A2780 cell line; (B) SKOV3 cell line.

Figure 7

Colony formation assay with or without 5‐aza‐2‐deoxycytidine. (A,B) A2780 cell line; (C,D), SKOV3 cell line.

Figure 8

Cell apoptosis induced by treatment with 5 μm 5‐aza‐2‐deoxycytidine. (A) Apoptosis of A2780 cells in the group without 5‐aza‐dC treatment (early apoptotic rate, 0.3; late apoptotic rate, 0.69); (B) apoptosis of A2780 cells in the 5 μm 5‐aza‐2‐deoxycytidine group (early apoptotic rate, 1.38; late apoptotic rate, 1.93); (C) apoptosis of SKOV3 cells in the group without 5‐aza‐dC treatment (early apoptotic rate, 0.44; late apoptotic rate, 0.99); (D) apoptosis of SKOV3 cells in the 5 μm 5‐aza‐dC group (early apoptotic rate, 0.57; late apoptotic rate, 1.02).