Boggess John F, Zhou Chunxiao, Bae-Jump Victoria L, Gehrig Paola A, Whang Young E
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Gynecol Oncol. 2006 Nov;103(2):417-24. doi: 10.1016/j.ygyno.2006.03.032. Epub 2006 May 11.
Given that prolonged exposure to unopposed estrogen is associated with endometrial cancer development and that the promoter region of the catalytic subunit of the telomerase enzyme, hTERT, contains putative estrogen response elements (EREs), we postulated that estrogen-receptor (ER)-mediated induction of telomerase activity may play an important role in endometrial carcinogenesis.
ER-positive and ER-negative endometrial cancer cell lines were used. ER alpha expression was reconstituted in ER-negative cell lines by transient transfection. Telomerase activity was assayed using a PCR-based telomeric repeat amplification protocol (TRAP) after exposure to estradiol (E2). hTERT mRNA expression was assessed by real-time RT-PCR. Gel shift assays using oligonucleotide probes encoding each ERE and transient expression assays using luciferase reporter plasmids containing varying lengths of the 5' promoter region of the hTERT gene were performed.
E2 induced both hTERT gene transcription and telomerase activity in the ER-positive cell lines, but not in the ER-negative cell lines. Transfection of ER alpha into ER-negative cell lines restored E2-induced hTERT gene transcription and telomerase activity. Gel shift assays revealed two EREs in the hTERT promoter that specifically bind to ER alpha. Luciferase assays demonstrated that at least the proximal ERE is responsible for transcriptional activation by ligand-stimulated ER alpha.
Telomerase activity and hTERT mRNA were increased in response to estrogen in an ER alpha-dependent fashion in endometrial cancer cells. Binding of complexed estrogen with ER alpha to the EREs found within the hTERT promoter suggests a possible mechanism for telomerase induction that may facilitate the malignant transformation of hormone-dependent endometrial cells.
鉴于长期暴露于无对抗的雌激素与子宫内膜癌的发生有关，且端粒酶催化亚基hTERT的启动子区域含有假定的雌激素反应元件（ERE），我们推测雌激素受体（ER）介导的端粒酶活性诱导可能在子宫内膜癌发生中起重要作用。
使用ER阳性和ER阴性子宫内膜癌细胞系。通过瞬时转染在ER阴性细胞系中重建ERα表达。在暴露于雌二醇（E2）后，使用基于PCR的端粒重复序列扩增协议（TRAP）测定端粒酶活性。通过实时RT-PCR评估hTERT mRNA表达。使用编码每个ERE的寡核苷酸探针进行凝胶迁移试验，并使用含有hTERT基因5'启动子区域不同长度的荧光素酶报告质粒进行瞬时表达试验。
E2诱导ER阳性细胞系中的hTERT基因转录和端粒酶活性，但不诱导ER阴性细胞系中的。将ERα转染到ER阴性细胞系中可恢复E2诱导的hTERT基因转录和端粒酶活性。凝胶迁移试验揭示了hTERT启动子中有两个ERE与ERα特异性结合。荧光素酶试验表明，至少近端ERE负责配体刺激的ERα的转录激活。
在子宫内膜癌细胞中，端粒酶活性和hTERT mRNA以ERα依赖的方式对雌激素作出反应而增加。复合雌激素与ERα结合到hTERT启动子内发现的ERE上，提示了一种可能的端粒酶诱导机制，这可能促进激素依赖性子宫内膜细胞的恶性转化。
