BRCA1 Inhibition of Telomerase Activity in Cultured Cells

Cell lines and culture.

Human prostate cancer (DU-145) and human breast cancer (T47D, MCF-7, and HCC1937) cell lines and mouse NIH 3T3 cells were originally obtained from the American Type Culture Collection (Rockville, Md.). The generation and characteristics of the DU-145 and T47D cell clones stably transfected with wild-type BRCA1 (wtBRCA1) and a breast cancer-associated point mutant BRCA1 (T300G [⁶¹Cys→Gly]) have been described previously (16, 18, 19). These are all single-cell-derived clones. The studies of these cell clones were performed after about 55 to 65 elapsed population doublings for DU-145 and 40 to 50 elapsed doublings for T47D as measured from the initiation of the cloning procedure.

The DU-145/wtBRCA1/Tet-Off cell line, which has a wtBRCA1 cDNA under the control of a tetracycline (TCN)-responsive promoter was described previously (17, 18). These cells exhibit low (basal) expression when cultured in the presence of TCN (2 μg/ml), but BRCA1 expression is rapidly induced by the removal of TCN from the medium. c-Myc^−/− and c-Myc^+/+ rat fibroblast cell lines were described previously (37). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% (vol/vol) (for DU-145) or 10% (vol/vol) (for all other cell types) fetal calf serum, l-glutamine (5 mM), nonessential amino acids (5 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml) (all obtained from BioWhittaker (Walkersville, Md.).

Reporters and expression plasmids.

The murine TERT promoter-Luc reporter vector (pGL3-354-Luc) was a gift from C. Giardina (University of Connecticut, Storrs, Conn.) (65). The E-box-Luc reporter plasmid contains a canonical E-box sequence upstream of a minimal promoter and luciferase reporter gene. The wild-type and mutant human Mad1 expression vectors have been described previously (4). The Mad1-Pro mutant (¹²Leu→Pro and ¹⁶Ala→Pro) inhibits the Mad1-mediated repression of c-Myc transcriptional activity. Wild-type and mutant BRCA1 cDNAs, cloned into the multicloning site of the pcDNA3 mammalian expression vector (Invitrogen), have been described previously (16, 18). The Sp1 expression vector (pCR3-Sp1) and the NF-κB expression vector (pCR3-NF-κB) were generous gifts from B. Gao (National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Md.). The human TERT promoter-luciferase reporter plasmid (pGL3-hTERT-Luc was provided by I. Horikawa (Center for Cancer Research, National Institutes of Health) (31).

Semiquantitative RT-PCR analysis.

The mRNA expression was determined by semiquantitative RT-PCR assays as described previously (16, 18). For each amplified product, the PCR conditions and cycle numbers were individually adjusted so that all reactions occurred within the linear range of product amplification. The sense and antisense PCR primer pairs (5′ to 3′ direction) and expected product sizes are listed in Table 1.

PCR products were analyzed by electrophoresis through 0.8% agarose gels containing 0.1 mg of ethidium bromide per ml, and gels were photographed under UV light. The mRNA levels were quantitated by densitometry of the cDNA bands and expressed relative to the control gene (β-actin).

Assays of telomerase activity.

Telomerase enzymatic activity was quantitated with the TRAPEZE telomerase detection kit (Intergen) according to the manufacturer's instructions. This assay is a modification of the original telomeric repeat amplification protocol (TRAP) (46), which utilizes the PCR to generate a ladder of products with six-base increments, starting at the position of 50 nucleotides. The PCR products were electrophoresed through a 7% polyacrylamide gel and visualized with SYBR Green I nucleic acid gel stain (FMC BioProducts). To monitor the efficacy of PCR amplification, 10 ng of internal control from phage DNA sequence together with 50 pmol of specific primers were added to the PCR mixture per reaction. The relative telomerase activity was determined by measuring the band intensities of telomerase ladders and comparing them with those of the internal standard. The band intensity was measured with Image picture analyzing software. The telomerase activity was expressed in terms of total product generated (TPG) units and normalized to that of the untransfected parental cells.

Assays of telomere length.

Telomere length was measured by Southern blotting of terminal restriction fragments of genomic DNA using the Telomere Length Assay Module (Pharmingen; kit catalog no. 45710K) according to the manufacturer's directions. Briefly, subconfluent proliferating cells were harvested, and genomic DNA was isolated and cleaved with a mixture of restriction enzymes HinfI and RsaI. This procedure results in small fragments of chromosomal DNA, with the exception of the telomeric and immediately subtelomeric DNA (terminal restriction fragments), which is not cut. The cleaved DNA was analyzed by electrophoresis on an 0.6% agarose gel and subjected to Southern transfer onto a nylon membrane. The membrane was hybridized using a 51-mer biotinylated telomere probe according to the manufacturer's protocol, washed three times with hybridization stringency wash buffer, and subjected to chemiluminescent detection using horseradish peroxidase conjugated to streptavidin. Biotinylated lambda DNA fragments digested with HindIII or BstEII (provided) were used as DNA size standards.

Telo-FISH assays.

Subconfluent proliferating cultures of DU-145 cells were transfected overnight with FLAG-wtBRCA1 or empty FLAG vector by using Lipofectamine (18) (10 μg of plasmid DNA per 100-mm dish). The cells were washed and postincubated for 4 days to allow telomere shortening. Telo-fluorescent in situ hybridization (FISH) assays to visualize telomeres in interphase cell populations were performed as described previously (67). Briefly, FISH analysis was performed with a commercial Cy3-conjugated telomere peptide nucleic acid probe per the manufacturer's (DAKO Corporation, Carpinteria, Calif.) instructions. Independent analysis of the stained nuclei was performed by two blinded observers using a Leica DMRBE microscope equipped with Cy3 and 4′,6′-diamidino-2-phenylindole (DAPI) filters. More than 100 interphase nuclei were visually evaluated, and digital images were obtained using a cooled CCD (charge-coupled device) camera.

Luciferase reporter assays.

Subconfluent proliferating cells in 24-well dishes were transfected overnight with 0.25 μg of each vector per well in the presence of Lipofectamine (Gibco Life Technologies). Appropriate quantities of the empty pcDNA3 vector were added to equalize the total transfected DNA content within each experiment. Cells were then washed to remove the excess plasmid DNA and Lipofectamine and were incubated for 24 h to allow expression of the transfected cDNAs. Luciferase activity was assayed 24 h after transfection, and the values were expressed relative to the positive control (endogenous reporter activity). All values were means ± standard errors of the means (SEMs) of quadruplicate wells. To ensure equality of transfection efficiencies within an experiment, cells were cotransfected with plasmid pRSV-β-galactosidase (β-Gal) to allow measurement of β-Gal activity by spectrophotometry. Each experiment was repeated at least twice to ensure the reproducibility of the results.

Immunoprecipitation.

Subconfluent proliferating cells in 150-cm² dishes were harvested and nuclear extracts were prepared as described previously (17). Each immunoprecipitation (IP) was carried out using 6 μg of antibody or antibody combination and 1,000 μg of nuclear extract protein. Precipitated proteins were collected using protein G beads, washed, and eluted in boiling Laemmli sample buffer, and the proteins were then subjected to Western blotting (see below). The IP antibodies were as follows: (i) BRCA1, combination of three mouse monoclonal antibodies (Ab-1 plus Ab-2 plus Ab-3; Oncogene Research Products), (ii) c-Myc, C-8 mouse monoclonal immunoglobulin G (IgG; Santa Cruz), and (iii) control nonimmune antibody, normal (nonimmune) mouse IgG.

Western blotting.

Western blotting assays were performed as described previously (17, 18). Equal aliquots of total cell protein (50 μg per lane) were electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels, transferred to nitrocellulose membranes (Millipore), and blotted using the following as primary antibodies: BRCA1 (C-20, rabbit polyclonal, 1:200; Santa Cruz); c-Myc (9E10 mouse monoclonal, 1:200; Santa Cruz); human TERT (K-370, rabbit polyclonal, 1:1,000; Calbiochem); HSP90 (H-114, rabbit polyclonal, 1:400; Santa Cruz); and α-actin (I-19, goat polyclonal, 1:500; Santa Cruz). Proteins were visualized using the enhanced chemiluminescence system (Amersham), with colored markers (Bio-Rad) as molecular size standards. Protein bands were quantitated by densitometry, and the values were expressed relative to α-actin as a control for loading and transfer.

Confocal laser scanning microscope analysis.

MCF-7 cells were cultured in an eight-well Lab-Tek chamber slide (Nunc, Naperville, Ill.) in 0.5 ml of culture medium per well. Subconfluent proliferating cells were fixed for 8 min at 37°C with freshly prepared 3.7% paraformaldehyde in fixation buffer (274 mM NaCl, 10 mM piperazine-N,N′-bis(2-ethanesulfonic acid [PIPES], 4 mM EGTA, 11 mM glucose in phosphate buffer). The fixation was followed by two rinses with Tris-buffered saline (TBS)-glycine. The cells were permeabilized at room temperature with 0.5% Triton X-100 in fixation buffer for 20 min and then washed three times with TBS for 5 min each. Samples were blocked with 1% goat serum and 2% bovine serum albumin in TBS at room temperature for 30 min. Cells were incubated with primary antibody (see below), diluted with blocking solution in a humidity chamber at 37°C for 20 min, rinsed, and then incubated with secondary antibody conjugated with Alexa Fluor, all performed under the same conditions as the primary antibody binding reaction.

The primary antibodies were as follows: (i) anti-BRCA1 (MS13, MS110 mouse monoclonal; kindly provided by R. Scully, Dana-Farber Cancer Institute, Boston, Mass.) and (ii) anti-c-Myc (C-15, rabbit polyclonal IgG; Santa Cruz). The secondary antibodies were goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 546 (red) (Molecular Probes Corporation). The cells were then washed twice with phosphate-buffered saline, and coverslips were applied using Gel/Mount (Biomedia, Foster City, Calif.). After immunocytochemical staining, the cells were examined with a Nikon confocal laser fluorescence inverted microscope with objective lenses (×60 with oil) (LSM 410; C. Zeiss, Oberkochen, Germany) using simultaneous lasers with excitation wavelengths of 543 and 488 nm for red and green, respectively, and detection using red and green narrow-band filters. Images were collected through the specimens every 2 μm in the vertical plane, overlaid to generate focus composite images, and stored as TIFF files. Figures were assembled from the TIFF files by using Adobe Photoshop software.

Chromatin IP (ChIP) assays.

Transient transfections of subconfluent proliferating DU-145 cells were carried out using Lipofectamine (Invitrogen) (10 μg of plasmid DNA per 100-mm dish). The total transfected DNA was maintained at a constant level in these transfection experiments by the addition of empty pcDNA3 plasmid DNA. At 24 h posttransfection, formaldehyde was added at a final concentration of 1% directly to the cell culture media. Fixation proceeded at 22°C for 10 min and was stopped by the addition of glycine to a final concentration of 0.125 M. The cells were collected by centrifugation and rinsed in cold phosphate-buffered saline. The cell pellets were resuspended in cell lysis buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40, and protease inhibitor cocktail [Sigma]), incubated on ice for 10 min, and then microcentrifuged (6,000 × g for 5 min at 4°C) to pellet the nuclei. The nuclei were then resuspended in nuclei lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1], and protease inhibitor cocktail) and incubated on ice for 10 min. The samples were sonicated on ice to an average length of approximately 600 bp and then microcentrifuged. The chromatin solution was precleared by the addition of Staphylococcus aureus protein A-positive cells for 15 min at 4°C. Prior to use, the Staph A cells were blocked with 1 μg of sheared salmon sperm DNA (10 μg/μl) and 1 μg of bovine serum albumin/μl for at least 4 h at 4°C.

The antibodies used for IPs included c-Myc, C-8 mouse monoclonal IgG (Santa Cruz), BRCA1, combination of three mouse monoclonal antibodies (Ab-1 plus Ab-2 plus Ab-3; Oncogene Research Products), and normal mouse IgG. Precleared chromatin from 10⁷ cells was incubated with specific immune monoclonal, normal mouse IgG, or no antibody, and rotated at 4°C for 12 to 16 h. IP, washing, and elution of immune complexes was carried out as previously described (5). Prior to the first wash, 20% of the supernatant from the reaction with no primary antibody was saved as total input chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. The cross-links were reversed by the addition of NaCl to a final concentration of 200 mM, and RNA was removed by the addition of 10 μg of RNase A per sample followed by incubation at 65°C for 4 to 5 h. The samples were then precipitated at −20°C overnight by the addition of 2.5 volumes of ethanol and pelleted by microcentrifugation.

The samples were resuspended in 100 μl of Tris-EDTA (pH 7.5), 25 μl of 5× proteinase K buffer (1.25% SDS, 50 mM Tris [pH 7.5], and 25 mM EDTA) and 1.5 μl of proteinase K (Boehringer Mannheim) and then incubated at 45°C for 2 h. The samples were phenol extracted and then precipitated with 1/10 volume of 3 M NaOAc (pH 5.3), 5 μg of glycogen, and 2.5 volumes of ethanol. The pellets were collected by microcentrifugation, resuspended in 20 μl of H₂O, and analyzed by PCR. The PCR mixtures contained 2 μl of immunoprecipitate or 2 μl of a 1:100 dilution of the total sample; 100 ng of each primer; 1.5 mM MgCl₂; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 1× thermophilic buffer (Invitrogen); and 2.0 U of Taq DNA polymerase (Invitrogen) in a total volume of 20 μl. Following 35 cycles of amplification, the PCR products were analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. The sequences of the primer pairs and the predicted sizes of the amplified DNA segments were as follows: pGL3-Luc, 5′-TCT TAC GCG GTG CTA G-3′ and 5′-ATG TTT TTG GCG TCT TCC-3′, 105-bp product; mTERT-Luc, 5′-TTC CCA GCT CAG GGC GAA-3′ and 5′ATG TTT TTG GCG TCT TCC-3′, 412-bp product; and hTERT-Luc, 5′-ATC AGG CCA GCG GCC AAA-3′ and 5′-ATG TTT TTG GCG TCT TCC-3′, 544-bp product.

Small interfering RNA.

Double stranded small interfering RNA (siRNA) to knock down endogenous BRCA1 protein levels (BRCA1-siRNA-3) and a scrambled-sequence (control) siRNA were chemically synthesized by Dharmacon, Inc. The sequences of these RNAs were as follows: BRCA1-siRNA-3, 5′-AATGCCAAAGTAGCTAATGTA-3′ and scrambled-sequence control siRNA, 5′-GTC ACG ATA AGA CAA TGA TAT-3′. For transfection of siRNAs, subconfluent proliferating MCF-7 cells cultured in 24-well dishes were treated with BRAC1-siRNA-3 or scrambled-sequence siRNA (as a control) (50 nM) using siPORT Amine transfection agent (Ambion) as per the manufacturer's instructions. The cells were harvested at different times following transfections and were subjected to Western blot analysis (100 μg of protein per lane) using antibodies against BRCA1 (C-20) and α-actin (I-19) (Santa Cruz).

Statistical analysis.

Where appropriate, statistical comparisons were carried out using the two-tailed Student's t test.

Overexpression of wtBRCA1 inhibits telomerase enzymatic activity.

Telomerase activity was measured in untransfected parental cells and in cell clones stably transfected with wtBRCA1 or empty vector (neo) (16, 18) by using a modification of the TRAP assay (45). The telomerase ladder intensity was divided by that of an internal standard, and this ratio was normalized to a value of 100% for the untransfected parental cells. wtBRCA1-transfected clones of both DU-145 human prostate cancer cells (Fig. 1A) and T47D human breast cancer cells (Fig. 1B) showed significantly reduced telomerase activities compared to the corresponding control clones or parental cells. As another control, the telomerase activity of DU-145 parental cells was virtually inhibited by heat treatment (100°C for 5 min) of the cell extract (Fig. 1A, right).

We also tested the effect of the wtBRCA1 gene on telomerase activity in HCC1937 breast cancer cells by using transient transfection assays. HCC1937 cells have no endogenous wtBRCA1 and contain a single copy of a mutant BRCA1 allele (5382insC) (59). As observed in the stable cell lines, transient expression of wtBRCA1 inhibited telomerase activity in HCC1937 cells compared with empty vector (pcDNA3) or untransfected control cells (Fig. 1C).

T300G is a tumor-associated point mutation of BRCA1 that encodes a full-length protein with a single-amino-acid substitution (⁶¹Cys→Gly) within the conserved amino-terminal RING domain (53). Previously, we showed that both wtBRCA1 and BRCA1 mutant T300G are overexpressed at similar levels in the same set of stably transfected DU-145 cell clones (18). In contrast to the wtBRCA1-transfected DU-145 cells, DU-145 clones stably transfected with the T300G mutant BRCA1 showed similar telomerase activity to that of the control cells and significantly greater telomerase activity than the wtBRCA1 cell clones (Fig. 1D).

wtBRCA1 causes down-regulation of TERT expression.

To investigate the mechanism(s) by which wtBRCA1 inhibits telomerase activity, we determined its effect on expression of a subset of the components of the telomerase holoenzyme, including TERT, TP1 (also known as TEP1 or TLP1), and HSP90. The mRNA and protein expression of these components were evaluated using semiquantitative RT-PCR analysis and Western blotting, respectively. Compared with parental and control transfected (neo) cells, the wtBRCA1-transfected DU-145 (Fig. 2A and B) and T47D (Fig. 2C and D) cell clones showed increased mRNA and protein levels of BRCA1 and decreased mRNA and protein levels of TERT. Similarly, HCC1937 breast cancer cells that were transiently transfected with wtBRCA1 showed increased expression of BRCA1 mRNA and decreased expression of TERT mRNA compared with untransfected or empty pcDNA3 vector-transfected cells (Fig. 2E). The BRCA1 5382insC mutation of HCC1937 cells results in BRCA1 protein truncation at amino acid 1755. However, the mutant BRCA1 mRNA is expressed, resulting in the appearance of an amplified cDNA segment, even in cells not transfected with wtBRCA1.

In contrast to TERT, stable wtBRCA1 expression had little or no effect on the expression of TP1 or HSP90 at the mRNA level (Fig. 3A) or on HSP90 expression at the protein level (Fig. 3B) in DU-145 cells. We were not able to detect TP1 protein in DU-145 cells. Since the c-Myc proto-oncogene product is a transcriptional activator of the telomerase gene (8, 63), we also compared the expression of c-Myc in wtBRCA1-transfected versus control DU-145 cell clones. This study revealed little or no difference in c-Myc mRNA or protein levels in parental, wtBRCA1, or neo clones of DU-145 cells (Fig. 3A and B). Thus, the reduction of TERT expression in wtBRCA1-transfected cell lines cannot be explained as being due to a reduction in c-Myc protein levels.

We also examined the expression of two telomere repeat binding factors, TRF1 and TRF2 (also known as TERF1 and TERF2, respectively) (54). Although TRF1 and TRF2 are not components of the telomerase enzyme, these factors are involved in the regulation of telomeres. In DU-145 cell clones, the stable expression of wtBRCA1 had no obvious effect on the mRNA levels, TRF1, or TRF2, as determined using semiquantitative RT-PCR analysis (Fig. 3C). In addition, wtBRCA1 had no effect on the mRNA levels of the human telomerase RNA (hTR) and dyskerin, a protein linked to the disease dyskeratosis congenita that is associated with the telomerase enzyme and with H/ACA small-nucleolar RNAs (40) (Fig. 3D).

Stable expression of wtBRCA1 (but not T300G mutant) causes telomere shortening.

To determine if the inhibition of TERT expression and telomerase enzymatic activity due to exogenous wtBRCA1 expression has functional consequences, we measured and compared telomere length in DU-145 cell clones stably transfected with wtBRCA1 or empty vector (neo). These cell lines consist of single-cell-derived clones (16, 18) and were estimated to have undergone about 55 to 65 population doublings between the original cloning and the performance of these experiments. The growth rates of the wtBRCA1 cell clones were only slightly less than those of the control (neo or parental) cell lines: the mean population doubling time (T_d) was 20 ± 1 h versus 18 ± 1 h, respectively. Telomere length was determined with a commercially available kit to measure the length of terminal restriction fragments of telomeric and subtelomeric DNA by Southern blot hybridization (see Materials and Methods). Untransfected parental DU-145 cells and neo clones exhibited telomeres with a mean length of about 3.0 to 3.3 kb, whereas wtBRCA1 cell clones showed telomeres that were shorter than 1.9 kb (the smallest DNA size marker) (see Fig. 4A). Several repeated experiments gave similar results.

On the other hand, DU-145 cell clones stably expressing the tumor-associated amino-terminal mutant BRCA1 T300G (estimated population doubling level of 55 to 65 since cloning) had telomeres of similar length to those of the parental cells and control (neo) cell clones (Fig. 4B). T300G cells exhibited a slightly faster growth rate than the control cells (T_d = 17 ± 1 h). Since the T300G BRCA1 mutant is well expressed in these DU-145 cell lines (18), our findings suggest that stable expression of exogenous wtBRCA1, but not of BRCA1 harboring the T300G mutation, leads to significant telomeric shortening in DU-145 cells.

Similar to DU-145, T47D cell clones stably expressing wtBRCA1 had significantly shorter telomeres than did the corresponding control (neo) cell clones (Fig. 4C). The T47D cell lines were also single-cell clones and were estimated to have undergone about 40 to 50 doublings between the initiation of cloning and the time of this study. The population doubling times for the wtBRCA1 and control T47D cell clones were 24 and 22 h, respectively. In this cell type, the control cell lines showed telomere populations of about 2.3 to 4.4 kb, while the wtBRCA1 cell lines had telomere population lengths of less than 2.0 kb.

Figure 4E shows the relative expression levels of BRCA1 in the different clonal types studied along with that of untransfected cell lines. Note that the levels of BRCA1 in DU-145 wyBRCA1 and DU-145 T300G cell clones are higher than the corresponding control (neo) clones but comparable to the endogenous BRCA1 levels in untransfected T47D and MCF-7 cells. T47D cells show higher basal BRCA1 levels than DU-145 but show slightly lower levels than MCF-7 cells, and the BRCA1 levels are significantly higher in T47D wtBRCA1 cell clones than in the control (neo) clones.

Transient expression of wtBRCA1 causes telomere erosion in DU-145 cells.

To assess the effect transient expression of exogenous wtBRCA1 on telomere length, subconfluent proliferating DU-145 cell cultures were transiently transfected with FLAG-wtBRCA1 versus empty FLAG vector (18), postincubated for 4 days to allow the telomere shortening to proceed, and processed for telo-FISH assays as described previously (67). This assay allows visualization of the telomeric signal by fluorescence microscopy of interphase cells. The telo-FISH assay revealed a significant loss of the telomere signal in cells transfected with FLAG-wtBRCA1 compared with empty vector (Fig. 4D), suggesting that overexpression of BRCA1 causes relatively rapid erosion of telomeres over a period of days. Previous studies revealed transfection efficiencies of about 80% for FLAG-wtBRCA1 in DU-145 cells transfected under similar conditions (18).

Time course of telomere shortening following induction of BRCA1.

To further investigate the time course of telomere shortening that occurs following wtBRCA1 overexpression, we utilized a DU-145 cell clone (designated DU-145/wtBRCA1/Tet-Off) that expresses wtBRCA1 under the control of a TCN-responsive promoter. The DU-145/wtBRCA1/Tet-Off cells have been described previously (18). For this experiment, DU-145/wtBRCA1/Tet-Off cells that had been cultured in the presence of doxycycline to suppress exogenous wtBRCA1 expression were subcultured in the absence (−TCN) or presence (+TCN) of doxycycline for up to 120 days, starting at time zero. The cells were collected periodically for analysis of telomere length and BRCA1 protein levels.

As demonstrated in Fig. 5B, the −TCN cells expressed higher levels of BRCA1 than the +TCN cells throughout the duration of the experiment, and MCF-7 cells expressed endogenous levels of BRCA1 protein comparable to those of the −TCN DU-145 cells and that were higher than the +TCN DU-145 cells. Consistent with the ability of DU-145 wtBRCA1 cell clones to continue to proliferate as stable cell lines, the continued overexpression of BRCA1 in the −TCN cells had no effect on the growth rate of DU-145 cells (Fig. 5C). In the control (+TCN) cells, there was little or no change in telomere length over the period of the experiment, except perhaps for a slight reduction in size of the longest telomere subpopulation (Fig. 5A). These cells showed at least three subpopulations of telomeres, with telomere restriction fragment sizes of roughly 2.3, 3.5, and 5.5 to 6 kb. When TCN was removed, the larger telomeres disappeared rapidly, while the 2.3-kb telomeres persisted for up to 31 or so days. By 59 days, nearly all of the telomere population was in the range of roughly 1.8 to 2 kb. These results suggest a rapid reduction in telomere length induced by BRCA1, with continued changes in the distribution of telomere lengths for at least 60 days. The possible significance of these findings is addressed in the Discussion.

wtBRCA1 blocks TERT promoter as well as c-Myc-dependent transcriptional activity.

To investigate the mechanism(s) of wtBRCA1 inhibition of TERT expression, we examined the effect of wtBRCA1 on TERT promoter activity by using a murine TERT promoter-luciferase reporter plasmid, pGL3-354-Luc (65). The 354-bp TERT promoter segment in this reporter contains NF-κB, Sp1, and E-box elements and is structurally and functionally very similar to the corresponding segment of the human TERT promoter. DU-145 cells were transiently transfected overnight with the pGL3-354-Luc reporter and wtBRCA1 or empty pcDNA3 vector, washed, and incubated for 24 h to allow BRCA1 and luciferase expression. As illustrated in Fig. 6A, wtBRCA1 strongly suppressed the TERT promoter activity (P < 0.001, two-tailed t test), while the empty pcDNA3 vector had little or no effect on TERT activity. In different experiments, TERT promoter activity was typically reduced to less than 10% of the control activity by wtBRCA1. As a negative control, the pGL3-Luc reporter missing the TERT promoter sequences gave no luciferase activity either in the absence or in the presence of cotransfected wtBRCA1 (Fig. 6A).

c-Myc-mediated transcriptional activation occurs through the interaction of Myc box 1 with a consensus DNA sequence (E-box) within the regulatory regions of various c-Myc target genes (47). To assess c-Myc-dependent transcriptional activity, we utilized a luciferase reporter plasmid driven by the canonical E-box DNA element (E-box-Luc). Similar to the findings obtained with pGL3-354-Luc, the E-box-Luc reporter was endogenously activated upon transfection into DU-145 cells, and its activation was significantly inhibited by cotransfection of wtBRCA1 but not of the empty pcDNA3 vector (P < 0.001) (Fig. 6B).

Mad1 (also known as Mxi1) antagonizes c-Myc transcriptional activity by converting the c-Myc-Max and E-box activator complex to a Max-Mad1 repressor complex (3, 4, 51). Mad1 causes the recruitment of transcriptional corepressors mSin3a and mSin3b along with histone deacetylases to the transcriptional complex. Cotransfection of Mad1 caused a reduction of pGL3-354-Luc activity (P < 0.001) to about 25% of the control value (Fig. 6C), suggesting that a high proportion of the endogenous TERT promoter activity is attributable to c-Myc. In contrast to wild-type Mad1, a c-Myc repression-defective Mad1 mutant (Mad1-Pro) (4) gave only a modest reduction of endogenous TERT activity. When c-Myc itself was cotransfected along with pGL3-354-Luc, there was a significant increase in reporter activity (P < 0.001), by more than twofold. This additional c-Myc-mediated TERT activation was blocked by either wtBRCA1 or Mad1, both of which reduced the TERT activity levels to less than the control values obtained in the absence of exogenous c-Myc (P < 0.001).

We performed similar assays with the E-box-Luc reporter system. In several independent assays, cotransfection of either wtBRCA1 or Mad1 (but not Mad1-Pro) reduced the reporter activity to about 15 to 20% of control, while c-Myc caused a 2- to 2.5-fold increase in reporter activity (Fig. 6D) (P < 0.001). When c-Myc was cotransfected along with either wtBRCA1 or Mad1, both wtBRCA1 and Mad1 reduced the residual E-box-Luc activity to significantly less than that observed in the absence of exogenous c-Myc (P < 0.001). In both the TERT promoter and E-box-Luc reporter experiments, there were no significant differences in transfection efficiencies under the various assay conditions, as determined by cotransfection of control plasmid pRSV-β-Gal.

Structural requirements for BRCA1 inhibition of the TERT promoter and Myc reporter.

We previously showed that the entire BRCA1 protein (including the amino and carboxyl termini) were required to inhibit the activity of an estrogen-dependent luciferase reporter (17). To assess the structural requirements for BRCA1-mediated inhibition of telomerase, we tested the ability of various mutated and truncated BRCA1 genes (see Fig. 7A) to inhibit TERT- and Myc-dependent reporter activity. The BRCA1 mutants tested included three cancer-associated mutations: the amino-terminal point mutation (T300G or ⁶¹Cys→Gly), a carboxyl terminal point mutation (C5365G or ¹⁷⁴⁹Pro→Arg), and the carboxyl-terminal truncation mutation (5382insC or 1755→term). The luciferase activity was normalized to the positive control (i.e., no BRCA1 vector transfected).

Studies utilizing the TERT promoter-reporter (pGL3-354-Luc) showed significant inhibition of reporter activity by wtBRCA1 as well as BRCA1 cDNAs encoding carboxyl-terminal mutants (C5365G and 5382insC), which yielded residual reporter activities of <20% of control (Fig. 7B) (P < 0.001). On the other hand, the amino-terminal point mutant T300G gave little or no inhibition of reporter activity, consistent with the earlier finding (Fig. 1D) that DU-145 T300G stable clones showed telomerase enzymatic activities similar to those of the parental cells and control (neo) cell clones. Studies that used deletion mutants revealed that a mutant containing only the first 302 amino acids of BRCA1 (ΔEcoRI) gave little or no inhibition of the TERT reporter, while mutants containing amino acids 1 to 771 (ΔKpnI) and amino acids 1 to 1313 (ΔBamHI) gave strong to nearly complete inhibition (P < 0.001).

The results obtained using the c-Myc responsive reporter (E-box-Luc) were generally similar to those obtained with the TERT reporter (Fig. 7C). Again, the carboxyl-terminal BRCA1 mutants and all but the most extreme truncated BRCA1 protein (ΔEcoRI) gave significant inhibition of E-box-Luc activity (P < 0.001). However, one difference was noted. In contrast to the minimal effect of BRCA1 T300G on TERT promoter activity, the T300G mutant gave significant inhibition of E-box-Luc activity (P < 0.001), with residual reporter activity of ≅25% of control. In each set of assays (pGL3-354-Luc and E-box-Luc), transfection efficiencies, as determined using control plasmid pRSV-β-Gal, were similar in all assay conditions. These findings suggest that the entire BRCA1 molecule is not required for effective suppression of TERT and c-Myc activity, and that only the amino-terminal half or so of the protein is required for these activities.

Lack of TERT promoter activity in c-Myc^−/− cell lines.

The finding that wtBRCA1 inhibits E-box activity, coupled with the finding that most of the TERT promoter activity is blocked by the c-Myc antagonist Mad1, suggest that the BRCA1 inhibition of TERT is due largely to inhibition of c-Myc transcriptional activity. To determine if c-Myc is required for BRCA1 inhibition of the TERT promoter, we compared the effect of wtBRCA1 on TERT and E-box-Luc activity in c-Myc^+/+ versus c-Myc^−/− rat fibroblast cell lines (37). As illustrated in Fig. 8A, the c-Myc^−/− cells had no detectable Myc and had much less TERT protein than did the control (c-Myc^+/+) cells.

wtBRCA1 strongly inhibited TERT reporter activity in c-Myc^+/+ fibroblasts (P < 0.001, two-tailed t test), just as it did in human carcinoma cells. TERT promoter activity was detected in the c-Myc^−/− cells but was only ≅10% of that found in the c-Myc^+/+ cells. However, this residual TERT activity in the c-Myc^−/− cells was inhibited by the transient expression of wtBRCA1 (P < 0.001). Interestingly, while transfection of the c-Myc gene into c-Myc^−/− cells caused a significant increase in TERT activity (P < 0.001), the resultant TERT activity remained considerably less than that in the c-Myc^+/+ cells (P < 0.001).

Similar findings were observed in assays utilizing the E-box luciferase reporter. There was little or no E-box activity in the c-Myc^−/− cells, and again, transient expression of c-Myc caused a modest increase in E-box activity in these cells, which was inhibited by the cotransfection of wtBRCA1 (Fig. 8C). These findings suggest that most of the TERT promoter activity in rat fibroblasts is due to c-Myc but that BRCA1 may also be able to suppress the TERT promoter independently of c-Myc.

c-Myc and BRCA1 associate with each other and colocalize in vivo.

We tested the ability of BRCA1 and c-Myc to associate with each other in vivo under endogenous conditions (i.e., in untransfected cells), by IP and Western blotting. As illustrated in Fig. 9A, IP of c-Myc in MCF-7 or T47D cells coprecipitated BRCA1 and vice versa. On the other hand, a control IP of MCF-7 cells, using normal mouse IgG, failed to precipitate BRCA1 or c-Myc.

We then performed confocal laser microscopy to determine if BRCA1 and c-Myc colocalize in MCF-7 cells under endogenous conditions. Both c-Myc and BRCA1 showed a largely granular nuclear distribution, with very little localization to the cytoplasm. The merged image revealed that a fraction of the c-Myc was colocalized with BRCA1 in a granular distribution within the nuclei of MCF-7 cells (Fig. 9B). However, under endogenous conditions, there was a clearly visible detectable fraction of c-Myc that had not colocalized with BRCA1. Taken together, these findings suggest that under endogenous conditions in proliferating cells, at least a fraction of the c-Myc and BRCA1 exist together in protein complexes.

Further analysis of BRCA1 regulation of the TERT promoter.

We performed several additional studies to further investigate the BRCA1-mediated inhibition of TERT promoter activity. Figure 10A shows a plasmid dose-response analysis of wtBRCA1 inhibition of pGL3-354-Luc activity. Under all conditions, the total transfected DNA content was kept constant by the addition of empty pcDNA3 vector. A wtBRCA1 dose of between 0.05 and 0.10 μg per well (50 to 100 ng of plasmid DNA per well) gave 50% inhibition of PGL3-354-Luc activity. A dose of ≥0.25 μg of wtBRCA1 per well gave nearly complete inhibition (>99%). We found similar results for BRCA1 inhibition of estrogen receptor activity (17).

Our previous experiments showed that BRCA1 inhibits both basal and c-Myc-stimulated TERT promoter activity, and they suggest that most of the basal TERT activity in DU-145 cells is due to endogenous c-Myc activity (Fig. 6). The mouse TERT promoter segment within the pGL3-354-Luc plasmid contains DNA elements for the binding of NF-κB, Sp1, and c-Myc (E-box) (31, 65). We tested the ability of wtBRCA1 to inhibit TERT promoter activity induced by NF-κB and Sp1. In transient transfection assays of DU-145 cells, exogenous NF-κB or exogenous Sp1 induced three- to fourfold increases in pGL3-354-Luc activity (Fig. 10B). However, unlike c-Myc, wtBRCA1 failed to inhibit NF-κB- or Sp1-induced pGL3-354-Luc activity. These findings suggest that the ability of BRCA1 to regulate TERT promoter activity is selective and does not extend to all transcription factors that can activate the promoter.

We next examined the ability of wtBRCA1 to inhibit mouse TERT promoter activity in other cell types than DU-145. In transient transfection assays, wtBRCA1 also strongly inhibited pGL3-354-Luc activity in HCC1937 and MCF-7 human breast cancer cells (Fig. 10C). And wtBRCA1 effectively inhibited mouse TERT promoter activity in several murine cell types, including normal mouse embryo fibroblasts and NIH 3T3 cells (Fig. 10D). The mouse and human TERT promoters exhibit a similar structure, with each promoter segment containing NF-κB, Sp1, and E-box sites (31, 65). Since most of our studies have utilized the mouse TERT reporter, we also tested the ability of wtBRCA1 to suppress the activity of a human TERT luciferase reporter, pGL3-hTERT-Luc, in a human cell line (DU-145). Similar to the results obtained using mouse TERT, wtBRCA1 strongly suppressed the activity of the human TERT promoter (Fig. 10E).

Finally, we recently developed siRNAs that specifically and nontoxically down-regulate BRCA1 protein levels over a 2- to 3-day interval (see Fig. 10F). We compared the effects of a single exposure (50 nM) to either a BRCA1-specific siRNA (BRCA1-siRNA)-3 or the corresponding control (scrambled sequence) siRNA, which has no effect on BRCA1 protein levels. In studies of both DU-145 and MCF-7 cells, untransfected cells and cells treated with scrambled-sequence siRNA showed similar levels of pGL3-354-Luc promoter activity (Fig. 10F). However, cells treated with BRCA1-siRNA-3 showed a two- to fourfold increase in promoter activity (P < 0.001), suggesting that knockdown of endogenous BRCA1 expression results in deregulated TERT promoter activity in human cancer cell lines.

BRCA1 is recruited to transiently transfected TERT promoters.

Since BRCA1 suppresses mouse and human TERT promoter activity, we sought to determine if BRCA1 is physically present at the TERT promoter segment within the mouse and human TERT-Luc reporters. Cells were transiently transfected with the appropriate reporter plasmid, and ChIP assays were carried out to detect the presence of BRCA1 and c-Myc. ChIP assays revealed the presence of endogenous BRCA1 and c-Myc at the mouse TERT and human TERT promoter segments within the TERT-Luc reporters (Fig. 11A and B, respectively). In cells transfected with (mouse TERT plus wtBRCA1) or with (human TERT plus wtBRCA1), the quantities of both BRCA1 and c-Myc associated with the promoters appeared to be increased, although the degree to which these assays can be considered to be quantitative is unclear.

A number of controls were included to establish the specificity of these assays. Assays performed using normal IgG rather than BRCA1 or c-Myc antibodies showed no PCR bands under any conditions. Transfections of control reporter plasmids (i.e., plasmids missing the TERT promoter segment) or transfections performed with only wtBRCA1 or empty pcDNA3 vector failed to yield any PCR bands in the IPs. These controls rule out the possibility that the PCR primers are amplifying nonspecific bands. As additional controls, the same sets of transfections and IPs were performed, except that the PCR primers were designed to amplify a DNA segment from within the multicloning site of the empty pGL3-Luc vector to within the luciferase gene. These control assays showed PCR bands in the appropriate input lanes, but there were no amplified bands corresponding to the BRCA1 or c-Myc IPs. These results indicate that neither BRCA1 nor c-Myc were bound nonspecifically to another portion of the reporter plasmid not containing the TERT promoter segment.

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