Telomerase activity in cultured normal peripheral blood lymphocytes. (A) The kinetics of telomerase activity in normal cultured peripheral blood lymphocytes was determined in 2 separate experiments by measuring time-dependent changes in telomerase activity from 3 healthy donors. Each time point was measured in triplicate for each individual. Telomerase activity was absent upon collection, very low during the first day in vitro, significantly increased at day 2, and then slightly decreased over time until day 7. (B) Androgens activate telomerase activity of cultured lymphocytes. Methyltrienolone (R1881) induced telomerase function in a dose-dependent fashion (n = 10, 5 men and 5 women; each measurement done in triplicate; data are combined results from 3 different experiments). 19-nortestosterone (19-NT) also induced telomerase function but not 6β-hydroxy-testosterone (n = 2, in 2 experiments; *P < .05; ***P < .001).

Androgens stimulate TERT gene expression. (A) Exposure of lymphocyte to androgen (R1881) was associated with higher TERT mRNA levels by RT-PCR (GADPH was used and loading control; results from one representative experiment). (B) To confirm these results, TERT expression was evaluated by real-time PCR in 2 separate experiments (n = 3): in lymphocytes, R1881 up-regulated TERT mRNA expression, an effect that was inhibited by tamoxifen (Tam). (C) Estradiol activates telomerase enzymatic activity in cultured lymphocytes (E2; n = 3, in 2 different experiments). (D) In a single separate experiment, lymphocytes were also cultured with hydrocortisone (n = 2). (E) In the same experiment, hydrocortisone (HY) inhibited cell cycle and at higher doses (10 μM) induced cell death by apoptosis (*P < .05; ***P < .001).

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).

siRNA “knockdown” of ERα but not ERβ abrogates increased telomerase activity induced by estrogen. In 5 separate experiments, peripheral blood lymphocytes were transfected with either scramble siRNA, siRNA for ERα (ESR1), for ERβ (ESR2), or no siRNA at all (mock) and cultured for 3 days in the presence of PHA, IL-2, and E₂ (1 μM). (A) Representative Western blot indicates that siRNA effectively knocked down ERα expression (> 85%). (B) RT-PCR of a representative experiment indicates that siRNA also knocked down ERβ (59% reduction). (C) Reduction in ERα expression correlated with significant decrease in telomerase activity (*P < .05).

Influence of androgen on telomerase activity of hematopoietic stem and progenitor cells. CD34⁺ cells were separated on immunomagnetic columns and cultured in long-term liquid media in the presence of methyltrienolone (R1881) for 8 days (n = 3), when they were harvested for flow and cell-cycle analyses and protein extraction. No changes in CD71 (transferrin receptor, erythroid marker) or CD33 (myelomonocytic marker) expression patterns were observed upon male steroid treatment.