Antibodies and Plasmids
TopBP1 (sc-22859) antibodies used for immunoprecipitation (IP) or immunoblot (IB) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to Serine 216 of Cdc25C, Serine 10 of histone H3, Rad 51, and γH2AX (Serine139) were from Cell Signaling (Beverly, MA, or Santa Cruz Biotechnology). Wild-type (wt) ERα, membrane or nuclear-targeted E domain (ligand-binding domain) of ERα, pRK5-HERCD533 (dom-neg enhanced green fluorescent protein [EGFR] from Axel Ullrich (Institute of Molecular Biology, A*STAR, Singapore) (Redemann et al., 1992
), and ERα
S522A constructs were previously described (Razandi et al., 2003a
; Pedram et al., 2006b
). Wild-type and S280AChk1 plasmids and phospho-Serine 280 Chk1 antibodies were from Dr. Emma Shtivelman (Bionovo, Emeryville, CA) (King et al., 2004
). Wtp53 and S15Ap53 were from Rainer Brockman via David Meek (University of Dundee, Dundee, United Kingdom) (Meek, 2002
). PMT2-AHAKT (dominant-negative AKT) was provided by Dr. Julian Downward (London Research Institute, London, United Kingdom). Flag-wt, S1159A, and S1159D TopBP1 constructs were from Dr. Weei-Chin Lin (University of Alabama at Birmingham, Birmingham, AL) (Liu, K. et al., 2006b
Cell Culture, Reagents, and Basic Experiments
All cell lines were from ATCC (Manassas, VA). MCF7 cells were grown using DMEM-F12, 10% FBS, and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA; 15240-062) at 37°C to 75% confluency in 100-mm dishes. In experiments where the effects of estrogen were determined, cells were cultured for 24 h in phenol red–free, charcoal-stripped FBS to remove any estrogenic effects, before E2 addition.
For experiments using random cycling cells, culture was done in the absence of serum for 2 h, before DNA damage± E2 addition. For S-phase synchronization, MCF7 cells were grown to ~60% confluence. Thymidine was added (final concentration 2 mM), and the cells were cultured at 37°C for 16 h. Medium was then removed, and cells were washed three times with PBS. Fresh medium without thymidine was added for 9 h at 37°C, followed by adding thymidine for 16 h. After washing, time-course studies of thymidine incorporation after release from the second thymidine incubation showed strong DNA synthesis at 6 h (S-phase). At this time ATR activity in response to DNA-damaging agents was determined.
Mouse mammary epithelial cells were isolated from 8-wk-old female C57/BL6 mice by enzymatic and mechanical methods and established as primary cultures for experiments. propyl-pyrazole-triol (PPT) and diarylpropionitrile (DPN) were from Tocris Chemicals (Ballwin, MO). Tyrphostin 1478 and LY294002 were from Calbiochem (La Jolla, CA). Small interfering RNAs (siRNAs) forTopBP1, and AKT were from Dharmacon (Boulder, CO). In general, E2 or other steroids or chemicals were added 20 min before DNA-damaging treatment. UV exposure (10 J/m2) was performed over 10 s using a Stratalinker 1800 (Stratagene, La Jolla, CA). Gamma radiation (IR) of cells was performed at our Radiation Therapy Center, after calculating the delivery of precise amounts of radiation (2 Gy) to cultured cells over 15 s. Hydroxyurea (HU), 1 mM, was added to the cells for 2 h, based on initial ATR kinase time-course studies.
The E domain of ERα was inserted into EGFP vectors (Clontech) targeting the E domain completely to the plasma membrane or nucleus as we described (Pedram et al., 2006b
), and the constructs were transfected into ER null HCC-1569 cells. Cells were synchronized to S-phase and exposed to brief UV in the presence or absence of E2
, and ATR activity was then determined.
Kinase Assays and 32Pi labeling
ATR kinase activity was determined in vitro. For kinase assays, both random cycling and S-phase–synchronized MCF7 cells were exposed to various treatments (e.g., brief UV±E2). MCF7 cells from each condition (~1 million) were then washed with DMEM/F12 medium and lysed in buffer (50 mM Tris-HCL, pH 7.5,100 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.5% Triton X-100, 40 mM β-glycerophosphate, 200 mM sodium orthovanadate, 40 mM PNPP, 100 mM PMSF, and protease inhibitor cocktail; Sigma, St. Louis, MO). The lysates were centrifuged at 14K rpm for 10 min, and the supernatants were exposed to 50 μl of suspended Protein A or G agarose beads in microcentrifuge tubes and rotated for 30 min at 4°C. This was done twice to ensure removal of endogenous IgG and nonspecific proteins from lysate. The cell lysate/protein G or A bead complex was centrifuged at 14,000 rpm for 30 s at 4°C. Without disturbing the pellet, the supernatant was transferred to a new tube. These precleared supernatants were stored on ice. For the ATR activity assay, 10 μl of ATR antibody (Santa Cruz Biotechnology, sc-1887or sc-21848) was conjugated to 50 μl of protein G beads (Sigma, P4691) for 2 h at room temperature, and the bead complex was washed. Then, 1 ml of precleared whole-cell extract from each experimental condition was added to the ATR antibody/protein G bead complex in lysis buffer (that also serves as the IP buffer), and rotated end-over-end overnight at 4°C. Beads were washed once with lysis buffer and twice with HEPES buffer (25 mM HEPES, 10 mM Mg-acetate). To the lysates/antibody/bead complexes, 40 μl of stock mixture, made up as 20 μl of 3 × kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgAc, 2 mM DTT, 40 mM ATP), and 10 μl of H2O, was added to 2 mg glutathione S-transferase (GST)-Phas1 in 9 μl of water, and 1 μl [γ-32P]ATP. Each tube was vortexed and then incubated at 30°C for 30 min. SDS-PAGE loading buffer (40 μl) was then added, vortexed, boiled for 5 min, and centrifuged at 14K rpm for 1 min. Samples of 40 μl from each condition were loaded onto a 10% gel. After electrophoresis, the gel was fixed, dried, and subjected to autoradiography, reflecting 32P incorporated into the Phas-1 protein. ATR activity was also determined by specific phosphorylation of endogenous p53 (Serine 15) and Chk1 (Serine 345), as immunoblots with phospho-specific antibodies (Santa Cruz Biotechnology). Total protein immunoblots serves as loading controls for many experiments and reflect additional gel separation of the same cell protein lysates.
Chk1activity was determined either using Serine 345 (Cell Signaling) phospho-Chk 1 antibodies for immunoblot, or as kinase activity using GST-Cdc25C as substrate. The in vitro kinase activity assay was similar to what was described for ATR. Immunoblots for Serine 280 Chk1 phosphorylation were also determined (antibody was a kind gift from Dr. Emma Shtivelman). Immunoprecipitated endogenous Cdc2 kinase from cells undergoing various conditions and p70 S6 kinase protein as exogenous substrate were used for in vitro Cdc2 kinase activity assays. Tyr15 phosphorylation of Cdc2 was determined by IB (antibody from Santa Cruz Biotechnology). Cdc2 kinase is also known as Cdk1 kinase.
32P-labeling of cellular protein was done in near confluent MCF7 cells transfected with Flag-wt or S1159ATopBP1 and recovered overnight. Transfected cells were placed for 18 h before labeling in fresh, phosphate-free DMEM (Invitrogen) supplemented with 5% charcoal-stripped FBS and incubated in 5% CO2 at 37°C. This medium was removed, and washed cells were pretreated with medium containing LY294002 for 20 min and then exposed to brief UV in the presence or absence of E2. Replacement with prewarmed medium containing 0.5 μCi/ml 32Pi (Perkin Elmer-Cetus) ± E2 ensued, and the cells were placed in a CO2 incubator inside a Plexiglas box for 2 h. Cells were washed to remove unincorporated 32Pi and lysed in the previously mentioned lysis buffer. Lysates were sonicated at 4°C for 10 min and then spun at 14K rpm for 5 min. Supernatants were placed in microcentrifuge tubes, and lysates were precleared with 50 μl of suspended agarose beads, for 30 min at 4°C while rotating. The lysate/bead complex in microcentrifuge tubes was centrifuged at 14K rpm for 30 s at 4°C. Without disturbing the pellet, the supernatant was transferred to a new tube and stored on ice. Flag antibody (20 μl, Santa Cruz Biotechnology) was conjugated to 50 μl of protein G beads (Sigma, P4691) for 2 h at room temperature. Then, 1 ml of precleared cell extract was added to the Flag antibody–protein G bead complex and rotated end-over-end overnight at 4°C. Beads were washed with PBS, and 50 μl of SDS-PAGE loading buffer was added. After boiling and centrifugation, 40 μl of each sample was loaded onto a 10% gel. Subsequently, the gel was fixed, dried, and subjected to autoradiography (using special intensifying screen for 32P).
IP of endogenous ATR, followed by immunoblot for TopBP1 was carried out in S-phase and in randomly cycling MCF7 cells under stated conditions. Whole-cell lysates were precleared by adding 1 ml of whole-cell lysate to 50 μl of protein A/G agarose beads at 4°C for 30 min. Beads were centrifuged at 3000 rpm for 30 s at 4°C. Supernatants (cell lysates) were transferred to a new microcentrifuge tube. To 1 ml of the above cell lysate, 10 μg of primary antibody (agarose conjugate) was added and incubated at 4°C overnight with end-to-end mixing. After centrifugation, pellets were washed three times with PBS. After final wash, the pellets were resuspended in 40 μl of 2× electrophoresis sample buffer, for separation by PAGE. Proteins from the gel were transferred to nitrocellulose and then exposed to TopBP1 antibodies for immunoblot, using the ExactaCruz kit (Santa Cruz Biotechnology) that features the elimination of light and heavy chain antibody detection. Similar techniques were used to assess Chk1:Claspin interactions. In some experiments, Flag antibodies were used. Cell localization of proteins and phosphorylation status was determined by immunofluorescent confocal microscopy after culturing the cells on glass coverslips before specific antibody staining, followed by FITC conjugated–second antibody.
DNA Repair and Viability Assays
S-phase and random cycling MCF7 cells were exposed to brief UV or IR±E2. The cells were allowed to proceed in DMEM-F12±E2 agarose, alkali-denatured, and subjected to electrophoresis. The cells were stained with DNA intercalating dye (SYBR green and visualized by epifluorescence (Trevigen, Gaithersburg, MD; UVDE Flare Assay Kit). Damaged, unrepaired DNA was seen as the “tail” of the Comet, quantified from 200 cells per condition by using Komet imaging software, version 5.5 (Andor Technology, South Windsor, CT). The experiment was repeated twice for mean ± SEM calculations of tail quantification. Cell viability was determined by MTT assay (Sigma) that measures mitochondrial dehydrogenase activity in viable cells. Random cycling MCF7 cells were exposed to DNA-damaging agents in the presence or absence of E2. At 24 h, the cells were incubated with medium containing MTT, 1 mg/ml, for 4 h at 37°C. MTT/formazan was extracted by overnight incubation at 37°C with 100 μl extraction buffer (20% SDS, 50% formamide adjusted to pH 4.7 with 0.02% acetic acid, and 0.025 N HCl). Optical densities at 570 nm were measured using extraction buffer as a blank.
Wild-type or S15Ap53 were expressed in HCC-1937 cells (p53 null, BRCA1 mutated, and ER negative), and the cells were recovered and then exposed to brief UV. DNA was obtained 6 h after treatment. In some cells, ERα was expressed, and the cells were incubated in the presence or absence of E2. RT-PCR for p21 was performed at 94°C for 3 min, followed by 26 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, using primers p21F (5′-CCAAGAGGAAGCCCTAATCC-3′) and p21R (5′-CCCTTCAAAGTGCCATCTGT-3′). As a control, the same reaction conditions were applied for PCR detection of the human GAPDH. The GAPDH primers were as follows: forward, 5′-AGCCACATCGCTCAGAACAC, and reverse, GAGGCATTGCTGATGATCTTG-3′, respectively.
S-phase MCF7 cells were exposed to UV±E2 and then fixed at 10 min, 2, 4, 6, or 8 h and exposed to photoproduct specific CPD fluorescent antibody (Sigma). The nuclear fluorescent signal was quantified and normalized to nuclear area determined by propidium iodide (PI) staining. Insets show PI staining of the same fields.
γH2AX and Rad51 Foci Formation
MCF7 cells were cultured in glass plates with phenol red–free DMEM-F12 containing charcoal-treated FBS. The cells were synchronized with 1 μM nocodazole for 16 h and then released and irradiated with UV 10 J/m2 in the presence or absence of 10 nM E2. Cells were fixed with 4% freshly prepared paraformaldehyde at room temperature, at 10 min, 2, 4, 6, and 8 h after UV. Cells were then briefly treated with 0.5% Triton X-100, washed four times with PBS, and incubated with 2% BSA for 1 h at room temperature. Then the primary antibodies Rad 51 (Santa Cruz Biotechnology), or γH2AX (Upstate Biotechnology, Lake Placid, NY) were incubated with cells overnight at 4°C. Cells then were washed and incubated with secondary antibody conjugated with FITC (Vector Laboratories, Burlingame, CA) at 1:100 dilution for 1 h, and visualized under fluorescent microscopy.
Preparation of Metaphase Chromosome Spreads from Adherent Cells
MCF7 cells were grown in DMEM-F12 with estrogen-free serum 2 d before performing the experiments involving chromosome spreads. Nonsynchronized cells were exposed to 2 Gy IR in the presence or absence of 10 nM E2 ± ICI182780 (ICI), (Faslodex, AstraZeneca, Wilmington, DE) and then were allowed to recover at 37°C for 48 h. For the last 3 h of incubation, colcemid was added to a final concentration of 1 μg/ml and incubated at 37°C. The cells were washed with PBS (without Ca2+ and Mg2+) and then detached with trypsin-EDTA. The cell pellet was treated with 0.075 M KCl solution and pipetted up and down to break clumps. After centrifugation, the supernatant was aspirated, leaving 200 μl of the hypotonic solution. After centrifugation, the pellet was resuspended in1 ml of methanol:glacial acetic acid (3:1) solution, and the procedure was repeated. Resuspending the pellet then occurred in a small volume of fixative (<500 μl). A drop of mounting solution with 1% Giemsa stain was added to each slide. The slides were examined under oil immersion and viewed at 10 × 100 magnification. Chromosomal damage (chromatid breaks, dicentric chromosomes, or condensation [ring formation]) was scored from 200 cells in each condition, and the study was repeated.
The microscopic images were obtained using a Nikon Eclipse TE-200 scope with magnification from 200 × to 400 × at room temperature. A Diagnostic Instruments camera (model 3.2.0; Sterling Heights, MI) was used in conjunction with Spot Advance software, to capture and transfer images to the computer. Rhodamine- (red) and FITC (green)-conjugated secondary antibodies (Vector Laboratories) were used for fluorescent visualization.