Breast cancer cell line MCF-7 and MDA-MB-231 were kindly provided by Professor Li Meng-feng (Sun Yat-sen University, China), and cultured with Dulbecco’s modified Eagle’s medium (DMEM, high glucose, Invitrogen, USA) supplemented with 10% fetal bovine serum (HyClone, USA) and 1% penicillin (100 IU/ml)/streptomycin (100 µg/ml) (complete media). Cells were cultured in incubator at 37˚C, 5% CO₂, and 95% humidity.

To establish stable cell lines, we used the pSUPER-retropuro plasmids obtained from Professor Li Mengfeng’s laboratory and the hTERT-targeted shRNA sequence were as follows: no.1, sense: 5’-gatcccc TTT CATCAGCA AGTTTGGAttcaaggaTCCAAACTTGCTGATGAAAtttt ta-3’ and anti-sense:’-agctta aaaaTTTCATCAGCAAGT TTGGAtctcttgaa TCAAACTTGCTGATG-AAAggg; no. 2, sense: 5’-gatccccAACCTTCCTCAGGACCCTGttcaa gagaCAGGGTCCTGAGAAGGTT ttttta-3’ and antisense: 5’-agcttaaaaaAACCTTCCTCAGGACCCTGtctct tgaaCAGGGTCCTGAGGAAGGTTggg-3’, the sequence were designed as previously described [11,12] and synthesized through Invitrogen. The vectors were reconstructed according to the protocols of Psu-PER-retropuro RNAi system (Oligo Engine, Seattle, USA) and recombinant retroviral vectors were produced by transient cotransfection as described previously [13]. Viral infections were done serially, and stable cell lines expression hTERT RNAi (s) were selected for 14 days with 0.5 mg/ml of puromycin 72 hr after transfection to establish a stable cell line. The sequence pSuper-retropuro after cloning was verified by enzymatic digestion and DNA sequencing.

The expression of hTERT mRNA was semi-quantitatively evaluated by RT-PCR. Briefly, total RNA from cells was isolated using Trizol^® reagent (Invitrogen, USA) according to the manufacturer’s instruction. Total RNA (2 µg) was used as the template in reverse-transcription using MMLV reverse transcriptase (Promega, USA) at 42˚C for 50 min and inactiviated at 95˚C for 10 min, 2 µl of RT reaction mixture and 10 µl Mix were mixed and amplified with PCR, The primer for hTERT were as follows: Forward 5’-ACCAAGCATTCCTGCTCAAGCT G-3’ and Reverse 5’-CGGCAGGTGTGCTGGACACT C-3’. The condition for PCR amplification of hTERT cNDA were denaturation at 94˚C for 10 min, followed by 35 cycles of denaturation at 94˚C for 30 sec, annealing at 68˚C for 45 sec and extension at 72˚C for 45 sec, and the last extension step was 72˚C for 10 min. 13 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control, the primers for GAPDH were as follows: Forward 5’-AATCCCATCACCATCTTCC A-3’ and Reverse 5’-CCTGCTTCACCACCTTCTTG-3’. Applying 10 min at 94˚C, 35 cycles of 30 sec at 94˚C, 30 sec at 56˚C and 30 sec at 72˚C for PCR amplication and the last extension step was 72˚C for 10 min [14]. The amplified products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. The relative band intensities were quantified applying Quality ONE^® and normalized to GAPDH in the same cDNA sample.

The telomeric repeat amplification protocol (TRAP) was performed with Telo TAGGG Telomerase PCR ELISA Kit (Roche, Germany, Cat No.11854666910). Samples were extracted following the standard protocol. Briefly, cell samples containing 2 × 10⁵ cells were transferred into a fresh Eppendorf tube and centrifuge at 3000 × g for 5 min at 4˚C, resuspended the cells in PBS and after repeated the centrifugation step, the pelleted cells were re-suspended in 200 µl Lysis reagent and centrifuged at 16000 × g for 20 min at 4˚C, then the supernatant were transferred to a fresh tube to perform the following TRAP reaction. Quantification of telomerase activity was performed as described in the previously published protocol [15,16]. All measurements were performed in trip-licates applying Synergy Multi-Detection Microplate Reader (BioTek, USA). Telomerase activity was normalized against protein concentrations of each sample.

To evaluate cell viability, we used the colorimetric [3-(4, 5-dimethylthiazol-2-yle) 2, 5-diphenyltetrazolium bromide] (MTT) assay. To perform MTT assay, the culture medium was removed and 20 µl of MTT solution (5 mg/ ml; Sigma Chemical Co. USA) were added to each well and incubated for 4 hours. Then the plates were centrifuged at 450 × g for 5 min and the medium containing MTT solution was removed, 150 µl of 100% d-i methyl sulfoxide (DMSO, Sigma Chemical Co, USA) were added and the plates were shaken at 100 rpm/min for 10 min on a plate shaker. The optical density (OD) was determined at a wavelength of 490 nm.

Cells trypsined and re-suspended in 0.33% Noble agar (BD, France) containing DMEM supplemented with 20% FBS were overlaid on the top of 0.66% base agar in DMEM supplemented with 10% FBS, at cell concentration of 1 × 10⁴ per well of a six-well plate, and incubated for 2 weeks. Colonies containing more than 50 cells were taken as positive colonies. Numbers of positive colonies were photographed and quantified.

For wound-healing migration assay, confluent monolayer cells were serum-free starved for 24 hr and washed with phosphate-buffered saline, a scrape in the form of a cross was made with a P-200 pipette tip, then the medium was replaced with DMEM supplemented with 5% FBS, wounded areas were marked for observation and photographed at indicated time after the scratch. Invasion assay were performed using 24-well polycarbonate filter (12- μm pore size) Transwell (Corning-Costar, USA) coated with matrigel (BD Pharmingen, USA), the upper sides of the membranes were pre-coated with Matrigel matrix, the coated insert was placed in each well filled with 600 µl of complete medium, the upper chambers contained a 200 µl suspension of cells (1 × 10⁵ cell/ml, in DMEM), the cells were incubated for 24 hr at 37˚C to allow invasion of the cells to the underside of the precoated filter. Non-invading cells on the upper surface were removed with a cotton swab. The filter were fixed, mounted and stained. The cells invaded through Matrigel were counted. Three invasion chambers were used for each experimental condition.

Cells (1 × 10⁴) were seeded in a 96-well micro-titer plate (Corning-Costar, USA) in a total volume of 200 µl per well and cultured for 18 hr to perform the following experiments of irradiation or chemotherapy. Irradiations of UV were performed at a dose of 40 J/cm² (UVP UV crosslink, USA), Adriamycin were added at a concentration of 1.0 mmol/L. After irradiation or chemotherapy, the plates were subsequently cultured for various periods (24 hr, 48 hr and 72 hr). Then the MTT assay was per formed as previously described, the radiosensitivity or chemosensitivity curve was drawn according to the OD value of each well.

The one-way ANOVA test was used to compare difference among hTERT-shRNA transfected groups, empty transfected group and the untransfected group (data from untransfected groups were designated as “control”). Differences were considered to be statistically significant if P-value < 0.05.

Stable transfectant cell lines were obtained by transfecting MCF-7 and MDA-MB-231 cells with pSuper-retropuro-hTERT-RNAi#1 (RNAi#1), pSuper-retro-puro-hTERT-RNAi#2 (RNAi#2) (transfected group) and pSuperretro-puro (empty-transfected group, Vector). Positive clones were confirmed by EcoRII and HindIII bi-enzyme digestion, which showed a 280 bp fragment, and DNA sequencing showed correct insertion site and sequence. After 2 weeks of puromycin selection, the stable transfected cell lines were established and expanded individually.

In the present study, the levels of hTERT mRNA expres sion and telomerase activity were quantitatively assessed by employing RT-PCR. As shown in Figure 1, there was a considerable reduction of hTERT mRNA in both transfected MCF-7 and MDA-MB-231 cell line compared with untransfected (MCF-7 or MDA-MB-231 cell line) or empty-transfected cells (P < 0.001). Reduction of hTERT mRNA resulted in a significant decrease in telomerase activity assessed by TRAP in both MCF-7 and MDA-MB-231 cell line (Figures 2(a) and (b), P < 0.005).

To determine the effect of telomerase activity on cellproliferation and clone formation we measured the growth rate of MCF-7 and MDA-MB-231 and their transfected stable cell lines by MTT assay and soft agar clone formation experiment respectively. In both cell lines, the hTERTRNAi-transfected stable cell lines proliferate slowly (Figures 3(a) and (b)) and form small and less clones (Figures 3(c) and (d)) compared with untransfected or empty-transfected cell lines, this indicated that inhibition of telomerase activity suppress cell proliferation and clone formation.

In this study, we use wound healing experiment and matrigel Transwell experiment to determine the ability of MCF-7 cells to migrate and MDA-MB-231 cells to in-

vade through matrigel respectively. After 24 hr culture, the gap between the scrap in the group of htert-RNAitransfected is much larger than the group of untransfected (see Figure 4(a)), which indicates that suppression of telomerase activity does inhibit breast cancer cell migration. As shown in Figures 4(b) and (c), the effective suppression of telomerase in MDA-MB-231 cells resulted in a significant reduction in the ability of invasion (P < 0.001). This implies that suppression of telomerase can inhibit breast cancer cell invasion.

We supposed that telomerase-based therapy combined with other adjuvant therapies such as radiotherapy or chemotherapy might enhance the effects of tumor suppression. So we treated each cell line with increasing doses of

ionizing radiation and Adriamycin, and assessed the cell viability by MTT assays. As shown in Figure 5, after UV radiation or Adriamycin-chemotherapy, the htert-RNAi #2 transfected cells tend to be less viable compared with untransfected or empty-transfected cells (P < 0.001).