Volume 35, Issue 3, Pages 327-339 (August 2009) Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage  Neil Johnson, Dongpo Cai, Richard D. Kennedy, Shailja Pathania, Mansi Arora, Yu-Chen Li, Alan D. D'Andrea, Jeffrey D. Parvin, Geoffrey I. Shapiro  Molecular Cell  Volume 35, Issue 3, Pages 327-339 (August 2009) DOI: 10.1016/j.molcel.2009.06.036 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Cdk1 Depletion Impairs S Phase Checkpoint Activation after DNA Damage (A) NCI-H1299 cells expressing shRNA targeting cdk1 were treated in the absence or presence of doxycycline prior to cisplatin (5 μg/ml) for the indicated times, fixed, and stained with propidium iodide. (B) Cells were grown as in (A), prior to IR (10 Gy). (Left) BrdU incorporation was measured 4 hr post-IR; the percent BrdU-positive cells is indicated for each condition. (Right) Percent of cells with S phase DNA content (determined by propidium iodide staining) that are BrdU positive or negative. (C) Cells were grown as in (A) and treated with cisplatin for 72 hr prior to collection for assessment of phosphohistone H3 staining by flow cytometry. (Left) Representative flow cytometry panels for cells treated with anti-FITC-conjugated phosphohistone H3 and counterstained with propidium iodide. (Right) Mean percent of phosphohistone H3-positive cells ± standard error (SE) over three experiments. (D) NCI-H1299 were treated as in (C) and subjected to a flow cytometry-based TUNEL assay. (Left) Representative flow cytometry panels for cells incorporating fluoresceinated dUTP in the presence of TdT, counterstained with propidium iodide. (Right) Mean percent of TUNEL-positive cells in G1, S, and G2/M ± SE over three experiments. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 After DNA Damage in Cdk1-Depleted Cells, Cdk2 Remains Active, and Phosphorylation of a Subset of ATR/ATM Substrates Is Compromised (A) NCI-H1299 cells inducibly expressing shRNA targeting cdk2 or cdk1 were grown with or without doxycycline and treated with 0, 2, or 5 μg/ml cisplatin for 24 hr prior to the preparation of lysates analyzed by western blotting. (B) (Left) Cells inducibly expressing shRNA targeting cdk1 were grown as in (A) and treated with vehicle or cisplatin. Lysates were subjected to mock or anti-cdk2 immunoprecipitation; immunoprecipitates were subjected to kinase assays using histone H1 as substrate and western blotting to monitor cdk2 recovery. (Right) Phosphorylated histone H1 was quantified with levels in vehicle-treated cells considered to represent 100% cdk2 activity. (C) (Left) Cells treated as in (B) were harvested and cyclin B1 immunoprecipitated. Cyclin B1-dependent kinase activity was measured by kinase assay using histone H1 as substrate and cyclin B1 and associated cdk1 and cdk2 levels measured by western blot. (Right) Quantification of phosphorylated histone H1, with levels in vehicle-treated cells considered to represent 100% kinase activity. (D) NCI-H1299 cells or A549 cells engineered to inducibly express shRNAs targeting cdk2 or cdk1 were treated as in (A), and lysates were analyzed by western blotting. (E) (Upper panel) Cells were grown as in (A) and left untreated or treated with 1 mM HU for 24 hr. (Lower panel) MCF7 and HeLa cells were treated with either scrambled (Sc) or cdk1 (K1) siRNA for 3 days. Cells were then left untreated or treated with 1 mM HU for 24 hr prior to the collection of lysates for western blotting. (F) NCI-H1299 cdk1 cells were grown as in (A) and left untreated or exposed to 10 Gy IR. Eight hours later, lysates were collected for western blotting. (G) Parental NCI-H1299 cells were treated with cisplatin for the indicated times and cell lysates analyzed by western blotting. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Cdk1 Depletion Does Not Affect DNA End Resection (A) NCI-H1299 cdk1 cells were grown in the absence or presence of doxycycline and treated with IR (10 Gy). Cells were fixed 6 hr post-IR and subjected to immunofluorescence and DAPI staining. (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least five phospho-ATM foci ± SE over three experiments. (B) Cells were treated as in (A). (Left) Representative foci-containing cells at 6 hr post-IR. (Right) Mean number of cells containing at least five BRCA1 or RPA32 foci at the indicated times post-IR ± SE over three experiments. (C) Cells were treated and fixed as in (A). (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least five ATR foci ± SE over three experiments. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Cdk1 Depletion Reduces BRCA1 Focus Formation (A) NCI-H1299 cdk1 cells were treated with cisplatin (5 μg/ml for 24 hr) and subjected to immunofluorescence and DAPI staining. (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least five ATR foci ± SE over three experiments. (B) Cells were grown and treated as in (A) and stained for BRCA1 (FITC), γ-H2AX (Texas Red), and DAPI. (Left) Representative foci-containing cells. (Right) Mean number of cells with at least five BRCA1-containing foci ± SE over three experiments. ∗p < 0.05 in the presence compared to the absence of doxycycline. (C) NCI-H1299 cdk1 cells were grown as in (A) and treated with vehicle (−) or cisplatin (+) for 24 hr, after which cells were fractionated and soluble (S) nuclear and chromatin fractions (pellet, P) isolated and subjected to western blotting with the indicated antibodies. (D) Parental NCI-H1299 cells were treated with DMSO or 2 μM RO-3306 either with vehicle or 5 μg/ml cisplatin for 24 hr, fixed, and stained with propidium iodide. (E) Parental NCI-H1299 cells were treated with DMSO (0 μM) or RO-3306 at the indicated concentrations and where indicated with 5 μg/ml cisplatin. Lysates were subjected to western blotting with the indicated antibodies. (F) Parental NCI-H1299 cells were treated as in (A) and stained with anti-BRCA1 antibody (FITC) or DAPI. Representative cells containing foci are shown. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Cdk1 Phosphorylates BRCA1 at S1497 and S1189/1191 and Promotes Formation of Foci (A) NCI-H1299 cdk1 cells were grown in the absence or presence of doxycycline and treated with vehicle or 5 μg/ml cisplatin. Whole-cell lysates were subjected to immunoprecipitation for BRCA1 followed by western blotting with the indicated antibodies. (B) Recombinant BRCA1-BARD1 protein was incubated with (+) or without (−) recombinant cyclin B1-cdk1 and kinase assay performed. (C) Overlapping GST-tagged BRCA1 fragments F1-6 were incubated with recombinant cyclin B1-cdk1 and phosphorylation assessed by kinase assay. Arrows indicate molecular weight and location of fragments. (D) Wild-type, S1497A, T1658N, and combined S1497A/T1658N double mutant (DM) GST-tagged F6 fragments were incubated with recombinant cyclin B1-cdk1 and kinase assay performed. Western blotting demonstrated equivalent amount of the F6 substrates in the respective kinase assays. (E) NCI-H1299 cdk1 cells were treated as in (A), and lysates were subjected to immunoprecipitation with anti-phospho BRCA1 pS1497 followed by western blotting with anti-BRCA1 antibody. (F) Wild-type, S1189A/S1191A GST-tagged F5 fragments were incubated with recombinant cyclin B1-cdk1 and kinase assay performed. Coomassie blue staining of protein demonstrated equivalent amount of the F5 substrates in the respective kinase assays. (G) Parental MBA-MB-436 cells or those expressing the indicated construct were left untreated or treated with 10 Gy IR. Six hours post-IR, cells were fixed and stained for BRCA1 (FITC), RPA32 (Texas Red), and DAPI. (Left) Representative images. (Right) Mean number of cells with at least five foci ± SE over three experiments. ∗p < 0.05 in the BRCA1 triple mutant compared to wild-type-expressing cells. In a separate experiment, ATR foci were also quantified. (H) Cells were treated as in (G) and lysates prepared at the indicated times and subjected to western blotting. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 Cdk1 Depletion Sensitizes Cells to DNA Damage (A) Western blots demonstrate NCI-H1299 engineered to inducibly express shRNA targeting cdk1 or cdk2. (Upper graphs) Cells were assessed for colony formation after vehicle or cisplatin treatment in the absence or presence of doxycycline. Mean survival from three experiments is expressed as a percentage of colonies formed ± SE relative to vehicle-treated cells in the absence (solid lines) or presence (broken lines) of doxycycline. (Lower panels and graphs) Representative plates are shown and mean survival is graphed after vehicle or 0.25 μg/ml cisplatin exposure, expressed as a percentage of colonies formed ± SE compared to vehicle-treated cells in the absence of doxycycline. (B) Similar experiments in A549 cells. (C) NCI-H1299 cdk1 cells were grown in the absence or presence of doxycycline and concomitantly transfected with either scrambled or BRCA1 siRNA for 3 days. Cells were subsequently treated with vehicle or 0.25 μg/ml cisplatin for 24 hr and replated for colony formation. Survival is expressed as a percentage of colonies formed ± SE compared to the corresponding vehicle-treated control over three experiments. (D) MDA-MB-436 cells as in Figure 5G were treated with vehicle or the indicated concentrations of cisplatin for 24 hr, replated for colony formation, and assessed 2 weeks later. Mean survival from three experiments is expressed as a percentage of colonies formed ± SE relative to vehicle-treated cells. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 7 Cdk1 Depletion or Inhibition Protects Nontransformed Cells from S Phase-Specific DNA-Damaging Agents (A) (Left) RPE and NCI-1299 cells were treated with cdk1 siRNA and cell-cycle profiles measured at the indicated times; the percentage of cells with G2/M DNA content is shown. (Right) Lysates were collected after siRNA transfection for 3 days to confirm knockdown of cdk1 expression by western blot. (B) RPE and NCI-1299 cells were treated with scrambled (Sc) or cdk1 (K1) siRNA and cell growth measured. Cell number was counted every day starting at day 0 (3 days posttransfection). (C) RPE and NCI-1299 cells were treated with scrambled or siRNA for 3 days prior to treatment with cisplatin for 24 hr followed by replating for assessment of colony formation after 14 days. Mean survival from three experiments is expressed as the percentage of colonies formed ± SE relative to vehicle-treated cells after treatment with scrambled siRNA (solid lines) or cdk1 siRNA (broken lines). (D) RPE and NCI-1299 cells treated as in (C). Bar graph shows cells treated with 0.5 μg/ml cisplatin and colony formation expressed as a percentage of the relative scrambled treated control. (E) Parental NCI-H1299, MCF7, and RPE cells were treated with 0.25 μg/ml cisplatin either alone (cis) or in combination with 2 μM RO-3306 (cis/RO) for 24 hr, replated, and assessed for colony formation after 14 days. Results represent the mean percentage ± SE of colonies formed relative to vehicle-treated cells over three experiments. Molecular Cell 2009 35, 327-339DOI: (10.1016/j.molcel.2009.06.036) Copyright © 2009 Elsevier Inc. Terms and Conditions