Cell lines and cell culture.
Most cell lines were derived from the human fibrosarcoma HT1080 cell line. Hypoxanthine phosphoribosyltransferase mutant (HPRT−
) HT1080 cells were selected by growth in 6-thioguanine (20 μM) and screened for a nonreverting HPRT
mutation. One surviving clone (HT1080/57) arose by spontaneous deletion in the single copy of the endogenous HPRT
gene and was used for further construction of cell lines. Five million HT1080/57 cells were electroporated with 1 μg of linearized plasmid pTet-ON (Clontech), which contains the gene for the rtTA protein, a fusion of the herpes simplex virus (HSV) VP16 activation domain and the reverse tetracycline repressor protein (20
). Clones resistant to G418 (600 μg/ml) were picked for analysis of rtTA expression using a luciferase reporter assay kit (BD Biosciences). One clone, HT1080/57-6, which gave about a 25-fold increase in luciferase activity upon addition of doxycycline (Dox; 2 μg/ml), was used for construction of the DIT and FLAH cell lines. HT1080/57-6 cells were used to construct FLAH cells as described previously (39
To construct DIT cells, 5 million HT1080/57-6 cells were electroporated with 1 μg of linearized plasmid pNEBR-R1 (New England Biolabs), which contains the genes for RheoReceptor-1 and RheoActivator (30
). Clones resistant to zeocin (300 μg/ml) were picked and analyzed for expression of these regulators using a luciferase reporter assay kit (BD Biosciences). One clone, RS11, which gave a 15-fold increase in luciferase activity upon addition of RSL1 (0.5 μM), was used for construction of the DIT cell lines. Five million RS11 cells were electroporated with 1 μg of linearized pDIT1 and then selected for colonies resistant to puromycin (2.5 μg/ml). Plasmid pDIT1, which has a pBluescript backbone with a selectable puromycin N
-acetyltransferase gene, contains an HPRT
minigene with a CAG95
repeat inserted in the middle of the intron between fused exons 1 and 2 and fused exons 3 to 9. The HPRT
minigene was modified so that the cytomegalovirus (CMV) promoter pTRE-CMVmini, which responds to the inducer doxycycline, controls its sense expression, and the pNEBRX1 promoter, which responds to the inducer RSL1, controls its antisense expression. Individual colonies were analyzed by Southern blotting to identify clones with a single integrated copy of the HPRT
minigene that was responsive to both inducers. Two cell lines, DIT7 and DIT3, were used in this study. Many of the colonies that were tested were responsive to doxycycline, but not to RSL1, even though they seemed to have an intact pNEBR-X1 promoter. Two such cell lines, DIT-NAT10 and DIT-NAT26, were used in this study.
The DH7 and DH10 cell lines were constructed by electroporating 1 μg of linearized pAH1 into 5 million FLHA25 cells and then selecting for colonies resistant to zeocin. In addition to a zeocin resistance gene, plasmid pAH1 contains an HPRT minigene that lacks a sense promoter but uses the constitutive pCMV promoter to drive antisense transcription.
The DIT-NR4 and DIT-NR5 cell lines were constructed by electroporating 1 μg of linearized pDIT-NR into 5 million RS11 cells, selecting for individual clones that were resistant to puromycin, and analyzing them by Southern blotting to identify clones with a single integrated copy of the plasmid. The pDIT-NR plasmid was derived from pDIT1 by excising the CAG repeat tract by NotI digestion, which removes the CAG tract along with about 130 bp of flanking sequences.
The DIT7-R cell lines were derived from DIT7 cells by selection for HPRT+ colonies, using hypoxanthine-aminopterin-thymidine (HAT) selection. HPRT+ colonies were screened for length of their contracted CAG repeats by gel electrophoresis of PCR products. The repeat tracts in DIT7-R15, DIT7-R20, and DIT7-R25 were determined by DNA sequencing.
The DITS-H5 and DITS-H8 cell lines were constructed by randomly integrating plasmid pDIT2 into HEK293 F-T cells (Invitrogen), which were modified from HEK293 cells, so that they express the rtTA regulator required for expression of the Tet-ON-inducible system. Plasmid pDIT2 contains a pBluescript backbone with a selectable puromycin N-acetyltransferase gene, along with one copy of the HPRT minigene with a CAG95 tract in its intron and two copies of the pTRE-CMVmini promoter, which flank the minigene and allow both sense and antisense transcription to be controlled by the inducer doxycycline.
In all cases, clones containing a single copy of the randomly integrated construct, as confirmed by Southern blotting, were used in this study (39
). All cell lines were grown at 37°C with 5% CO2
in Dulbecco's modified Eagle's medium-F-12 medium (DMEM/F-12) supplemented with 10% fetal bovine serum and 1% MEM nonessential amino acids.
Induction of transcription.
Cells were initially plated in the absence of transcription inducers. The time at which inducers were added to the cultures was defined as day 0. To induce sense transcription in HT1080-derived cell lines, we added doxycycline to final concentrations of 200 ng/ml for DIT and DIT-NR cells and 2,000 ng/ml for DIT-NAT, FLAH, and DH cells. Because the half-life of doxycycline in medium is about 24 h, we added half the starting amount of doxycycline directly into the medium each day until the treatment was completed. To induce antisense transcription fully in cells driven by the Rheo-Switch inducible promoter, RSL1 was added to the medium to a final concentration of 500 nM. No additional RSL1 was required for treatments up to 5 days. In the HEK293-derived DITS-H cells, we induced both sense and antisense transcription simultaneously by addition of 2,000 ng/ml doxycycline.
Viable cell measurements.
Adherent cells (attached to the plate) are operationally defined as viable, and nonadherent cells (present in the medium) are operationally defined as dead. Adherent cells typically contain fewer than 4% of cells that stain with propidium iodide (PI), indicating that >96% of adherent cells are viable. The number of dead cells was determined by counting the nonadherent cells in the medium. The number of viable cells was counted after detachment of adherent cells from the dish by trypsin treatment. The percentage of viable cells was calculated as the number of adherent cells divided by the total number of cells, which is the sum of adherent and nonadherent cells. The data in various figures have not been corrected for the small percentage (<4%) of dead cells in the adherent population.
Total RNA was extracted from about 1 million cells using RNeasy minikits (Qiagen). Induction of HPRT
sense or antisense transcription was measured on RNA extracted 1 day after addition of doxycycline or RSL1 to the medium. The levels of sense and antisense transcripts were measured separately using strand-specific real-time reverse transcription-PCR (RT-PCR), as illustrated in Fig. H. To measure the level of sense transcript, we first synthesized cDNA from the sense transcript, using reverse transcriptase and the SSP-H8 primer, which links the HPRT
sense-strand-specific H8 sequence to the SSP universal primer, whose sequence is not present in the genome. Reverse transcriptase was then inactivated by incubation at 95°C for 10 min. The cDNA was then amplified using the HPRT
-specific H7 primer and the SSP universal primer and quantified by real-time PCR. For measurement of antisense transcripts, we synthesized cDNA from the antisense transcript, using reverse transcriptase and the SSP-H7 primer, in which the universal SSP primer is linked to the antisense-strand-specific HPRT
primer H7. The “antisense” cDNA was then amplified using a mixture of the H7 and SSP primers and quantified by real-time PCR. Sequences of the primers are listed in Tables and . For strand-specific real-time RT-PCR, total RNA (50 ng per reaction) was reverse transcribed at 50°C for 30 min and assayed using the SYBR green PCR kit (Qiagen). Results were normalized to the concentration of β-actin RNA, which was determined the same way. HPRT
sense or antisense transcript levels were expressed relative to the RNA level in untreated DIT7 cells, which was arbitrarily defined as 1 (55
). The conditions for real-time PCR were 95°C for 15 min, followed by 45 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 30 s.
FIG. 1. Cell lines for assessing effects of sense and antisense transcription. In all cells, the CAG tract is centered in the 2.1-kb intron in the single, randomly integrated HPRT minigene and the repeat is 1.6 kb downstream of the sense promoter and 2.5 kb upstream (more ...)
Sequences of real-time RT-PCR primers and siRNAs used in this study
Sequences of strand-specific real-time RT-PCR primers for HPRT
Chemical and siRNA treatments.
For chemical treatments, 250,000 DIT7 cells were plated in each well of a 6-well plate on day −1. On day 0, we treated the cells with chemicals at concentrations shown in Table . When necessary, we induced convergent transcription by adding doxycycline and RSL1 along with the chemicals on day 0. Because many of the chemicals were dissolved in dimethyl sulfoxide (DMSO), a small amount was carried over into the treated cell cultures. We showed that DMSO at those concentrations had no effect on cell viability, except for serum-starved cells, where DMSO caused some toxicity (see Fig. ). The siRNAs (Dharmacon or Invitrogen) used in this study are listed in Table . For siRNA treatments, we used the procedure described previously (42
). Briefly, about 100,000 DIT7 cells were plated in each well of a 6-well plate on day −3. On day −2, we transfected cells with siRNAs at a final concentration of 200 nM, using Oligofectamine (Invitrogen). Treatments with 200 nM vimentin siRNA served as controls. On day 0, the cells were again transfected with siRNA, and cultures were then grown in the presence or absence of doxycycline plus RSL1. We evaluated knockdown of target gene expression and sense and antisense HPRT
transcription by analyzing total RNA isolated on day 1.
Effects of chemical inhibitors of ATR and ATM pathway components on DIT7 cell viability after induction of convergent transcription
FIG. 6. Apoptosis in nonproliferating cells. (A) Convergent transcription and cell death in serum-starved DIT7 cells in the presence and absence of zVAD. Serum-starved DIT7 cells were treated with DMSO, doxycycline plus RSL1 (Dox + RSL1), or doxycycline (more ...) Contraction assay.
One million DIT cells were plated in each well of a 6-well plate on day −3. On days −2, −1, and 0, cells were refed with fresh medium daily until they reached confluence. On days 1, 3, and 5, doxycycline (200 ng/ml), RSL1 (500 nM), or both were added. Medium containing the inducers was removed on days 2, 4, 6, 7, and 8 and replaced with fresh medium lacking the inducers. For contraction assays in the presence of 20 μM zVAD [benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone], zVAD was present from day 0, when inducers were added, until day 6, when the inducers were removed. On day 9, HPRT+ cells were selected by plating 500,000 DIT cells on 100-mm dishes in HAT medium (0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymine) for 2 weeks. Contraction frequencies were calculated as the number of HPRT+ colonies divided by the number of viable cells; they are the average of at least eight experiments. A few dozen individual HPRT+ colonies were grown up, and the lengths of their repeat tracts were assayed by gel electrophoresis and DNA sequencing; all contained shortened CAG repeats, ranging from CAG7 to CAG35.
In the fluorescein isothioncyanate (FITC)-annexin V-propidium iodide (PI) assay (BD Biosciences), different densities of cells were plated in each well of 6-well plate on day −1. On days 0, 1, 2, 3, and 4, the inducers doxycyline and RSL1 were added for 5-, 4-, 3-, 2-, and 1-day treatments, respectively. On day 5, adherent cells were trypsinized, collected together with nonadherent cells by brief centrifugation (3,000 rpm for 3 min), and resuspended in binding buffer (BD Biosciences) at a concentration of 1 × 106 cells/ml. About 100,000 cells were treated with FITC, annexin V, and PI according to the manufacturer's instructions. Annexin V is a 35- to 36-kDa Ca2+ dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, which is translocated from the inner to the outer leaflet of the plasma membrane in early apoptotic cells. Viable cells have intact membranes and thus exclude PI, while dead cells are permeable to it. Viable cells are both annexin V and PI negative, early apoptotic cells are annexin V positive but PI negative, and dead cells are both annexin V and PI positive. Cells were incubated at room temperature for 15 min before analysis using a FACSCantoII fluorescence-activated cell sorter (BD Biosciences). Frequencies of apoptosis were calculated as the number of apoptotic cells divided by the total number of cells, which is the sum of viable cells, apoptotic cells, and dead cells.
In the Vybrant apoptosis assay (Invitrogen), different densities of cells were plated in each well of 6-well plate on day −1. On days 0, 1, 2, 3, and 4, the inducers doxycyline and RSL1 were added for 5-, 4-, 3-, 2-, and 1-day treatments, respectively. On day 5, the medium was removed and the cells were washed once with 1 ml of phosphate-buffered saline (PBS). Hoechst 33342 and PI were then added according to the manufacturer's instructions. Cells were incubated on ice for 30 min before observation by fluorescence microscopy. Hoechst 33342 stains the condensed chromatin of apoptotic cells more brightly than the chromatin of normal cells. Thus, dead cells are red and bright blue cells, apoptotic cells are bright blue but not red cells, and viable cells are light blue. Frequencies of apoptosis were calculated as the number of apoptotic cells divided by the total number of cells, which is the sum of viable, apoptotic, and dead cells.
Cell cycle assays.
To analyze the distribution of cells in the cell cycle, 250,000 DIT7 cells were plated in each well of a 6-well plate on day −1. On day 0, cells were refed with medium and transcription inducers were added. On day 2, the distribution of cells in the cell cycle was determined using two methods. For the flow cytometry assay (BrdU [bromodeoxyuridine] flow kit; BD Biosciences), BrdU was added to the culture medium at a final concentration of 3 mg/ml 1 h before cells were collected. The cells were then treated according to the manufacturer's instructions. Briefly, cell pellets were washed once with 1 ml of staining buffer. Cells were fixed by resuspension in 100 μl of Cytofix/Cytoperm buffer and incubation at room temperature for 20 min. Cells were washed once with 1 ml of BD Perm/Wash buffer, resuspended in 100 μl of Cytofix/Cytoperm buffer, incubated for 5 min at room temperature, and washed again. To expose BrdU for antibody binding, cells were resuspended in 100 μl of diluted DNase (300 μg/ml), incubated at 37°C for 1 h, and then washed once. Cells were then resuspended in 50 μl of BD Perm/Washer buffer containing diluted (1:50) fluorescent anti-BrdU antibody and incubated at room temperature for 20 min. Cells were washed once with Perm/Wash buffer and resuspended in 1 ml binding buffer containing 20 μl of 7-amino-actinomycin D, which stains DNA. The suspensions were stored at 4°C overnight before analysis by flow cytometry using the FACSCantoII (BD Biosciences). As per the manufacturer's recommendation, cells with values of 110 or higher for BrdU incorporation in the cytometry plot (y axis) were defined as S-phase cells, cells with values higher than about 100 on the x axis, which represents the relative cellular DNA level, were defined as G2/M-phase cells, and cells with values lower than about 100 on the x axis were defined as G0/G1-phase cells.
For the cell cycle staining assay (BD Biosciences cell cycle kit), BrdU was added to the culture medium at a final concentration of 4 mg/ml and incubated at 37°C for 1 h. To fix cells, 250 μl of fixation buffer was added to 3 ml of the medium and incubated at room temperature for 15 min. Cells were washed twice with PBS, followed by incubation with 400 μl of cold Perm buffer III at room temperature for 10 min. Cells were washed twice with PBS and incubated with 400 μl of stain buffer at room temperature for 30 min. To expose BrdU for antibody binding, cells were incubated in 500 μl of PBS containing 0.3 mg/ml DNase at 37°C for 1 h, followed by one wash with PBS. Cells were then stained at room temperature for 15 min with Alexa Fluor 488 mouse anti-BrdU antibody (1:50) for BrdU, Alexa Fluor 647 rabbit anti-histone H3 (S28P) antibody (1:50) for histone H3, and Hoechst 33342 (0.02 mg/ml) for DNA. The conjugated antibody against BrdU generates green fluorescence; thus, cells at S phase are green. The conjugated antibody against histone H3 (S28P), which is specifically phosphorylated at M phase, generates red fluorescence; thus, M-phase cells are red. G-phase cells, which are not bound by antibody against BrdU or antibody against histone H3 (S28P), are blue due to Hoechst staining.
For immunofluorescence microscopy, we grew DIT7 cells on poly-l-lysine-coated coverslips, so that they were at about 50% confluence at the time of analysis. After treatment, we fixed cells in methanol for 20 min at −20°C and then rehydrated and washed them with PBS. Fixed cells were incubated with the specific primary antibody in PBS containing 2% goat serum for 16 h at 4°C and then incubated with the corresponding secondary antibody in PBS with 2% goat serum for 1 h at room temperature. Finally, cells were mounted with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Molecular Probes). The cells were observed by fluorescence microscopy (Nikon Eclipse TE2000-U). In this study, we used rabbit polyclonal antibodies against cleaved caspase 3 (Asp175) (1:50), ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related)-S428P (1:2,000), and CHK1-S345P (1:1,000) and mouse monoclonal antibodies against p53-S15P (1:100) and ATM-S1981P (1:5,000) (Cell Signaling Technologies). Secondary antibodies for immunostaining included Alexa Fluor 488 goat anti-rabbit IgG (1:1,000) or Alexa Fluor 569 goat anti-mouse IgG (1:1,000) (Molecular Probes).
Cells were counted in randomly selected fields on two to three coverslips for each of three different experimentally treated cell populations. For each coverslip, at least 1,000 total cells were evaluated and the results for multiple coverslips were summed. Percentages of positive cells were averaged for the three cell populations and displayed along with their standard deviations (SDs).
DIT7 cells were treated for 48 h in the absence or presence of doxycycline plus RSL1 and then chemically cross-linked by the addition of fresh formaldehyde solution to a final concentration of 1%, followed by gentle shaking at room temperature for 10 min. Cross-linking was stopped by addition of 2 M glycine after rinsing cells twice with cold PBS. Cells were collected using a silicon scraper and then lysed and sonicated to solubilize and shear cross-linked DNA using a Virsonic 600 sonicator. Sonication was carried out at 4°C at power setting 4 for 10 10-s pulses with 30-s pauses between pulses. The whole-cell extract was then precleared by addition of 50 μl protein A beads, 10 μl IgG, 10 μl 5% bovine serum albumin (BSA), and 5 g of sheared salmon sperm DNA and centrifuged at 13,000 rpm for 5 min at 4°C in a benchtop centrifuge. After centrifugation, the supernatant was incubated overnight at 4°C with 30 μl of protein A agarose beads, 3 μg of individual antibodies, 1 μl 5% BSA, and 25 μg of salmon sperm DNA. Beads were washed four times with chromatin immunoprecipitation (ChIP) buffer and once with Tris-EDTA (TE) containing 1 mM dithiothreitol. Bound complexes were eluted from the beads using elution buffer (0.5% SDS, 0.1% NaHCO3), and cross-linking was reversed by overnight incubation at 65°C. Immunoprecipitated DNA and whole-cell extract DNA were then purified using a Qiagen PCR purification kit (Qiagen). The antibodies used for the ChIP assay were directed against RNAP II (Abcam), ATR (Abcam), ATRIP (ATR-interacting protein) (Abcam), TOPBP1 (topoisomerase II binding protein 1) (Abcam), RPA70 (Cell Signaling Technologies), and nonspecific IgG antibodies (Sigma). Immunoprecipitated DNA was quantified by real-time PCR at two sites: ChIP-1, which is adjacent to the CAG repeat tract; and ChIP-2, which is about 1 kb away. The PCR product at ChIP-1 is 82 bp, and the product at ChIP-2 is 106 bp. Enrichment of ChIP-1 DNA in the specific antibody pulldown relative to the IgG pulldown under induced (doxycycline plus RSL1) versus uninduced conditions was calculated from the PCR results as follows: ChIP-1 = [(antibody induced/antibody uninduced)/(IgG induced/IgG uninduced)]. Enrichment of ChIP-2 DNA is calculated as ChIP-2 = [(antibody induced/antibody uninduced)/(IgG induced/IgG uninduced)].
Statistical analyses of significance were conducted using Student's t test to compare the means and standard deviations derived from multiple experiments.