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Molecular and Cellular Biology, December 2003, p. 8668-8690, Vol. 23, No. 23
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.23.8668-8690.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

BRCA1 Inhibition of Telomerase Activity in Cultured Cells

Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040,¹ Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461,³ Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20057²

Received 28 May 2003/ Returned for modification 10 July 2003/ Accepted 15 August 2003

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

	INTRODUCTION

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Telomerase is a ribonucleoprotein enzyme that synthesizes telomeres, i.e., DNA repeats that cap and protect the ends of chromosomes (42). The core of the mammalian telomerase holoenzyme is the catalytic subunit, telomerase reverse transcriptase (TERT; also known as hEST2), which is a reverse transcriptase that adds hexameric DNA repeats (TTAGGG) that correspond to the telomerase RNA component known as TERC (also known as hTR) (12, 20, 44). Mammalian telomerase has been found to have several additional components, including two heat shock proteins (HSP90 and HSP23) and the telomerase-associated protein TP1 (also known as TEP1/TLP1) (28-30). HSP90 functions as an activator of telomerase catalytic activity (30), but the precise function of TP1 is unclear. While TP1 is not required for telomerase activity, it is thought to function as a structure component, contributing to the stability of the telomerase protein-RNA complex (33).

Telomerase function has been closely linked to cellular immortalization, an event that is necessary but not sufficient for cellular transformation (2, 13). In genetic complementation assays to determine the minimal essential requirements for the transformation of human mammary epithelial cells and fibroblasts, TERT can cooperate with SV40 large T antigen and H-Ras to induce transformation (14, 25). However, TERT could not substitute for the c-Myc oncogene in the rat embryonic fibroblast transformation assay (24). In adult human cells normally subject to the development of replicative senescence, the telomerase activity levels are very low to undetectable. However, the vast majority of cancer cell lines express high levels of telomerase activity, which presumably allows for continued cell proliferation (26, 34). The telomerase activities present in human cancer cells usually correlate with the expression of TERT more than with the other components of the telomerase enzyme (38, 58).

The breast cancer susceptibility gene BRCA1 on human chromosome 17q21 encodes an 1,863-amino-acid protein with a conserved amino-terminal RING finger domain and an acidic carboxyl-terminal transcriptional activation domain (39, 41). Inherited mutations of this gene confer an increased risk for breast and ovarian cancers in women and possibly for prostate cancer in men (21, 22, 57). BRCA1 appears to function as a classic tumor suppressor, since BRCA1 mutation-associated cancers exhibit loss of the wild-type allele in the tumor cells (10, 45). The molecular pathways by which BRCA1 suppresses tumorigenesis have not been fully elucidated. However, recent studies suggest that the BRCA1 tumor suppressor activity may be linked to its ability to regulate cell cycle progression (27, 56) and to its participation in certain DNA damage signaling and repair pathways (11, 23, 43, 61). BRCA1 can also positively or negatively modulate a variety of transcriptional pathways, in part due to direct physical interactions with various transcriptional regulatory proteins (15-17, 32, 52, 60, 64, 66).

	MATERIALS AND METHODS

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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 [⁶¹CysGly]) 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 (¹²LeuPro and ¹⁶AlaPro) 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-B expression vector (pCR3-NF-B) 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.

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-ß-galactosidase (ß-Gal) to allow measurement of ß-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 -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 -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 (x60 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 x 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 5x 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; 1x 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 -actin (I-19) (Santa Cruz).

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

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
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).

View larger version (55K): [in this window] [in a new window]   FIG. 1. Overexpression of wtBRCA1 inhibits telomerase activity. (A) Stable expression of BRCA1 inhibits telomerase activity in DU-145 human prostate cancer cells. TRAP assays were performed using subconfluent proliferating cultures of DU-145 parental cells and three cell lines each of wtBRCA1- and control (neo)-transfected clones. Telomerase enzymatic activities were calculated as TPG units and normalized to a value of 100% for the untransfected parental cells. Telomerase activities of wtBRCA1 cell clones were significantly smaller than those of the control(parental plus neo) cells (P < 0.001, two-tailed t test). Note: In each panel, the data shown are representative of at least two independent experiments. (B) Stable expression of BRCA1 inhibits telomerase activity in T47D human breast cancer cells. TRAP assays were performed usingsubconfluent proliferating cultures of T47D parental cells and two lines each of wtBRCA1- and control (neo)-transfected T47D cell clones. (C) Transient transfection of wtBRCA1 inhibits telomerase activity in HCC1937 human breast cancer cells. Subconfluent proliferating HCC1937 cells were transfected overnight with wtBRCA1 or the empty pcDNA3 vector (10 µg of plasmid DNA per 100-mm culture dish) using Lipofectamine, washed to remove the excess vector and Lipofectamine, incubated for 24 h to allow BRCA1 expression, and subjected to TRAP assays to measure telomerase enzymatic activity. (D) In contrast to wtBRCA1, stable expression of a tumor-associated mutant BRCA1 (T300G) fails to suppress telomerase activity in DU-145 cells. Assays were performed using parental cells and two clones each of wtBRCA1 and T300G-transfected cells.

T300G is a tumor-associated point mutation of BRCA1 that encodes a full-length protein with a single-amino-acid substitution (⁶¹CysGly) 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.

View larger version (112K): [in this window] [in a new window]   FIG. 2. Overexpression of wtBRCA1 causes the down-regulation of the expression of the catalytic subunit of telomerase (TERT) at the mRNA and protein levels. Subconfluent proliferating cells were harvested for RNA isolation for semiquantitative RT-PCR assays (panels A and C) or for preparation of whole-cell lysates for Western blotting (panels B and D). TERT expression was compared in control cells (parental, neo) versus wtBRCA1-transfected clones of DU-145 (A and B) or T47D (C and D) cells. (E) Semiquantitative RT-PCR assays of HCC1937 breast cancer cells transiently transfected with vehicle only, wtBRCA1, or empty pcDNA3 vector. Amplified cDNA segments and protein bands were quantitated by densitometry and expressed relative to ß-actin or -actin, respectively, normalized to a value of 100% for the vector-transfected control cells. In panels A and B, the TERT levels were significantly lower and the BRCA1 levels were significantly higher (relative to the actin levels) in the wtBRCA1-transfected cell lines than those of the corresponding control (parental plus neo) cell lines (P < 0.001, two-tailed t tests).

View larger version (102K): [in this window] [in a new window]   FIG. 3. BRCA1 does not inhibit the expression of several other components of the telomerase enzyme, the telomerase transcriptional regulator c-Myc, or several telomere repeat binding proteins. Subconfluent proliferating DU-145 cells were harvested for semiquantitative RT-PCR assays (A and C) or Western blotting (B), as described in the Fig. 2 legend. In panels A and B, there were no significant differences in the levels of TP1, HSP90, c-Myc (relative to actin levels) in the wtBRCA1 versus control (parental plus neo) cell lines (P > 0.1), nor were there significant differences in the expression of TRF1, TRF2, hTR (the human telomerase RNA component), or dyskerin in wtBRCA1 versus control cells (C).

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.

View larger version (103K): [in this window] [in a new window]   FIG. 4. Expression of exogenous wtBRCA1 but not tumor-associated mutant BRCA1 (T300G) causes telomere shortening. (A through C) Telomere shortening in cell lines stably overexpressing BRCA1. Subconfluent proliferating DU-145 or T47D cell clones were harvested and assayed for telomere length by Southern blot hybridization using the Telomere Length Assay Module (Pharmingen) (see Materials and Methods section for details). In panel A, parental DU-145 cells and three clones each of control (neo)-transfected and wtBRCA1-transfected cell clones were tested. In panel B, parental cells and two clones each of neo, wtBRCA1, and BRCA1-T300G-transfected DU-145 cells were assayed. In panel C, three clones each of control (neo)- and wtBRCA1-transfected T47D cell clones were analyzed. (D) Rapid telomere erosion in DU-145 cells transiently transfected with wtBRCA1. Subconfluent proliferating DU-145 cells were transiently transfected with FLAG-wtBRCA1 or the empty FLAG vector (18), washed, and postincubated for 4 days. Telo-FISH analysis was then performed as described previously (67) (see Materials and Methods). The red fluorescence represents the telomere signal. Note: about 50 to 100 cells in each transfected population were examined by two observers blinded to cell treatment, and each observer noted a significant reduction in the telomeric signal in the FLAG-wtBRCA1-transfected cells. Representative fields from each transfected cell population are shown. (E) Comparative BRCA1 protein levels in cell clones and untransfected cell lines. Subconfluent proliferating cultures of DU-145 and T47D cell clones and untransfected T47D and MCF-7 parental cell lines were harvested for Western blotting to detect BRCA1 and -actin (control for loading and transfer).

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.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Transient expression of wtBRCA1 causes time-dependent telomere shortening in DU-145 cells. DU-145/wtBRCA1/Tet-Off cells that had been maintained in medium containing TCN (2 µg/ml) were divided into two groups, +TCN and -TCN, starting at time zero. The cells were subcultured at a fixed inoculation number (104 cells/100-mm dish) every 7 to 10 days; periodically, the cultures were harvested for analysis of telomere length (Southern blot hybridization) (A) and BRCA1 and -actin protein levels (Western blotting) (B). The corresponding cell numbers at the time of subculture and the number of elapsed population doublings since removal of TCN are shown in (C) for cells cultured without (-) or with (+) TCN.

View larger version (31K): [in this window] [in a new window]   FIG. 6. wtBRCA1 inhibits TERT promoter and c-Myc transcriptional activity. Subconfluent proliferating DU-145 cells were transfected overnight with 0.25 µg of each vector per well, using Lipofectamine, washed to remove the Lipofectamine and excess vector, and incubated for another 24 h prior to harvesting for luciferase measurements. TERT promoter activity was measured using the TERT promoter-reporter plasmid pGL3-354-Luc (A and C). c-Myc-dependent transcriptional activity was determined using a reporter containing the c-Myc binding DNA element (E-box-Luc) (B and D). Luciferase values are expressed relative to the positive control (reporter only) and are means ± SEMs of quadruplicate wells. Notes: The total transfected DNA content was maintained constant by the addition of the appropriate quantity of empty vector. To monitor transfection efficiencies, control plasmid pRSV-ß-Gal was cotransfected, and the ß-Gal activities were measured spectrophotometrically. Assays are representative of at least two independent experiments.

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-ß-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 ⁶¹CysGly), a carboxyl terminal point mutation (C5365G or ¹⁷⁴⁹ProArg), and the carboxyl-terminal truncation mutation (5382insC or 1755term). The luciferase activity was normalized to the positive control (i.e., no BRCA1 vector transfected).

View larger version (35K): [in this window] [in a new window]   FIG. 7. Ability of mutated and deleted forms of BRCA1 to inhibit the TERT promoter and a c-Myc-responsive reporter. Assays were performed using DU-145 cells, as described in the Fig. 6 legend. (A) Schematic diagrams of the different BRCA1 cDNAs tested. (B and C) The effects of transient transfection of these BRCA1 cDNAs on TERT promoter and E-box-Luc activity, respectively. Luciferase values are expressed relative to the positive control (reporter only) and are means ± SEMs of quadruplicate wells. To control for transfection efficiency, plasmid pRSV-ß-Gal was cotransfected, and the ß-Gal activities were measured. The results are representative of at least two independent experiments.

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 (EcoRI) 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-ß-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.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Loss of TERT promoter activity in c-Myc-/- rat fibroblasts. c-Myc+/+ (wild type) and c-Myc-/- rat fibroblasts were assayed for wtBRCA1-mediated inhibition of TERT promoter activity using TERT promoter-reporter plasmid pGL3-354-Luc (B) or of c-Myc-dependent transcriptional activity using reporter plasmid E-box-Luc (C). Luciferase values are expressed relative to the positive control (reporter only) of the c-Myc+/+ cells and are means ± SEMs of quadruplicate wells. Western blots in (A) show the endogenous TERT protein levels in the c-Myc-/- and c-Myc+/+ cells. Note: The total transfected DNA content was maintained constant by the addition of the appropriate quantity of empty vector.

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.

View larger version (61K): [in this window] [in a new window]   FIG. 9. In vivo association and colocalization of BRCA1 and c-Myc. (A) In vivo association of BRCA1 and c-Myc by IP-Western blotting. Subconfluent proliferating cultures of MCF-7 or T47D human breast cancer cells were harvested, and nuclear lysates were subjected to BRCA1 IPc-Myc Western blotting or vice versa. As a control, MCF-7 nuclear lysates were subjected to IP with control nonimmune antibody (normal mouse IgG). See Materials and Methods for details. (B) Colocalization of BRCA1 and c-Myc in MCF-7 cells by confocal laser microscopy. Assays were performed as described in Materials and Methods.

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).

View larger version (37K): [in this window] [in a new window]   FIG.10. Further analysis of BRCA1 regulation of the TERT promoter. (A) Plasmid dose dependence of wtBRCA1 inhibition of TERT promoter activity. TERT promoter activity was measured using the pGL3-354-Luc reporter as described in the Fig. 6 legend, except that the dose of wtBRCA1 plasmid was varied from 0 to 5 µg per well. The total amount of DNA transfected was kept constant by the addition of empty pcDNA3 vector. The open bar represents the luciferase activity in the absence of transfection of wtBRCA1 or pcDNA3. (B) BRCA1 has little or no effect on Sp1- or NF-B-driven TERT promoter activity. TERT promoter activity was measured as described in the legend of Fig. 6 with the indicated expression vectors (0.25 µg per well for each vector). The total amount of transfected DNA was kept constant by the addition of empty pcDNA3 vector. (C) BRCA1 inhibits TERT promoter activity in MCF-7 and HCC1937 human breast cancer cells. Assays were performed as described in the legend of Fig. 6. (D) BRCA1 inhibits mouse TERT promoter activity in mouse cell lines. Assays were performed as described in the legend of Fig. 6 using normal mouse embryo fibroblasts and using the NIH 3T3 mouse cell line. (E) BRCA1 inhibits human TERT promoter activity in DU-145 human prostate cancer cells. Assays were performed as described in the legend of Fig. 6, except that a human TERT promoter reporter (pGL3-hTERT-Luc) was utilized. (F) Effect of BRCA1 knockdown by siRNA on TERT promoter activity. Subconfluent proliferating DU-145 or MCF-7 cells were transfected with BRCA1-siRNA-3 or a scrambled-sequence (control) siRNA (50 nM), incubated for 3 days to allow the loss of BRCA1 protein, and then transfected with pGL3-354-Luc and wtBRCA1 overnight. The cells were then washed, postincubated for 24 h to allow gene expression, and harvested for measurement of luciferase activity. The Western blots show the time course for reduction of BRCA1 protein levels in subconfluent proliferating MCF-7 cells transfected with a single dose of BRCA1-siRNA-3 (50 nM) or scrambled-sequence siRNA (50 nM). *, Statistically significant difference (P < 0.001, two-tailed t test) compared with the scrambled-sequence siRNA or no siRNA controls. Note: in panels A through E, the transfection efficiencies were monitored using the pRSV-ß-Gal control plasmid and were similarly high under all different experimental conditions.

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-B, 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.

View larger version (41K): [in this window] [in a new window]   FIG.11. Association of BRCA1 with exogenous mouse and human TERT promoters in vivo. (A and B) Mouse TERT ChIP assays. DU-145 cells were transiently transfected with pcDNA3, wtBRCA1, pGL3-354-Luc plasmid (mTERT), wtBRCA1 plus mTERT, the empty pGL3-Luc reporter plasmid (pGL3), or wtBRCA1 plus pGL3. The cells were treated with formaldehyde to cross-link protein to DNA; and nuclear extracts were prepared and chromatin sheared to an average length of 600 bp. The cross-linked chromatin was purified and subjected to IP using anti-BRCA1, anti-c-Myc, or control IgG. In panel A, the immunoprecipitated DNA was analyzed by PCR using primers designed to specifically recognize the exogenous mTERT expression plasmid promoter region. The PCR products were analyzed by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. In panel B, the same immunoprecipitated DNA used in panel A was analyzed by PCR using PCR primers specifically designed to recognize the empty pGL3 luciferase vector. The diagrams on the right of panels A and B illustrate the locations of the primers (arrows) within the reporter plasmids. MCS denotes the multiple cloning site of the vector. (C and D) Human TERT ChIP assays. DU-145 cells were transiently transfected with pcDNA3, wtBRCA1, the human TERT luciferase reporter plasmid (hTERT), wtBRCA1 plus hTERT, empty pGL3-Luc reporter plasmid (pGL3), or wtBRCA1 plus pGL3. ChIP was then carried out as described above. In panel C, the immunoprecipitated DNA was analyzed by PCR using primers designed to specifically recognize the exogenous human TERT luciferase expression plasmid promoter region. PCR products were analyzed by electrophoresis on a 1.5% agarose gel and visualized by ethidium bromide staining. In Panel D, the same immunoprecipitated DNA used in panel C was analyzed by PCR with PCR primers specifically designed to recognize the empty pGL3-Luc vector. The diagrams on the right of panels A and B illustrate the locations of the primers (arrows) within the reporter plasmids.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our findings suggest that overexpression of the BRCA1 gene inhibits TERT expression and telomerase enzymatic activity in various cell types and causes telomere shortening. On the other hand, BRCA1 had little or no effect on the expression of a subset of other components of the telomerase holoenzyme (TP1, HSP90, hTR, and dyskerin), c-Myc, or two telomeric repeat-binding proteins (TRF1 and TRF2). The inhibition of TERT expression is due, in part, to the inhibition of c-Myc-mediated transactivation of the TERT promoter, as demonstrated using TERT promoter and E-box-luciferase reporter assays. Moreover, while BRCA1 inhibited c-Myc-induced TERT promoter activity, it did not inhibit Sp1- or NF-B-induced promoter activity.

It is unlikely that inhibition of cell proliferation contributed significantly to the reduced TERT expression and telomerase activity or the shortened telomeres observed in wtBRCA1-transfected cell lines. Thus, the BRCA1-overexpressing DU-145 and T47D cell clones had only a slight reduction in doubling times compared with the control (neo) clones or parental cells, and induction of BRCA1 overexpression in the TCN-regulated DU-145 cell line had no effect on cell proliferation. The reduction in TERT promoter activity and c-Myc E-box activity occurred rapidly upon expression of wtBRCA1. Thus, each of these was observed in transient transfection assays in which the end points were determined 24 h after an overnight transfection of the wtBRCA1 vector. Similarly, in HCC1937 breast cancer cells, which contain only mutant BRCA1 (5382insC), wtBRCA1 inhibited telomerase enzymatic activity in a transient transfection assay. In these cells, transient transfection of wtBRCA1 has no effect on cell proliferation and protects these cells against cytotoxicity and apoptosis induced by a DNA damaging agent (adriamycin) (18). Human primary cell cultures usually exhibit very low TERT expression and telomerase activities. Whether or not the mechanism of telomerase suppression in human primary cells is related to that in wtBRCA1-overexpressing cancer cells remains to be determined.

Stable DU-145 and T47D wtBRCA1 cell clones were able to maintain telomeres, although these were significantly shorter than in the control clones. The ability to maintain short telomeres may be explained by residual telomerase activity, since neither TERT expression nor telomerase activity was abolished by wtBRCA1. Alternatively, the telomere maintenance could be due to a telomerase-independent mechanism for the alternative lengthening of telomeres, a process that is often observed in tumor cells (6, 9).

The studies of DU-145/wtBRCA1/Tet-Off cells suggest that up-regulation of BRCA1 causes progressive shortening of telomeres over about 60 days (60 doublings) but that the initial phase of the loss of longer subpopulations of telomeres occurred very rapidly, over 3 days (three to four doublings). This finding was consistent with evidence of significant telomere erosion only 4 days after transient transfection of a wtBRCA1 gene in DU-145 cells demonstrated by the telo-FISH assay. The rapidity of telomere loss induced by exogenous BRCA1 suggest the possibility that telomerase inhibition is not the only mechanism by which BRCA1 causes telomere shortening. Similarly rapid telomere shortening (0.85 kb per day) due to telomere degradation was observed in cells expressing a TRF2 protein targeted to telomeres (1). And very rapid telomere loss was precipitated by exogenous RB94 (an isoform of RB1 lacking amino acids 1 to 112) in telomerase-positive bladder cancer and immortalized urothelial cells (67). Whether BRCA1 promotes telomere degradation is conjectural, but this seems likely since rapid telomere shortening is not characteristic of cells with absent or very low telomerase activity.

It is interesting that the RMN complex (Rad50-M_re11-p95NBS1), which is implicated in DNA double-strand break repair, was found to interact with TRF2 and to localize at telomeres (69). It was also reported that BRCA1 interacts and colocalizes with this complex following DNA damage (61, 68). It remains to be discovered if BRCA1 is present at telomeres or exerts a telomerase-independent telomere regulatory function.

Unlike normal fibroblasts, DU-145 and T47D wtBRCA1 clones and DU-145/wtBRCA1/Tet-Off cells induced to overexpress BRCA1 were able to proliferate in long-term culture without entering into senescence or apoptosis, despite severe telomere shortening. Critically short telomeres may cause senescence, in part, by activating a p53/p21^WAF1/Cip1 pathway and/or a p16/Rb1 pathway (55). DU-145 cells contain inactivating mutations in both p53 (double point mutation) and Rb1 (deletion of exon 21), and T47D cells contain mutant p53 but have wild-type Rb1. Thus, defects in telomere damage signaling pathways may contribute to the ability of these cells to survive and grow despite having very short telomeres. Our studies suggest that wtBRCA1 does not alter the expression of the telomere repeat binding factors TRF1 and TRF2 (at least at the mRNA level). However, given that the telomeres are considerably shorter in wtBRCA1 than control cell clones, it is possible that the same levels of TRF1 and TRF2 could help to protect the shorter telomeres.

Interestingly, subclones derived from immortal populations of HeLa cells (which had stable telomere populations with a mean TRF length of 3.7 kb), showed various telomere lengths, the smallest of which was about 1.7 kb (7), similar to that observed in DU-145/wtBRCA1/Tet-Off cells that were induced to express BRCA1.

The studies using c-Myc^-/- fibroblasts revealed a very low level of TERT promoter activity (10% of that found in c-Myc^+/+ cells), which was further suppressed by wtBRCA1. This finding suggests that wtBRCA1 can also cause c-Myc-independent inhibition of TERT promoter activity, although most of the endogenous, BRCA1-suppressible activity appears to be c-Myc dependent in the cell types studied. While exogenous c-Myc caused a significant increase in TERT and E-box-Luc activity in c-Myc^-/- cells, the activity levels in these cells were well below the endogenous activity levels in c-Myc^+/+ cells. This finding suggests that the c-Myc^-/- cells have developed an additional mechanism(s) to down-regulate the TERT/Myc activity.

Our findings are consistent with a previous study in which BRCA1 was found to interact with c-Myc and to repress c-Myc transcriptional and transformational activity (60). In that study, the helix-loop-helix region of c-Myc, the same region involved in c-Myc-Max heterodimerization, was required for the BRCA1-c-Myc interaction. Amino acids 175 to 303 and 443 to 511 of BRCA1 were required for the interaction with c-Myc, as determined by glutathione S-transferase capture assays (60). In another study, it was reported that BRCA1 inhibits c-Myc-inducible TERT promoter activity through a three-way interaction involving BRCA1, c-Myc, and Nmi (N-Myc interactor) (35). In that study, wtBRCA1 was insufficient to inhibit TERT promoter activity, and addition of exogenous Nmi was required to inhibit TERT. In our study, wtBRCA1 alone was sufficient to inhibit TERT promoter activity, TERT expression, and telomerase activity. The finding that wtBRCA1 inhibited TERT promoter activity in c-Myc^-/- rat fibroblasts suggests that BRCA1 can also inhibit c-Myc-independent TERT promoter activity. However, a caveat is that unlike the case in most human primary cells, TERT and telomerase are constitutively expressed in rodent cells, thus indicating that their regulation in rodents and humans is not identical.

We found that the carboxyl terminus of BRCA1 is dispensable for inhibition of TERT and E-box reporter activity. BRCA1 genes encoding amino acids 1 to 771 (KpnI) and 1 to 1313 (BamHI) (but not amino acids 1 to 302 (EcoRI]) partially or strongly inhibited these reporters. However, one mutant BRCA1 (T300G) failed to inhibit telomerase or TERT-Luc activity but gave significant, though incomplete, inhibition of E-box-Luc activity. T300G is a breast cancer-associated mutant that codes for a full-length protein with a point mutation (⁶¹CysGly) that disrupts the RING domain. This finding suggests that an intact RING domain is required for inhibition of TERT. The T300G mutation (which affects BRCA1 amino acid 61) should not disrupt the BRCA1-c-Myc interaction, which involves amino acids 175 to 303 and 443 to 511 of BRCA1 (60). These findings suggest that inhibition of the c-Myc transactivation function is insufficient to inhibit TERT or that more complete inhibition of c-Myc is required to inhibit the TERT promoter. They also suggest that BRCA1 binding to c-Myc is necessary but not sufficient to suppress TERT promoter activity. Similarly, we found that the T300G mutation did not disrupt BRCA1 binding to the estrogen receptor but inhibited its ability to prevent estrogen receptor transcriptional activity (17).

Most of these studies were conducted using a BRCA1 overexpression model. However, several considerations suggest that the findings may be relevant to the physiological regulation of TERT. First, knockdown of BRCA1 significantly increased TERT promoter activity in MCF-7 cells, although the extent was modest (30 to 35%). The small extent of the increase in TERT activity may be due, in part, to the partial recovery of BRCA1 protein levels by the time the cells were harvested for luciferase assays. The BRCA1 knockdown model may mimic some sporadic breast and ovarian cancers; a significant proportion of these tumors exhibit loss of BRCA1 expression (50, 62). Secondly, endogenous BRCA1 was detectable at transiently transfected mouse and human TERT promoters, and the quantity of BRCA1 at the promoter sites was increased in cells transfected with a wtBRCA1 gene. Interestingly, the quantity of endogenous c-Myc at the TERT promoter was also increased in cells transfected with wtBRCA1. These findings suggest that BRCA1 may recruit c-Myc to the TERT promoter or stabilize its interaction with the promoter, although the precise mechanism remains to be determined.

Finally, an overexpression model may be very relevant to understanding some of the physiological actions of BRCA1 as a breast cancer tumor suppressor. Thus, during normal mouse development, BRCA1 is especially highly expressed in proliferating mammary epithelial cells that are undergoing differentiation during puberty and pregnancy (36, 48, 49). And cultured mammary epithelial cells induced to undergo differentiation in vitro also show greatly increased BRCA1 expression. It has been suggested that overexpression of BRCA1 during these particular periods may contribute to its tumor suppressor activity.

FIG. 4—Continued.

	ACKNOWLEDGMENTS

 
This work was supported in part by USPHS grants RO1-ES09169, RO1-CA82599, and RO1-CA80000; U.S. Army Breast Cancer grant DAMD17-99-1-9254; and the Susan G. Komen Breast Cancer Foundation.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Oncology, Lombardi Cancer Center, Georgetown University, Preclinical Sciences Bldg., Room GM12B, 3970 Reservoir Rd., NW, Washington, DC 20007. Phone: (202) 687-7695. Fax: (202) 687-7505. E-mail: emr36{at}georgetown.edu.

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