Rap1 is indispensable for TRF2 function in etoposide-induced DNA damage response in gastric cancer cell line

Abstract

The telomeric protein TRF2, involving in telomeric and extratelomeric DNA damage response, has been previously reported to facilitate multidrug resistance (MDR) in gastric cancer cells by interfering ATM-dependent DNA damage response induced by anticancer drugs. Rap1 is the TRF2-interacting protein in the shelterin complex. Complex formation between Rap1 and TRF2 is essential for their function in telomere and end protection. Here we focus on the effects of Rap1 on TRF2 function in DNA damage response induced by anticancer drugs. Both Rap1 and TRF2 expression were upregulated in SGC7901 and its MDR variant SGC7901/VCR after etoposide treatment, which was more marked in SGC7901/VCR than in SGC7901. Rap1 silencing by siRNA in SGC7901/VCR partially reversed the etoposide resistance. And Rap1 silencing partially reversed the TRF2-mediated resistance to etoposide in SGC7901. Rap1 silencing did not affect the TRF2 upregulation induced by etoposide, but eliminated the inhibition effect of TRF2 on ATM expression and ATM phosphorylation at serine 1981 (ATM pS1981). Furthermore, phosphorylation of ATM targets, including γH2AX and serine 15 (S15) on p53, were increased in Rap1 silencing cells in response to etoposide. Thus, we confirm that Rap1, interacting with TRF2 in the shelterin complex, also has an important role in TRF2-mediated DNA damage response in gastric cancer cells treated by etoposide.

Figure 1

TRF2 and Rap1 upregulation induced by etoposide in gastric cancer cells. (a) Protein expression levels of TRF2 and Rap1 detected by western blot in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. (b) Relative mRNA expression levels of TRF2 and Rap1 detected by real-time PCR analysis in SGC7901 and SGC7901/VCR after treated with 20 μg/ml of etoposide for 6 h. *P<0.0001 vs untreated-SGC7901; **P<0.0001 vs untreated-SGC7901/VCR; ***P<0.0001 vs etoposide-SGC7901.

Figure 2

Rap1 is involved in TRF2-mediated etoposide resistance in gastric cancer cells. (a) Rap1 siRNA vectors were transfected into SGC7901/VCR, respectively. Rap1 expression after treated with 20 μg/ml of etoposide for 6 h was detected by western blot and real-time PCR analysis. *P<0.0001 vs SGC7901/VCR and SGC7901/VCR-Cont. (b) Rap1 siRNA vector and TRF2 eukaryotic expression vector were cotransfected into SGC7901. TRF2 and Rap1 expression after treated with 20 μg/ml of etoposide for 6 h were detected by western blot and real-time PCR analysis. *P<0.0001 and **P<0.0001 vs SGC7901 and SGC7901-Cont. (c) IC₅₀ values (μg/ml) of gastric cancer cells for etoposide. The sensitivity of gastric cancer cells to etoposide was evaluated using MTT assay as described in Materials and Methods section. The concentration of etoposide that caused a 50% reduction in number of colonies (IC₅₀) was calculated. *P<0.01 vs SGC7901/VCR and SGC7901/VCR-Cont; **P<0.01 vs SGC7901–TRF2 and SGC7901-Cont.

Figure 3

Downregulation of Rap1 eliminated the inhibition effects of TRF2 on etoposide-induced ATM activation. (a) TRF2 eukaryotic expression vector was transfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. (b) Rap1 siRNA vector and TRF2 eukaryotic expression vector were cotransfected into SGC7901. Expression of ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot. (c) Rap1 siRNA vector was transfected into SGC7901/VCR. Expression of TRF2, Rap1, ATM, ATM pS1981, γH2AX and p53 pS15 after treated with 20 μg/ml of etoposide for 6 h were detected by western blot.