Involvement of the ER pathway in androgen-mediated activation of telomerase. (A) Schematic representation of the TERT gene promoter region, emphasizing ERE at position −2677 and Sp1-ERE at position −873. The first base of TERT promoter is −1. (B) In 2 independent experiments, 4-hydroxy-tamoxifen (4-OH-Tam) inhibited both methyltrienolone (R1881)– and estradiol (E2)–dependent activation of telomerase activity (n = 4; each individual was measured in triplicate). Tamoxifen alone at 1 μM did not influence telomerase activity. (C) One microgram of testosterone and/or 1 μM flutamide, an androgen receptor inhibitor, was added to lymphocyte culture for 3 days and telomerase activity measured. Addition of testosterone alone increased telomerase activity but not flutamide alone. However, flutamide did not inhibit testosterone effects on telomerase activity. (D) Western blot of cell lysates of lymphocytes, MCF-7, a breast cancer cell line, and LNCaP (a prostate cancer cells line). AR was not expressed by lymphocytes (representative, n = 3) but was present in MCF-7 and LNCaP cell lines. ERα had low expression in lymphocytes in comparison with MCF-7 lines but was absent in LNCaP. Actin was used as loading control. (E) Expression of ERβ was measured in the same cells using RT-PCR (GADPH was used as expression control). ERβ was more expressed in lymphocytes than in MCF-7 and LNCaP cell lines. Blanks represent the negative control for the RT-PCR reaction. (F) RT-PCR for CYP19 mRNA expression in peripheral blood lymphocytes of healthy male (subjects A and C) and female (B) subjects cultured in the presence or absence of testosterone 1 μM. CYP19 was to be expressed in lymphocytes in both sexes, but mainly in male subjects, but was not affected but addition of testosterone to cell culture. GADPH was used as expression control (*P < .05; **P < .01).