Cdk1 Participates in BRCA1-Dependent S Phase Checkpoint Control in Response to DNA Damage

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NIH Public Access Author Manuscript Mol Cell. Author manuscript; available in PMC 2011 January 20.

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Published in final edited form as: Mol Cell. 2009 August 14; 35(3): 327–339. doi:10.1016/j.molcel.2009.06.036.

CDK1 PARTICIPATES IN BRCA1-DEPENDENT S PHASE CHECKPOINT CONTROL IN RESPONSE TO DNA DAMAGE Neil Johnson1, Dongpo Cai1, Richard D. Kennedy2,#, Shailja Pathania3, Mansi Arora4, YuChen Li1, Alan D. D’Andrea5, Jeffrey D. Parvin4, and Geoffrey I. Shapiro1,6,* 1 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA 2

Department of Radiation Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA

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Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA

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Department of Biomedical Informatics and the Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA

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Department of Pediatrics, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02115, USA 6

Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA

SUMMARY

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Cdk2 and cdk1 are individually dispensable for cell cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small cell lung cancer cells sensitized them to DNA damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in non-transformed cells, and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1 proficient cancer cells to DNA damaging treatments by disrupting BRCA1 function.

INTRODUCTION Cell cycle progression, controlled by cyclin-dependent kinases (cdks), is a tightly regulated process in eukaryotic cells. Genomic integrity is maintained through the precise activation of cdks and the correctly timed coordination of DNA synthesis. Cdk2 and cdk1 together direct S and G2 phase transit, while cdk1 governs the G2/M transition and mitotic progression. Individual shRNA-mediated depletion of these cdks from transformed cells indicates that they can easily compensate for one another (Cai et al., 2006b; L’Italien et al., 2006). A

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Correspondence: [email protected]. #Present address: Almac Diagnostics Ltd., Craigavon, BT63 5QD, UK Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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decrease in cyclin A-cdk2 complexes after cdk2 depletion results in an increase in cyclin Acdk1 complex formation. Similarly, after cdk1 depletion, cyclin B-cdk2 complexes are formed; although G2 progression is slowed, cells continue to proliferate. Therefore, in a variety of cancer cell types, both S phase delay and G2 arrest require combined cdk2 and cdk1 depletion.

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Exposure to genotoxic insults results in the activation of checkpoint cascades which downregulate cdk activities and impose cell cycle arrest that prevents the propagation of damaged DNA. Delay of cell cycle progression is initiated by DNA damage-induced activation of the phosphatidylinositol 3-kinase-like protein kinases ATM (ataxiatelangiectasia mutated) and ATR (ATM and Rad3-related) (Abraham, 2001). ATR is the primary sensor of events that cause replication stress, such as stalling of replication forks or formation of single strand DNA breaks (SSB). ATR is recruited to sites of single-stranded DNA coated with replication protein A (RPA) via ATR-interacting protein (ATRIP), and phosphorylates substrates that mediate checkpoint control and DNA repair, including the checkpoint kinase Chk1. ATM is recruited to double stranded DNA breaks (DSB) induced by ionizing radiation (IR) by the Mre11-Rad50-Nbs1 (MRN) complex, and upon activation, also phosphorylates multiple substrates, including Chk2. Additionally, during the S and G2 phases, the processing of double-strand breaks by end resection generates RPA-coated single-stranded DNA, resulting in ATM-dependent ATR activation (Jazayeri et al., 2006). Activated Chk1 and Chk2 antagonize the function of Cdc25 phosphatases, allowing accumulation of inhibitory phosphates on Thr14 and Tyr15 of cdks, resulting in inhibition of cdk activity and delayed cell cycle progression (Bartek and Lukas, 2003), providing time for DNA repair. BRCA1 is a critical component of ATM and ATR-mediated checkpoint signaling and is hyperphosphorylated by ATM and ATR in response to DNA damage. BRCA1 serves as a scaffold that facilitates the ability of ATM/ATR to phosphorylate a subset of substrates including Chk1 and Chk2 (Foray et al., 2003; Yarden et al., 2002). However, BRCA1 is dispensable for the phosphorylation of chromatin-associated substrates, including H2AX and Rad17. Nonetheless, reduced activation of Chk1 and Chk2 cause BRCA1-deficient cells to display aberrant checkpoint control, resulting in failure to inhibit cdk activity and cell cycle progression after DNA damage. Coupled with compromised repair by homologous recombination, this accounts for their favorable response to DNA damaging and crosslinking agents (Quinn et al., 2003).

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Several studies have implicated cdk2 or cdk1 not just as the terminal targets of checkpoint signaling cascades, when their kinase activity is inhibited, but also in the initiation of checkpoint signaling events. For example, treatment with the cdk inhibitor roscovitine has been shown to compromise Chk1 phosphorylation following IR during the S and G2 phases (Deans et al., 2006; Jazayeri et al., 2006). There is also precedence in budding yeast, where cdk1 (cdc28) is required for activation of DNA end resection and ultimately the Mec1 (ATR homolog)-dependent DNA damage checkpoint following a DSB (Ira et al., 2004). However, it has not known whether cdk1-mediated control of end resection is conserved in mammalian cells, or whether there is a role for cdk1 in the initiation of checkpoint cascades at sites of ssDNA generated in response to agents that cause stalled replication forks or otherwise directly activate ATR. Although cdk2 and cdk1 can compensate for one another during cell cycle progression, allowing cells depleted of a single cdk to proliferate, it is unclear whether they play nonoverlapping roles during DNA damage-induced checkpoint control. Here, we demonstrate that selective cdk1 depletion causes abrogation of S phase checkpoint cell cycle arrest in non-small cell lung cancer (NSCLC) cells in response to both cisplatin and IR. Cdk1 is

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required for the efficient recruitment of BRCA1 to sites of DNA damage, so that compromise of its activity results in the failure of ATM and ATR to phosphorylate a subset of substrates. Consequently, cancer cells depleted of cdk1 are sensitized to DNA damaging treatments.

RESULTS Cdk1 Depletion Abrogates S Phase Checkpoint Activation

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To determine if individual depletion of either cdk2 or cdk1 would affect checkpoint control in response to DNA damaging agents, we utilized NCI-H1299 NSCLC cell lines engineered to express shRNA targeting either cdk2 or cdk1 upon doxycycline induction (Cai et al., 2006b), and assessed the degree of S phase cell cycle arrest after exposure to cisplatin or IR in the absence or presence of doxycycline. In the absence of doxycycline in both sets of cells, cisplatin treatment for 24 hours caused cells to accumulate in S phase (Figures 1A and S1). Cdk2 depletion did not affect the degree of S phase accumulation (Figure S1). In contrast, S phase accumulation was markedly reduced when cdk1 was depleted (Figure 1A). To further assess the degree of S phase arrest after cdk1 depletion, cells were exposed to IR and BrdU incorporation was measured 4 hours later (Figure 1B). In the absence of doxycycline, there was an overall accumulation of cells with S phase DNA content, but a marked decrease in the percentage of cells incorporating BrdU and undergoing DNA synthesis. In contrast, when cdk1 was depleted, cells continued to synthesize DNA and incorporate BrdU, despite IR treatment. There was also a reduction in the overall accumulation of cells with S phase DNA content, compared to cells expressing normal levels of cdk1. Similarly, when BrdU incorporation was measured by immunofluorescence, there was a marked reduction in the number of BrdU positive cells after IR in the absence of doxycycline, and no reduction in the number of BrdU positive cells in the presence of doxycycline (Figure S2). Therefore, the S phase checkpoint is not properly activated when cdk1 levels are reduced.

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In the absence of doxycycline, continued exposure to cisplatin for an additional 24 or 48 hours (48 and 72-hour time points) resulted in accumulation of cells in G2/M (Figure 1A). Cells depleted of cdk1 had reduced accumulation in G2/M at 48 and 72 hours, with increased sub-G1 DNA content, suggestive of cell death. To determine whether the reduced G2/M content observed in the absence of cdk1 was related to abrogation of the G2 checkpoint, we assessed levels of the mitotic marker phospho-histone H3 in vehicle and cisplatin-treated cells in the absence or presence of doxycycline (Figure 1C). In the absence of doxycycline, a small percentage of NCI-H1299 cells stained positively for phosphohistone H3, which increased slightly after exposure to cisplatin for 72 hours. Therefore, at this time point after cisplatin exposure, NCI-H1299 cells are predominantly arrested at G2, with limited G2 checkpoint abrogation. In the presence of doxycycline, baseline levels of phospho-histone H3 were lower, consistent with the small degree of G2 accumulation caused by cdk1 depletion in these cells (Cai et al., 2006b). Staining for phospho-histone H3 did not increase after cisplatin exposure, suggesting that cdk1 depletion did not afford G2 checkpoint escape into mitosis after DNA damage. Rather, it is more likely that cells are dying from the S and G2 phases. To assess this possibility, we performed concomitant TUNEL and propidium iodide staining by flow cytometry. As shown in Figure 1D, in the absence of doxycycline, continuous cisplatin treatment for 72 hours produced a small percentage of TUNEL-positive cells (~9%). In contrast, the degree of TUNEL-positivity in cdk1-depleted was substantially greater (~35%), with the majority of dying cells demonstrating G2 and S-phase DNA content. In summary, cdk1 depletion abrogates the S phase DNA damage checkpoint, resulting in the death of cells from the G2 and S phases. Mol Cell. Author manuscript; available in PMC 2011 January 20.

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Cdk1-Depleted Cells Maintain High Cdk2 Activity in the Presence of DNA Damage

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Having established that cdk1-depleted cells fail to activate the S phase checkpoint after DNA damage, we investigated how cell cycle progression is driven under these conditions. After cdk1 depletion, cyclin B1-cdk1 complexes are replaced by cyclin B1-cdk2 complexes that presumably mediate S and G2/M progression in undamaged cells (Cai et al., 2006b; L’Italien et al., 2006). We therefore examined the effects of cdk1 depletion on cdk2 activity in NCI-H1299 cells that had been exposed to cisplatin. We first measured the levels of cdk1 and cdk2 phosphorylated at Tyr15, indicative of cdks inhibited after DNA damage because of cytoplasmic sequestration or degradation of cdc25 phosphatases. As shown in Figure 2A, in the absence of doxycycline, both Tyr15-phosphorylated cdk2 and cdk1 accumulate in response to cisplatin treatment. After depletion of cdk2, Tyr15 phosphorylation of cdk1 increased to the same degree as in cells in which cdk2 levels were intact (Figure 2A, lefthand panel). However, in the absence of cdk1, phosphorylation of cdk2 at Tyr15 was compromised, indicating cdk2 activity was not reduced after DNA damage treatment (Figure 2A, right-hand panel). To directly measure cdk2 activity after cisplatin treatment, immunoprecipitated cdk2 kinase assays were performed. Consistent with the levels of pTyr15-cdk2 after cisplatin treatment, in the absence of doxycycline, there was a 2.5 fold reduction in cdk2 kinase activity. However, in the presence of doxycycline, after cdk1 depletion, cdk2 activity was only reduced 1.1-fold (Figure 2B). Similar results were obtained when immunoprecipitated cyclin B1-kinase activity was assayed (Figure 2C). In the absence of doxycycline, cyclin B1 was found only in complex with cdk1, and with cdk1 levels replete, there was a 2.3 fold reduction in cyclin B1-dependent kinase activity in cisplatin-treated cells. In contrast, in cdk1-depleted cells, cyclin B1 was found to coimmunoprecipitate with cdk2. Although cyclin B1-cdk2 complexes had reduced kinase activity compared to cyclin B1-cdk1 complexes in untreated conditions (1.3 fold reduction), cisplatin did not reduce cyclin B1-cdk2 kinase activity to the same degree (1.2 fold reduction) as cyclin B1-cdk1 in the absence of doxycycline. Taken together with the cell cycle analyses shown in Figure 1, our results indicate that in the absence of cdk1, compromised S phase arrest results from a failure to inhibit cdk2, in complex with either cyclin A or cyclin B. Cdk1 Depletion Compromises the Phosphorylation of ATM/ATR Substrates

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To investigate checkpoint signaling further, we measured the levels of proteins that mediate the DNA damage response. Cdk1 depletion did not affect the protein levels of ATM (not shown), ATR or BRCA1 (Figure 2D). We also assessed levels of phosphorylated ATM/ ATR substrates after cisplatin treatment in NCI-H1299 cells inducibly depleted of either cdk2 or cdk1. In the absence of doxycycline, cisplatin treatment at 2 or 5 μg/ml results in phosphorylation of Chk1 and RPA32. Cisplatin also caused accumulation of the higher molecular weight, ubiquitinated form of FANCD2 and hyperphosphorylated BRCA1. Levels of phosphorylated Chk2 and γ-H2AX also increased with cisplatin treatment. Depletion of cdk2 did not affect the levels of these phosphorylated proteins after exposure to cisplatin. In contrast, after cdk1 depletion, levels of phosphorylated Chk1 and RPA32, as well as of ubiquitinated FANCD2, were compromised, while phosphorylation of BRCA1, Chk2, Rad17 and H2AX remained intact. Similar effects of cdk1 depletion were also seen in A549 cells after cisplatin treatment (Figure 2D), as well as in both cell lines after hydroxyurea (HU) treatment (Figure 2E, upper panels). Furthermore, MCF7 and HeLa cells subjected to siRNA-mediated cdk1 depletion and HU treatment also had reduced Chk1 and RPA32 phosphorylation and FANCD2 ubiquitination (Figure 2E, lower panels). In addition to cisplatin and hydroxyurea, which primarily induce SSB’s and activate ATR, we examined substrate phosphorylation after DSB formation resulting from IR treatment, during which ATM is primarily activated. Treatment with IR resulted in phosphorylation of Chk1, Chk2, SMC1 and H2AX in NCI-H1299 cells with normal cdk levels and in cdk2-depleted cells Mol Cell. Author manuscript; available in PMC 2011 January 20.

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(Figure 2F, left hand panel). However, cdk1 depletion resulted in marked reduction in phosphorylation of Chk1, Chk2 and SMC1 but did not affect H2AX phosphorylation (Figure 2F, right hand panel). These data indicate that in a variety of cellular backgrounds, cdk1 is required for ATM and ATR to efficiently phosphorylate a subset of their substrates, whereas other substrates, including Rad17 and H2AX, can still be phosphorylated. Our data suggest that cdk1 activity is required to initiate checkpoint signaling, so that its activity is not downregulated until relatively late after DNA damage exposure. We therefore measured levels of phosphorylated Chk1, γ-H2AX and Tyr-15-cdk1 in parental NCI-H1299 cells treated with cisplatin in a time-course experiment (Figure 2G). There was an increase in the level of phosphorylated Chk1 levels by 8 hours after cisplatin treatment, and an increase in the level of γ-H2AX by 12 hours. It was not until 16 hours after cisplatin treatment that an increase in the levels of phosphorylated Tyr15-cdk1 could be detected (Figure 2G). Thus, cdk1 remained active when initial signaling events required for the DNA damage response were taking place. DNA End Resection is Not Compromised by Selective Cdk1 Depletion or Inhibition

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During DNA damage checkpoint activation in Saccharomyces cerevisiae, cdk1 (cdc28) is required for resection of DSB ends and for the recruitment of the single-stranded DNA binding complex, RPA (Ira et al., 2004). RPA binding is essential for the association and activation of the ATR homologue, Mec1. To determine if cdk1 plays a similar role in mammalian cells, we examined the effect of cdk1 depletion on ATM, RPA32 and ATR focus formation after IR treatment in NCI-H1299 cells. As shown in Figure 3, cdk1 depletion did not affect these processes. Phosphorylated ATM is efficiently recruited to sites of DNA damage (Figure 3A), and is followed by end resection, as evidenced by the formation of RPA32 foci (Sartori et al., 2007). These are first detected 4 hours post-IR and maximally detected at 6 hours. The time course of RPA32 recruitment was not affected in the absence of cdk1 (Figure 3B). Furthermore, ATR recruitment to sites of damage was also unaffected by cdk1 depletion (Figure 3C), in both BrdU-positive and negative cells (Figure S3).

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Previous work in mammalian cells indicated that roscovitine treatment compromises RPA focus formation (Jazayeri et al., 2006). Since roscovitine inhibits both cdk2 and cdk1 (McClue et al., 2002), it is likely that both cdks contribute to DNA end resection. We investigated this further by analyzing RPA32 focus formation after treatment with RO-3306 (Vassilev et al., 2006) and AZ703 (Cai et al., 2006a), cdk inhibitor compounds with selectivity for cdk1 (10-fold over cdk2) and equipotency for cdk2 and cdk1, respectively. RPA32 focus formation following IR was only compromised by concomitant AZ703 treatment (Figure S4). Cdk1 Depletion or Inhibition Reduces Formation of BRCA1 Foci Because BRCA1 is required for the phosphorylation of a number of ATR and ATM substrates (Foray et al., 2003; Yarden et al., 2002), we measured the efficiency of BRCA1 to form foci in cdk1-depleted cells. We found that the number of cells containing at least five BRCA1 foci was reduced 2.8 fold in the absence of cdk1 in untreated cells, and by 3.7- (p = 0.00075) fold at 6 hours after IR treatment, (Figure 3B). Following the disappearance of smaller S phase BRCA1 foci in untreated cells (Scully et al., 1997a) larger BRCA1containing DNA damage foci were observed 2 hours post-IR treatment, and maximally detected by 6 hours. After IR in cdk1-depleted cells, cells containing BRCA1 foci could not be detected until 3 hours post-IR treatment. The percentage of BRCA1 foci-containing cells remained low throughout the time course, suggesting that cdk1 is required for efficient BRCA1 recruitment to chromatin.

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We also examined ATR and BRCA1 focus formation after cisplatin treatment in NCIH1299 cells. In the absence of cdk1, ATR focus formation was uncompromised; however, the number of cells containing at least five BRCA1 foci was reduced by 7.4-fold (p = 0.0086), similar to results seen after IR (Figures 4A, B, S5A). As expected, FANCD2 focus formation is also compromised (Figure S5B). HeLa cells treated with an siRNA pool targeting cdk1 also had less BRCA1 foci in untreated (4-fold reduction, p = 0.006) and hydroxyurea treated cells (4-fold reduction, p = 0.0023) (Figure S5C). To further validate these data, we isolated soluble nuclear and chromatin fractions from NCI-H1299 cells treated with vehicle or cisplatin in the presence or absence of doxycycline (Figure 4C). Cells treated with cisplatin had an increase in the amount of BRCA1 protein associated with chromatin. In cdk1-depleted cells, cisplatin did not result in the detection of BRCA1 in the chromatin fraction, but remained in the soluble fraction. Downstream of BRCA1 activity, ubiquitinated FANCD2 was present only at low levels in doxycyclinetreated cells, with compromised recruitment to chromatin after cisplatin. In contrast, ATR and Orc2 chromatin association were not affected by doxycycline treatment.

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To determine if these results could be replicated using a small molecule cdk1 inhibitor, we utilized RO-3306. The cdk1 depletion phenotype, manifested by a small increase in the G2/ M cell cycle fraction, could be reproduced by treatment with 2 μM RO-3306 in NCI-H1299 cells, which abrogated S phase accumulation (Figure 4D), compromised Chk1 phosphorylation (Figure 4E) and reduced the number of cells with at least five BRCA1 foci by 2.3-fold (49% to 21% cells with foci; p = 0.029) (Figure 4F) in response to cisplatin. Cdk1 Phosphorylates BRCA1 at S1497 and S1189/S1191 and Mutation Reduces Formation of Foci

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To further investigate how cdk1 depletion may affect BRCA1 focus formation at sites of damaged DNA, we determined whether cdk1 directly interacts with BRCA1 in cells. When BRCA1 was immunoprecipitated from cells, we found that cdk1, but not cdk2, could be coimmunoprecipitated. Additionally, increased levels of cdk1 were associated with BRCA1 after DNA damage (Figure 5A). We next determined if recombinant cyclin B1-cdk1 could phosphorylate a recombinant BRCA1-BARD1 substrate in an in vitro kinase assay. BRCA1 was strongly phosphorylated by cdk1, whereas BARD1 was phosphorylated to a lesser degree (Figure 5B). Regions of the BRCA1 protein phosphorylated were identified by incubation of recombinant cyclin B1-cdk1 holoenzyme with overlapping GST-tagged short peptide fragments spanning the amino-to carboxy-terminus of the BRCA1 protein (F1–F6) in kinase assays. The F1–4 fragments, representing amino acids1–1067, had undetectable levels of phosphorylation, whereas the F5 peptide (amino acids 1005–1313) and the F6 carboxy-terminal fragment (amino acids 1314–1863) were strongly phosphorylated (Figure 5C). Analysis of the amino acid sequence of the F6 fragment revealed two possible cdk phosphorylation motifs involving S1497 (Ruffner et al., 1999) and T1658. We mutated these sites and assessed the phosphorylation levels of the mutant fragments. Mutation of serine 1497 to alanine resulted in loss of phosphorylation (5.7 fold reduction measured by densitometry) and mutation of threonine 1658 to asparagine slightly reduced phosphorylation (0.2 fold) (Figure 5D), indicating S1497 is the predominant cdk1 phosphorylation site in the F6 region fragment. Furthermore, in intact cells, cdk1 depletion results in reduction of BRCA1 phosphorylated at S1497, demonstrated with a phosphospecific antibody (Figure 5E). We also analyzed the F5 region for cdk consensus sites and found a double phosphorylation motif at S1189 and S1191. When these serine residues were both mutated to alanine, phosphorylation was also reduced 5-fold (Figure 5F).

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Because loss of cdk1 activity resulted in reduction in the amount of BRCA1 protein forming foci following DNA damage, we measured the ability of both wild type and mutated HAtagged full length BRCA1 protein to form DNA damage-induced foci in reconstituted MDA-MB-436 BRCA1-mutant cells (Elstrodt et al., 2006). As shown in Figure 5G, following IR, 62% of cells expressing wild-type BRCA1 had at least 5 detectable BRCA1containing foci. In cells engineered to express BRCA1 harboring S1497A and S1189A/ S1191A mutations, there was a 4.7-fold reduction in BRCA1-foci containing cells (p = 0.0003). In contrast, RPA32 and ATR focus formation was intact (Figure 5G and Figure S6). Additionally, compared to cells expressing wild-type BRCA1, phosphorylation of both Chk2 and Chk1 is compromised after IR in triple-mutant cells (Figure 5H). Similar results were obtained in 293T and HeLa cells transfected with wild-type BRCA1 or BRCA1 mutated at cdk1 phosphorylation sites (Figure S7). Cdk1 Depletion or Inhibition Selectively Sensitizes Cancer Cells to DNA Damaging Treatments

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The disruption of BRCA1 function and checkpoint control mediated by cdk1-depletion or inhibition is expected to translate to sensitization of cells to DNA damaging treatments. We therefore assessed colony formation after cisplatin treatment in NCI-H1299 cells before and after depletion of either cdk1 or cdk2. Long-term cdk2 depletion had only a modest effect on colony formation after exposure to vehicle and did not further compromise colony formation after cisplatin treatment (Figure 6A). In contrast, cells that were depleted of cdk1 demonstrated reduced colony formation by approximately 70% after exposure to vehicle, consistent with gradual G2/M slowing observed in these cells. Additionally, cdk1-depleted cells were significantly more sensitive to cisplatin compared to cells expressing uncompromised levels, with the LC50 of cisplatin reduced four-fold in the presence of doxycycline (p = 0.004). A549 NSCLC cells engineered to inducibly express shRNA targeting cdk1 were also more sensitive to cisplatin in the presence of doxycycline (3.4-fold; p = 0.02), while cdk2 depletion did not affect cell survival (Figure 6B). As with NCI-H1299 cells, the effects of cdk1 depletion after cisplatin treatment were superimposed on the reduced colony formation afforded by cdk1 depletion alone (reduction by 58% compared to cells expressing normal cdk1 levels). We also measured the ability of cdk1 depletion to sensitize NCI-H1299 cells to other DNA damaging agents. Cdk1 depleted cells were 6-(p = 0.003), 2.5- (p = 0.02) and 4.2- (p = 0.01) fold more sensitive to adriamycin, mitomycin C and IR treatment, respectively, than cells expressing normal cdk1 levels (Figure S8).

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If cdk1 depletion sensitizes cells to cisplatin primarily through abrogation of BRCA1 function, then cdk1 depletion should not sensitize BRCA1-deficient cells to DNA damage. As shown in Figure 6C, in the absence of doxycycline, BRCA1 depletion sensitized cells to cisplatin treatment to a similar degree as cdk1 depletion. However, there was no further reduction in colony formation after cisplatin treatment in cells that were depleted of BRCA1 and cdk1 together. Additionally, reconstitution of MDA-MB-436 cells with wild-type BRCA1 makes them less sensitive to cisplatin (LC50 increased from 0.009 μg/ml in cells expressing vector control to 0.018 μg/ml in cells expressing wild-type BRCA1), whereas expression of BRCA1 harboring S1497A and S1189A/S1191A mutations only slightly alters the sensitivity of BRCA1-mutant cells (LC50 0.011 μg/ml). Furthermore, at 0.0125 and 0.025 μg/ml cisplatin, colony formation of cells expressing wild-type BRCA1 is 1.8- and 3-fold increased compared to cells expressing triple mutant BRCA1, respectively (p = 0.0027 and 0.0138) (Figure 6D). The targeting of proteins involved in DNA damage response pathways carries the risk of sensitizing both transformed and non-transformed cells to DNA damaging treatments. To Mol Cell. Author manuscript; available in PMC 2011 January 20.

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assess this possibility for cdk1, we determined the effects of cdk1 knockdown in combination with cisplatin in non-transformed retinal pigment epithelial (RPE) cells compared to NCI-H1299 cells. Consistent with previous data, siRNA-mediated depletion of cdk1 in NCI-H1299 caused only a small increase in G2/M content and mediated increased sensitivity to cisplatin compared to cells treated with scrambled siRNA (Figure 7A, C, D). In contrast, siRNA-mediated depletion of cdk1 in RPE cells led to a substantial increase (39%) in the percentage of cells with G2/M phase DNA content 3 days after transfection (Figure 7A) and inhibited cell growth (Figure 7B). Cdk1-depleted RPE cells were also 2-fold more resistant to cisplatin treatment compared to scrambled siRNA-treated cells (p = 0.04; Figure 7C, D). These results were confirmed with small molecule cdk1 inhibition. NCI-H1299 and MCF7 cells were 5- and 3-fold more sensitive to cisplatin treatment when concomitantly treated with RO-3306 compared to cisplatin alone, measured by colony formation assay (p values 0.003 and 0.0032, respectively; Figure 7E). However, RPE cells are not sensitized to cisplatin by concomitant treatment with RO-3306. Thus, in the context of these in vitro models, cdk1 depletion or inhibition can sensitize transformed cycling cells but protects non-transformed G2/M arrested cells from S phase DNA damaging treatments.

DISCUSSION NIH-PA Author Manuscript

Cdks 1 and 2 have been linked to the initiation of checkpoint control in mammalian cells, (Deans et al., 2006; Jazayeri et al., 2006; Maude and Enders, 2005) although their individual roles have not been clarified. Here, we have shown that cdk1 phosphorylates BRCA1 at S1497 and S1189/S1191, both part of evolutionarily conserved motifs (Abel et al., 1995). Cdk1-mediated phosphorylation is necessary for efficient formation and/or maintenance of BRCA1-containing foci at the sites of DNA strand breaks and facilitates ATR/ATMmediated phosphorylation of a subset of substrates required for the activation of DNA damage checkpoint signaling. BRCA1 has been proposed to play a scaffolding role facilitating the ATM- and ATRmediated phosphorylation of substrates not bound to chromatin (Foray et al., 2003). Consistent with this, DNA-associated substrates, including H2AX and Rad17, were phosphorylated after DNA damage even in the absence of cdk1. In cdk1-depleted cells the phosphorylation of Chk1 was compromised by cisplatin, hydroxyurea and IR treatment, which activate ATR either directly in response to SSBs or via ATM-dependent regulation after DSBs. The phosphorylation of Chk2 was also reduced in response to IR, which activates ATM-Chk2 signaling.

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The reduced phosphorylation of Chk1 and Chk2 after IR in cdk1-depleted cells is consistent with the known requirement of BRCA1 for Chk1 and Chk2 activation following DNA damage (Foray et al., 2003; Yarden et al., 2002). Additionally, cdk inhibition mediated by roscovitine has been shown to abrogate Chk1 activation in response to IR in osteosarcoma and breast carcinoma cells (Deans et al., 2006; Jazayeri et al., 2006). Dual inhibition of cdk1 and cdk2 by roscovitine or AZ703 (Figure S4) also reduces the formation of RPA foci after IR, suggesting that cdk inhibition compromises DNA end resection following DSB formation. However, after selective cdk1 depletion or RO-3306-mediated inhibition, formation of RPA32- and subsequent ATR-containing foci is intact, likely because of compensatory cdk2 activity. In addition, under conditions of replicative stress and SSBs induced by cisplatin or hydroxyurea, when DNA end resection is not required, cdk1 depletion still results in compromised Chk1 phosphorylation. The mechanism accounting for reduced Chk1 and Chk2 phosphorylation after cdk1 depletion or inhibition is reduced phosphorylation of BRCA1, an event that cannot be compensated by cdk2, resulting in inefficient and diminished formation of BRCA1 foci, and consequently compromised ATM/ ATR-mediated checkpoint signaling.

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The requirement for cdk1 in BRCA1-dependent ATR-Chk1 and ATM-Chk2 signaling suggests these cascades are regulated in a cell cycle-dependent manner and occur primarily in the S and G2 phases. Furthermore, the association of BRCA1 with the MRN complex is restricted to S and G2 and is dependent on cdk activity (Chen et al., 2008). During most of G1, when cdk1 activity is lower, BRCA1 would not be expected to efficiently form foci. A low level of Chk2 phosphorylation may occur without robust BRCA1 focus formation, adequate to facilitate p53 activation and G1 arrest. Additionally, during this time, repair mechanisms other than homologous recombination predominate that do not require the activity of ATR or BRCA1, such as non-homologous end joining.

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The effects of combined cdk1 depletion and cisplatin in non-transformed RPE cells suggest a therapeutic window for cdk inhibitor-chemotherapy combinations. In these cells, cdk1 depletion induced more potent G2/M arrest, suggesting the ability of cdk2 to compensate for cdk1 in cell proliferation is far less than in transformed cells. This is consistent with the inability of other family members to compensate for cdk1 loss in the cdk1−/− mouse embryo (Santamaria et al., 2007). Because cdk1-depleted RPE cells were G2/M arrested at the time of treatment with an S phase DNA damaging agent, fewer cells died from treatment than cdk1-expressing that were cycling. Furthermore, the cdk1 depletion phenotype in both the initiation of DNA damage-induced checkpoint control and the selective sensitization of transformed cells was confirmed using the small molecule inhibitor RO-3306. Cdk1 inhibition may therefore represent a reasonable strategy to use in concert with DNA damage against BRCA1-proficient cancer cells.

EXPERIMENTAL PROCEDURES Cell lines and reagents Cell lines were obtained from the American Type Culture Collection (Manassas, VA). NCIH1299 and A549 cells expressing shRNA for either cdk1 or cdk2 were generated and maintained as previously described (Cai et al., 2006b). Inducible cdk knockdown was achieved by pretreatment of cells with 5 μg/mL doxycycline for 3 days. Pooled transfectants of MDA-MB-436 cells were selected and maintained in 800 μg/ml neomycin. RO-3306 was purchased from Calbiochem, and AZ703 was obtained from AstraZeneca. Cisplatin (Roche Pharmaceuticals) was supplied as a 1 mg/ml solution in 0.9% NaCl. siRNA, Plasmids, mutagenesis and purification of proteins and chromatin

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siRNA targeting BRCA1 was 5′CAGGAAATGGCTGAACTAGAA 3′ (Qiagen). F1–6 BRCA1 fragment GST proteins were prepared as previously described (Scully et al., 1997b). Both the F5 and F6 fragments and a BRCA1-HA tagged plasmid (Quinn et al., 2003) were used for mutagenesis, carried out using a Quickchange kit (Stratagene). After nuclear isolation, the soluble nuclear fraction and insoluble chromatin were extracted as previously described (Mendez and Stillman, 2000). The chromatin fraction was sonicated prior to gel electrophoresis and Western blotting. Orc2 served as a chromatin fraction control. Immunoprecipitation and in vitro kinase assays Immunoprecipitated or recombinant cdk1/cyclin B1 (Cell Signaling Technology) were incubated with full-length recombinant BRCA1-BARD1 (Starita et al., 2004), BRCA1 fragment proteins or histone H1 as substrates. Immunoprecipitation and kinase assays were performed as previously described (Cai et al., 2006a). Colony formation assay Cells were treated with or without doxycycline for 3 days or siRNA for 3 days, exposed to increasing concentrations of chemotherapy for 24 hours and replated in the presence or Mol Cell. Author manuscript; available in PMC 2011 January 20.

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absence of doxycycline at 1 × 104/10 cm dish. After 2 weeks, cells were fixed (3:1::methanol:acetic acid), rinsed and stained with 0.4% crystal violet and colonies counted. Western blot analysis Whole-cell lysates were prepared in lysis buffer (Cell Signaling Technology), containing protease and phosphatase inhibitors. Nuclear extracts were prepared using NE-PER Nuclear Extraction Reagents (Pierce). Protein concentrations were determined by Bradford assay (Bio-Rad), and equivalent amounts (10–50 μg) were subjected to SDS-PAGE. Western blotting was carried out with antibodies recognizing Chk1 [pS317], Chk2, Chk2 [pT68] (Cell Signaling Technology); BRCA1, H2AX [pS139] (Upstate Biotechnology); RPA32 [pS4/8] (Bethyl Labs); HA (Abcam); and all others from Santa Cruz Biotechnology. Immunofluoresence microscopy

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Preparation of cells was performed as described previously (Garcia-Higuera et al., 2001). Primary antibodies recognizing ATR, ATM [pS1981], BRCA1, H2AX [pS139] and HA (Covance) were followed by secondary antibodies conjugated to FITC or Texas Red (Jackson ImmunoResearch Laboratories). For BrdU analyses, cells were treated with nucleases and an anti-BrdU antibody (Amersham) as previously described (Sartori et al., 2007). Images were acquired using an Axioplan 2 imaging microscope (Carl Zeiss) equipped with a digital camera and processed using Openlab software. Fluorescence-activated cell sorting analysis, detection of apoptosis, BrdU and phosphorylated histone H3 by flow cytometry For cell cycle analysis, fixed cells were stained with propidium iodide and analyzed for DNA content using ModFit (Verity Software House) or CellQuest software (BD Biosciences). For apoptosis assays, a fluorescein detection kit was used (Promega). BrdU content was measured as previously described (Cai et al., 2006a). For detection of phosphorylated histone H3 (pHH3), ethanol-fixed cells were treated in 0.25% Triton X-100, washed, incubated in PBS containing 1% BSA and 0.75 μg of anti-pHH3 (Upstate Biotechnology), rinsed, resuspended in FITC-conjugated secondary antibody, treated with RNase A and counterstained with propidium iodide. Statistical analysis—The two-tailed, unpaired Student’s t test was used. P < 0.05 was considered statistically significant.

Supplementary Material NIH-PA Author Manuscript

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments This work was supported by NIH Grants R01 CA090687, P50 CA089393 [Dana-Farber/Harvard Cancer Center (DF/HCC) Specialized Program of Research Excellence (SPORE) in Breast Cancer] and P20 CA090578 (DF/HCC SPORE in Lung Cancer) (GIS), as well as Susan G. Komen Post-Doctoral Fellowship Award KG080773 (NJ and GIS), NIH Grant CA111480 (JDP) and an AstraZeneca Sponsored Research Agreement.

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Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001;15:2177–2196. [PubMed: 11544175] Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 2003;3:421– 429. [PubMed: 12781359] Cai D, Byth KF, Shapiro GI. AZ703, an imidazo[1,2-a]pyridine inhibitor of cyclin-dependent kinases 1 and 2, induces E2F-1-dependent apoptosis enhanced by depletion of cyclin-dependent kinase 9. Cancer Res 2006a;66:435–444. [PubMed: 16397259] Cai D, Latham VM Jr, Zhang X, Shapiro GI. Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells. Cancer Res 2006b;66:9270– 9280. [PubMed: 16982772] Chen L, Nievera CJ, Lee AY, Wu X. Cell cycle-dependent complex formation of BRCA1.CtIP.MRN is important for DNA double-strand break repair. J Biol Chem 2008;283:7713–7720. [PubMed: 18171670] Deans AJ, Khanna KK, McNees CJ, Mercurio C, Heierhorst J, McArthur GA. Cyclin-Dependent Kinase 2 Functions in Normal DNA Repair and Is a Therapeutic Target in BRCA1-Deficient Cancers. Cancer Res 2006;66:8219–8226. [PubMed: 16912201] Elstrodt F, Hollestelle A, Nagel JH, Gorin M, Wasielewski M, van den Ouweland A, Merajver SD, Ethier SP, Schutte M. BRCA1 mutation analysis of 41 human breast cancer cell lines reveals three new deleterious mutants. Cancer Res 2006;66:41–45. [PubMed: 16397213] Foray N, Marot D, Gabriel A, Randrianarison V, Carr AM, Perricaudet M, Ashworth A, Jeggo P. A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein. Embo J 2003;22:2860–2871. [PubMed: 12773400] Garcia-Higuera I, Taniguchi T, Ganesan S, Meyn MS, Timmers C, Hejna J, Grompe M, D’Andrea AD. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell 2001;7:249–262. [PubMed: 11239454] Ira G, Pellicioli A, Balijja A, Wang X, Fiorani S, Carotenuto W, Liberi G, Bressan D, Wan L, Hollingsworth NM, et al. DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 2004;431:1011–1017. [PubMed: 15496928] Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J, Jackson SP. ATM- and cell cycledependent regulation of ATR in response to DNA double-strand breaks. Nat Cell Biol 2006;8:37– 45. [PubMed: 16327781] L’Italien L, Tanudji M, Russell L, Schebye XM. Unmasking the redundancy between Cdk1 and Cdk2 at G2 phase in human cancer cell lines. Cell Cycle 2006;5:984–993. [PubMed: 16687918] Maude SL, Enders GH. Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response. Cancer Res 2005;65:780–786. [PubMed: 15705874] McClue SJ, Blake D, Clarke R, Cowan A, Cummings L, Fischer PM, MacKenzie M, Melville J, Stewart K, Wang S, et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 2002;102:463–468. [PubMed: 12432547] Mendez J, Stillman B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol 2000;20:8602–8612. [PubMed: 11046155] Quinn JE, Kennedy RD, Mullan PB, Gilmore PM, Carty M, Johnston PG, Harkin DP. BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res 2003;63:6221–6228. [PubMed: 14559807] Ruffner H, Jiang W, Craig AG, Hunter T, Verma IM. BRCA1 is phosphorylated at serine 1497 in vivo at a cyclin-dependent kinase 2 phosphorylation site. Mol Cell Biol 1999;19:4843–4854. [PubMed: 10373534] Santamaria D, Barriere C, Cerqueira A, Hunt S, Tardy C, Newton K, Caceres JF, Dubus P, Malumbres M, Barbacid M. Cdk1 is sufficient to drive the mammalian cell cycle. Nature 2007;448:811–815. [PubMed: 17700700] Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J, Baer R, Lukas J, Jackson SP. Human CtIP promotes DNA end resection. Nature 2007;450:509–514. [PubMed: 17965729]

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Figure 1. Cdk1 depletion impairs S phase checkpoint activation after DNA damage

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(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 hours 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 hours prior to collection for assessment of phospho-histone H3 staining by flow cytometry. (Left) Representative flow cytometry panels for cells treated with anti-FITC-conjugated phospho-histone H3 and counterstained with propidium iodide. (Right) Mean percent of phospho-histone 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.

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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 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 hours 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)

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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 hydroxyurea (HU) for 24 hours. 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 hydroxyurea (HU) for 24 hours 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.

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NIH-PA Author Manuscript Figure 3. Cdk1 depletion does not affect DNA end resection

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(A) NCI-H1299 cdk1 cells were grown in the absence or presence of doxycycline and treated with IR (10 Gy). Cells were fixed 6 hours post IR and subjected to immunofluorescence and DAPI staining. (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least 5 phospho-ATM foci ± SE over three experiments. (B) Cells were treated as in A and fixed at the time points indicated post -IR. (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least 5 BRCA1 or RPA32 foci ± 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 5 ATR foci ± SE over three experiments.

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NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 4. Cdk1 depletion reduces BRCA1 focus formation

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(A) NCI-H1299 cdk1 cells were treated with cisplatin (5 μg/ml for 24 hours), and subjected to immunofluorescence and Dapi staining. (Left) Representative foci-containing cells. (Right) Mean number of cells containing at least 5 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 5 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 hours, 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 hours, 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.

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Figure 5. Cdk1 phosphorylates BRCA1 at S1497 and S1189/1191 and promotes formation of foci

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(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 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.

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(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 5 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.

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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 vehicletreated 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 hours 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.

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(D) MDA-MB-436 cells as in Figure 5G were treated with vehicle or the indicated concentrations of cisplatin for 24 hrs, 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.

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Figure 7. Cdk1 depletion is mimicked by a small molecule cdk1 inhibitor and protects nontransformed cells from S phase specific DNA damaging agents

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(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 three days prior to treatment with cisplatin for 24 hours followed by replating for assessment of colony formation after 14 days. Mean survival from 3 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 hours, 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.

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