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. 2000 Oct;20(20):7751-63.
doi: 10.1128/MCB.20.20.7751-7763.2000.

RB-dependent S-phase response to DNA damage

K E Knudsen  1 , D Booth, S Naderi, Z Sever-Chroneos, A F Fribourg, I C Hunton, J R Feramisco, J Y Wang, E S Knudsen
Affiliations

RB-dependent S-phase response to DNA damage

K E Knudsen et al. Mol Cell Biol. 2000 Oct.

Abstract

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.

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Figures

FIG. 1
FIG. 1
CDDP inhibits DNA synthesis and induces RB dephosphorylation in S-phase cells. (a) Rat-1 cells were arrested in Aph (2 μg/ml, 24 h) and then treated with CDDP (50 μM) or vehicle for an additional 3 h. Cells were washed extensively to remove Aph and CDDP and then collected at 2, 4, or 6 h after washing. The DNA content of each population of cells was determined by flow cytometry. Pretreatment with CDDP blocked S-phase progression in Rat-1 cells. (b) Rat-1 cells were arrested in Aph (2 μg/ml, 24 h) and then treated with CDDP (50 μM) or vehicle for an additional 3 h. Cells were washed extensively to remove Aph and CDDP and then labeled with BrdU for 3 h. The cells were then fixed and reacted with anti-BrdU antibody. The percentage of cells that incorporated BrdU was determined by counting at least 100 cells per experiment. Data shown are means and standard deviations from three independent experiments. Representative fields of BrdU-labeled cells are shown. (c) The status of RB phosphorylation in Rat-1 cells that were serum starved for 72 h (Qui; lane 1), asynchronously growing (Asy; lane 2), Aph arrested (2 μg/ml, 24 h) (APH; lane 3), or Aph arrested and CDDP treated (50 μM, 3 h) (CDDP + APH; lane 4) was examined by immunoprecipitation of total cell lysate with anti-RB 851 followed by immunoblotting with anti-RB 851.
FIG. 2
FIG. 2
Inhibition of DNA synthesis by CDDP requires RB. MEFs were prepared by mating Rb-heterozygous mice and harvesting embryos at day 10 to 12 of gestation. The genotype of each embryo was determined by PCR of head DNA. Rb+/+ or Rb−/− MEFs from the same litter were used between passages 2 and 6. (a) Rb+/+ and Rb−/− MEFs were cultured in 0.1% serum for 72 h. (Left) Quiescent cells were stimulated with 10% FBS for 26 h (10%) or for only 30 min and then transferred into 0% FBS (0%) and labeled with BrdU. The stained cells were counted (see Materials and Methods), and the percentage of BrdU-positive cells in 10% FBS was set at 100. (Right) In parallel, quiescent cells were treated with the indicated concentration of CDDP for 16 h and then stimulated with 10% FBS and labeled with BrdU for 26 h. The BrdU-positive cells were counted, and the value was normalized to the percentage of BrdU-positive cells in untreated cells, which was set to 100%. (b) (Left) Rb+/+ or Rb−/− MEFs were arrested in early S phase by Aph as described in Materials and Methods. The Aph-arrested cells were beyond the G1 restriction point, as shown by a serum-independent DNA synthesis following release from the Aph block. After the Aph was washed away, BrdU was added to cells with the indicated concentration of FBS for 4 h. BrdU-positive cells were counted, and the percentage of BrdU-positive cells in 10% FBS was set as 100. (Right) Aph-arrested S-phase cells were treated with the indicated concentrations of CDDP for 16 h. Cells were then washed extensively to remove both Aph and CDDP and labeled with BrdU in 10% FBS for 4 h. The BrdU-positive cells were counted, and the value was normalized to that of CDDP-untreated culture, which was set to 100%. (c) MEFs of either Rb+/+ or Rb−/− genotype were synchronized in S phase by sequential serum stimulation from quiescence for 16 h followed by incubation in HU (1 mM) for 10 h. The S-phase cells were then subjected to either 0 or 32 μM CDDP for 16 h and washed extensively in medium for release from the HU block. BrdU was added to the medium, and cells were labeled for 4 h to monitor progression through S phase. Cells were fixed and stained for the incorporation of BrdU. Data shown are relative percentages of cells staining positive for BrdU incorporation from two independent experiments. (d) Rb+/+ or Rb−/− MEFs were synchronized in S phase using Aph, treated with 0 to 32 μM CDDP for 16 h, and then washed for release for 4 h. Cells were fixed and subjected to staining with the ICR4 antibody, which detects platinated DNA. Representative pictures shown were captured with identical exposure times.
FIG. 3
FIG. 3
Dephosphorylation of RB in S phase precedes the inhibition of DNA replication. (a) Lysates were prepared from either asynchronously growing Rb−/− 3T3 cells (lane 1) or Rb+/+ MEFs which were synchronized in either quiescence (Qui; lane 2) or Aph (lane 3) and then treated with vehicle (lane 4) or treated with 32 μM CDDP (lane 5) for 12 h. RB was recovered from the lysates by immunoprecipitation, resolved by SDS-PAGE, and detected by immunoblotting. (b) Lysates were prepared from either asynchronously growing Rb−/− 3T3 cells (lane 1) or Rb+/+ MEFs that were synchronized in quiescence (lane 2) or S phase using Aph (lane 3) and treated with 32 μM CDDP for 0 (lane 4), 4 (lane 5), 8 (lane 6), or 12 (lane 7) h. RB was recovered from the lysates by immunoprecipitation, resolved by SDS-PAGE, and detected by immunoblotting. (c) Cells cultured as described for panel b were released from the Aph block, and BrdU incorporation was determined from a 4-h labeling. Data shown are relative percentages of BrdU-positive cells from two independent experiments.
FIG. 4
FIG. 4
p21-defective cells exhibit a partial response to CDDP. (a) Aph-blocked wild-type (top), Rb−/− (middle), and p21Cip1−/− (bottom) MEFs were subjected to increasing doses (0 to 40 μM) of CDDP for 16 h. Cells were released from Aph and labeled with BrdU for 4 h. The relative percent BrdU incorporation shown is the average from one to three experiments. (b) Lysates were prepared from either asynchronous Rb−/− 3T3 cells (lane 1) or p21Cip−/− MEFs that were synchronized in quiescence (lane 2), blocked in Aph (lane 3), and treated with 32 μM CDDP for 0 (lane 4), 4 (lane 5), 8 (lane 6), 12 (lane 7), or 16 (lane 8) h. RB was recovered using immunoprecipitation, resolved by SDS-PAGE, and detected by immunoblotting. (c) Lysates were prepared from Rb+/+ MEFs that were synchronized with Aph (lane 1) and treated with 32 μM CDDP for 16 h (lane 2). Equal amounts of protein were resolved by SDS-PAGE, and p21Cip1 was detected by immunoblotting. (d) Lysates were prepared from Rb+/+ and p21Cip−/− MEFs that were synchronized in quiescence (lanes 1 and 4), blocked in Aph (lanes 2 and 5), and treated with 32 μM CDDP for 16 h (lanes 3 and 6). Cdk2 complexes were recovered by immunoprecipitation and analyzed in in vitro kinase assays using histone H1 as a substrate. Kinase activity was detected by autoradiography, and the immunoprecipitated Cdk2 was detected by immunoblotting.
FIG. 5
FIG. 5
CDDP inhibits DNA synthesis in mid-S-phase cells. (a) Asynchronously proliferating Rb+/+ MEFs were subjected to vehicle (lanes 1, 3, 5, and 7) or 64 μM CDDP (lanes 2, 4, 6, and 8) for the indicated time. Cells were harvested, lysates were prepared, and RB was immunoprecipitated, resolved by SDS-PAGE, and detected by immunoblotting. (b) Asynchronously growing Rb+/+ MEFs were treated with CDDP for the indicated times (panel 1, no BrdU; panel 2, 0 h; panel 3, 6 h; panel 4, 8 h) with BrdU pulse-labeling during the final hour of treatment. Cells were fixed and processed for BrdU incorporation to detect DNA synthesis and stained with propidium iodide (PI) to determine cell cycle phase by flow cytometry. Shown are representative scatter plots with the log fluorescein isothiocyanate (FITC) anti-BrdU staining versus propidium iodide staining. (c) Asynchronously growing Rb+/+ (panels 1 and 2) or Rb−/− (panels 3 and 4) MEFs were treated with vehicle (panels 1 and 3) or 64 μM CDDP (panels 2 and 4) for 8 h with BrdU pulse-labeling during the final hour of treatment. Cells were fixed and processed for BrdU incorporation to detect DNA synthesis and stained with propidium iodide to determine cell cycle phase by flow cytometry. Shown are representative scatter plots with the log FITC anti-BrdU staining versus propidium iodide staining. (d) The percentage of S-phase cells (greater than 2N but less than 4N DNA content) exhibiting DNA synthesis (BrdU positive) was determined. Data shown are from three independent experiments. (e) Asynchronously growing wild-type and Rb−/− MEFs were treated with either IR (50 Gy), 5 μM etoposide, or 4 μg of mitomycin C (MMC) per ml. Following treatment, cells were pulsed with BrdU for 1 h and subsequently fixed and processed for bivariate flow cytometry. The percentage of S-phase cells (with DNA content between 2N and 4N) that incorporated BrdU was determined.
FIG. 5
FIG. 5
CDDP inhibits DNA synthesis in mid-S-phase cells. (a) Asynchronously proliferating Rb+/+ MEFs were subjected to vehicle (lanes 1, 3, 5, and 7) or 64 μM CDDP (lanes 2, 4, 6, and 8) for the indicated time. Cells were harvested, lysates were prepared, and RB was immunoprecipitated, resolved by SDS-PAGE, and detected by immunoblotting. (b) Asynchronously growing Rb+/+ MEFs were treated with CDDP for the indicated times (panel 1, no BrdU; panel 2, 0 h; panel 3, 6 h; panel 4, 8 h) with BrdU pulse-labeling during the final hour of treatment. Cells were fixed and processed for BrdU incorporation to detect DNA synthesis and stained with propidium iodide (PI) to determine cell cycle phase by flow cytometry. Shown are representative scatter plots with the log fluorescein isothiocyanate (FITC) anti-BrdU staining versus propidium iodide staining. (c) Asynchronously growing Rb+/+ (panels 1 and 2) or Rb−/− (panels 3 and 4) MEFs were treated with vehicle (panels 1 and 3) or 64 μM CDDP (panels 2 and 4) for 8 h with BrdU pulse-labeling during the final hour of treatment. Cells were fixed and processed for BrdU incorporation to detect DNA synthesis and stained with propidium iodide to determine cell cycle phase by flow cytometry. Shown are representative scatter plots with the log FITC anti-BrdU staining versus propidium iodide staining. (d) The percentage of S-phase cells (greater than 2N but less than 4N DNA content) exhibiting DNA synthesis (BrdU positive) was determined. Data shown are from three independent experiments. (e) Asynchronously growing wild-type and Rb−/− MEFs were treated with either IR (50 Gy), 5 μM etoposide, or 4 μg of mitomycin C (MMC) per ml. Following treatment, cells were pulsed with BrdU for 1 h and subsequently fixed and processed for bivariate flow cytometry. The percentage of S-phase cells (with DNA content between 2N and 4N) that incorporated BrdU was determined.
FIG. 6
FIG. 6
Active RB is sufficient to block recovery from S-phase block. (a) Rb−/− MEFs were synchronized in S phase by sequential serum stimulation from quiescence for 16 h followed by incubation in Aph for 10 h. These S-phase cells were then microinjected with the indicated plasmids (50 ng/μl) and a plasmid encoding nuclear GFP (50 ng/μl). After 16 h (to allow for expression of the plasmid-encoded protein), the cells were washed extensively in medium for release from Aph block. BrdU was added to the medium, and cells were labeled for 4 h to monitor progression through S phase. Cells were fixed and stained for the incorporation of BrdU. Representative images are shown. (b) Data shown are percentages of GFP-positive cells staining positive for BrdU incorporation from three independent experiments with more than 40 injected cells per experiment. Control reflects the percentage of uninjected cells staining positively for BrdU incorporation from the same coverslips.
FIG. 7
FIG. 7
RB-dependent S-phase arrest correlates with inhibition of cyclin A but not E2F. (a) Rb+/+ and Rb−/− MEFs were cotransfected with the 3×E2FLUC and CMV-betagal reporter constructs. Transfected cells were synchronized posttransfection with Aph and treated with 0 or 32 μM CDDP. Cells were harvested after 16 h of treatment, and reporter assays were performed. Data shown are averages of at least four independent experiments. (b) Asynchronously growing Rb+/+ MEFs were treated with 0 or 64 μM CDDP for 8 h; TSA (200 ng/ml) or vehicle was also added for the indicated times. BrdU was added to the cells for the last hour of treatment, and the incorporation of BrdU was determined by staining with anti-BrDU antibody followed by FACS analysis (Materials and Methods). The percent of S-phase cells (with DNA content between 2N and 4N) that incorporated BrdU was determined and shown for each of the experimental time points. (c) Rb+/+ and Rb−/− MEFs were synchronized in Aph and treated with 0 or 32 μM CDDP for 16 h. Lysates were harvested, equal amounts of protein were resolved by SDS-PAGE, and cyclin A, cyclin E, and Cdk2 were detected by immunoblotting.
FIG. 8
FIG. 8
RB prevents cell death following DNA damage. (a) Rb+/+ MEFs either asynchronously proliferating (Asy; panel 1) or treated with 32 μM CDDP for 16 h and then cultured for a total 36 (panel 2) or 144 (panel 3) h after CDDP addition were harvested, fixed, and stained with propidium iodide. Shown are representative histograms of two independent experiments. (b) Rb+/+ or Rb−/− MEFs were treated with 0, 16, or 32 μM CDDP. After 16 h of treatment, CDDP was washed from the cells. The cells were then cultured for the indicated times after CDDP addition, at which point they were fixed and stained with crystal violet (left panel). The relative number of cells was determined by counting random fields of crystal violet stained dishes (right panels). The data are from duplicate plates and representative of three independent experiments.
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