Speedy (Spy1) is a novel cell cycle regulator that binds and activates cdk2, and was originally identified as a suppressor of Rad1 deficiency in Schizosaccharomyces pombe. Here we demonstrate that overexpression of human Spy1 enhances mammalian cell viability during cellular responses to DNA damage induced by genotoxic agents such as camptothecin, cisplatin, and hydroxyurea. Clonogenic survival assays and comet assays also show that Spy1 expression enhances cell survival after DNA damage. Consistent with Spy1 having a role in the DNA damage response, endogenous Spy1 protein levels are up-regulated in response to DNA damage induced by camptothecin, cisplatin, or hydroxyurea. We found that Spy1 can activate cdk2 during the DNA damage response and that expression of a dominant-negative form of cdk2 overrides Spy1 function in damaged cells. Lastly, ablation of endogenous Spy1 expression using small interference RNA results in hypersensitization of cells to DNA damage. Together, these results demonstrate that human Spy1 mediates protection of mammalian cells against DNA damage.

DNA damage-inducible cell cycle checkpoints are complex signal transduction networks composed of cellular responses to genotoxic stresses. Some of these responses include cell cycle arrest while DNA damage is repaired, and induction of apoptosis if the cell is too heavily damaged for appropriate repair. The integrity of these checkpoint pathways is critical for the maintenance of genomic stability and cellular recovery from genotoxic damage. Many checkpoint proteins have been identified through the investigation of yeast genetics. In the fission yeast, S. pombe, both DNA replication and DNA damage checkpoints require Hus1, Rad1, Rad3, Rad9, Rad17, and Rad26 gene products (1, 2, 3, 4, 5, 6, 7). Specifically, the cell cycle checkpoint gene Rad1 is required to ensure that mitosis does not occur in the presence of DNA damage or in the absence of complete DNA replication (8, 9).

A Rad1-deficient strain of S. pombe that fails to arrest normally at the G2/M boundary upon DNA damage (10) was used previously to screen a Xenopus laevis total ovary cDNA library for plasmids that conferred resistance to UV radiation. Expression of the X-Spy13 gene product was found to partially rescue fission yeast under both UV and γ-radiation conditions (11), indicating that Spy1 complements a Rad1 deficiency in yeast. We recently isolated the human homologue of Speedy and have demonstrated that human Spy1 plays an essential role in the normal cell cycle progression of mammalian cells through its ability to bind and activate cdk2 (12).

To gain insight into the function of Spy1 during the DNA damage response, we have undertaken a biochemical and cellular analysis of human Spy1 with agents that induce DNA damage. In this report we demonstrate that Spy1 expression in mammalian cells enhances survival under conditions of genotoxic damage, including treatments of CPT, cisplatin, and HU. Clonogenic studies show that Spy1 expression enhances cell viability and that Spy1-expressing cells have a significant decrease in damaged DNA as compared with their mock counterparts. We also show that cdk2 is required for Spy1-enhanced cell viability and that Spy1 activates cdk2 under conditions of genotoxic stress. Endogenous Spy1 proteins are up-regulated in response to cellular treatment with CPT, cisplatin, and HU. Lastly, we show that ablation of endogenous Spy1 expression using siRNA results in an increased sensitivity to DNA damage. Together, these results indicate that human Spy1 actively participates in cellular responses to genotoxic stress.

Cell Culture.

293T cells (human embryonic kidney) were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 10% CO2. COS-1 cells (SV40-transformed African green monkey kidney) were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO2. NIH3T3 cells (mouse embryonic fibroblast) were cultured in DMEM supplemented with 10% calf serum at 37°C in 10% CO2.

Viability Assays.

Cells grown on collagen-coated glass coverslips (for 293T cells) or glass coverslips (for COS-1 and NIH3T3 cells) were transfected by the calcium phosphate precipitation method with 10–20 μg of Myc-tagged Spy1 DNA or 1–2 μg GFP vector (pEGFP-C1). Forty-eight h after transfection cells were fixed with 3% paraformaldehyde and permeabilized with blocking solution (0.1% Triton X-100, 0.2 m glycine, and 2.5% fetal bovine serum in PBS). Myc-tagged Spy1 protein was visualized by use of c-Myc (9E10) antisera (Santa Cruz) with antimouse IgG (fab-specific)-FITC conjugate antisera (Sigma). Hoechst dye 33342 (1 μg/ml) was used to detect nuclei. For quantitation of cell survival, 40 fields at ×40 magnification were examined per sample by indirect immunofluorescence. Cells positive for Myc-Spy1 expression were counted and scored as a percentage of untreated Myc-Spy1-expressing cells. Cells positive for GFP expression were counted and scored as a percentage of untreated GFP-expressing cells (visualized by GFP). Mock cells were counted and scored as a percentage of untreated mock cells (visualized by Hoechst dye). Results shown represent the average of three experiments ± SD.

Drug Treatment.

CPT (Sigma; 10 mm stock solution in DMSO) was added directly to cell medium 24 h after transfection, unless otherwise indicated. After treatment, cells were either fixed in 3% paraformaldehyde for immunofluorescence studies or harvested in 1% NP40 buffer for SDS-PAGE analysis.

Cisplatin (Sigma; 20 mm stock solution in 50:50 H2O/DMSO) was added directly to cell medium 24 h after transfection. Cells were treated with 10 μm, 20 μm, or 40 μm cisplatin concentrations for 1 h, and then fresh medium was added (10% fetal bovine serum in DMEM). Cells were then incubated for an additional 15 h and then fixed in 3% paraformaldehyde for immunofluorescence studies.

HU (Sigma; 1 m stock solution in H2O) was added directly to cell medium 24 h after transfection. Cells were treated with 1 mm, 2 mm, or 3 mm HU concentrations for 16 h. Cells were then fixed in 3% paraformaldehyde for immunofluorescence studies.

Clonogenic Survival Assays.

Long-term survival was determined by a colony-forming assay. NIH3T3 cells were seeded at a density of 2 × 105 cells/100-mm plate. Cells were transfected with the calcium phosphate precipitation method using 10 μg of DNA per construct (empty vector serves as mock, and Myc-tagged Spy1 vector serves as Spy1). Cell lysates were also examined for Spy1 expression. Twenty-four h after transfection, cells were split out onto multiple 60-mm plates (with duplicate plates per data point). Twenty-four h after splitting, cells were treated with 0, 5, 10, 15, and 20 μm CPT for 15 min. Immediately after treatment, cell monolayers were rinsed twice and then refed with fresh medium. Every 2 days cells were refed with fresh medium. The cultures were incubated for 14 days, and then fixed and stained with 0.1% crystal violet solution. Colonies consisting of >50 cells were counted, and surviving fractions were determined based on plating efficiencies. Results are the average of two independent experiments performed in duplicate.

For the time course survival experiment, NIH3T3 cells were seeded, transfected, and split as described above. Cell lysates were also examined for Spy1 expression. Twenty-four h after splitting, cells were treated with 10 μm CPT for 15 min (or with DMSO for 15 min for untreated samples). Immediately after treatment, cell monolayers were rinsed twice and then refed with fresh medium. Every 2 days cells were counted by Coulter Counter, and then cells were refed with fresh medium. Loss of viability over time (post-CPT treatment) was determined as the ratio of cells counted from treated samples over cells counted from untreated samples. Results are the mean and SE for three independent experiments with two replicates per experiment.

Comet Assays.

NIH3T3 cells were seeded at a density of 2 × 105 cells/100-mm plate. Cells were transfected with the calcium phosphate precipitation methods as described above. Forty-eight h after transfection, cells were treated with 10 μm CPT for 15 min (or with DMSO for 15 min for untreated samples). Immediately after treatment, cell monolayers were rinsed twice and then refed with fresh medium. Four days after CPT treatment, cells were harvested by trypsin and then rinsed twice with PBS. For the alkaline comet assay (13), microscope slides were precoated with 75 μl of 1% agarose (at 65°C) dropped onto a slide, covered with a glass coverslip, and allowed to set overnight at 4°C. Pelleted cells were resupended with an equal volume of PBS. Seventy-five μl of this cell suspension and 75 μl of 1% agarose were mixed by pipetting and then loaded onto each precoated slide, and then covered with a glass coverslip until set. The coverslips were removed, and the slides were immersed in ice-cold lysis solution [2.5 m NaCl, 0.1 m EDTA, 10 mm Tris-HCl, and 1% Triton X-100 (pH 10)] for 1 h. Slides were placed horizontally in an electrophoresis tank. The reservoirs were filled with ice-cold electrophoresis buffer solution (0.3 m NaOH and 1 mm EDTA) until just covering the slides. Slides were allowed to stand in the buffer for 20 min (to allow DNA unwinding), after which electrophoresis was carried out for 15 min at 25 V. After electrophoresis, the slides were removed and washed three times (5 min each) in ice-cold neutralization buffer [0.4 m Tris-HCl (pH 7.5)]. Twenty μl of a 1 μg/ml solution of 4′,6-diamidino-2-phenylindole was pipetted onto slide, and then covered with a glass coverslip. Slides were examined at ×40 magnification using a fluorescence microscope. One-hundred cells were examined per sample from two independent experiments.

cdk2-DN.

293T cells were seeded at a density of 1 × 105 cells/plate in 10-cm dishes. Cells were transfected by the calcium phosphate precipitation method with either empty vector (pCS3+MT) ± HA-cdk2-DN (pCMVcdk2D145N; Ref. 14) or Myc-Spy1 ± HA-cdk2-DN (pCMVcdk2D145N; 2.5 μg total DNA). Twenty-eight h after transfection cells were treated with or without 0.01 mm CPT for 20 h. Forty-eight h after transfection cells were trypsinized and counted by trypan blue exclusion (12). Each assay was performed in triplicate, and each experiment was performed three times. Results shown are of a representative experiment ± SE.

SDS-PAGE Analysis.

293T cells were harvested in PBS, and then lysed in 1% NP40 buffer [1% NP40, 200 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin]. Lysate samples were analyzed by Bradford assay to quantitate protein concentrations. Each sample was normalized to 1 mg/ml protein concentration. Lysates were precleared with Protein A-Sepharose beads and then subjected to 2 μg of cdk2 (M-2) antisera (Santa Cruz) for immunoprecipitations. After the addition of Protein A-Sepharose beads, samples were washed with 0.5% NP40 buffer [0.5% NP40, 200 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin]. Samples were then boiled for 5 min in 2× sample buffer and analyzed by 10% SDS-PAGE.

Immunoblots.

SDS-PAGE analyzed samples were transferred to nitrocellulose membrane and then blocked in 2% nonfat milk in TBS-T with gentle rotation at 4°C overnight. Primary antibodies [c-Myc (9E10), Santa Cruz; cdk2 (d-12), Santa Cruz; actin (C-2), Santa Cruz; human Spy1, Pocono Rabbit Farm and Laboratory] were used at 1:2000 concentration in 2% nonfat milk/TBS-T solution. Secondary antibodies (antimouse immunoglobulin-horseradish peroxidase conjugate and antirabbit immunoglobulin-horseradish peroxidase conjugate, both from Amersham) were used at 1:5000 concentration in 2% nonfat milk/TBS-T solution. An Enhanced chemiluminescence kit (Amersham) was used to detect protein expression.

cdk2 Kinase Assay.

For detection of phosphorylation of histone H1 in vitro by cdk2, 293T cells were transfected by the calcium phosphate precipitation method with 5 μg of either empty vector (pCS3+MT) or Myc-Spy1. Twenty-four h after transfection cells were exposed to 0.1 mm HU, 0.025 mm CPT, or 20 μm cisplatin for an additional 24 h. Both untreated and treated samples were harvested 48 h after transfection. Proteins were immunoprecipitated with cdk2 (d-12; Santa Cruz) antibody after lysis in 0.1% NP40 buffer. Protein content was standardized as determined by the Bradford assay. The immunoprecipitates were incubated at 30°C for 10 min in 50 μl of kinase buffer [50 mm Tris-HCl (pH 7.5), 20 mm EGTA, 10 mm MgCl2, 1 mm DTT, 50 μm ATP, and 10 μCi [γ-32P]ATP] with the addition of 4 μg histone H1 as substrate. The reaction was terminated by the addition of 2× sample buffer. Samples were then analyzed by 10% SDS-PAGE followed by autoradiography.

Human Speedy Antisera.

The human Spy1 antibody was obtained through Pocono Rabbit Farm and Laboratory. The human Spy1 antibody specifically detects an endogenous protein of Mr ∼34,000 (12).

siRNA.

RNA interference assays were conducted through the construction of siSpy1. The siSpy1 oligo (-GTACGAAATTTTTCCATGG-) was synthesized, purified, and duplexed by Dharmacon Research. As a negative control a luciferase GL2 duplex siRNA (siControl) was used (d-1000-Lu-5) from Dharmacon Research.

293T cells (1 × 105 cells/ml) were seeded in 10-cm dishes. Cells were transfected by the calcium phosphate precipitation method with 0.6 nm siSpy1 or siControl duplex. Twenty-four h after transfection, siSpy1 and siControl plates were counted by trypan blue exclusion. Cells were replated in triplicate at a density of 1 × 105 cells/ml in 60-mm dishes and incubated at 10% CO2 for 12 h before treatment with various doses of CPT. Twelve h after CPT treatment, cells were recounted. Survival after CPT treatment was expressed as a fraction of each sample over the DMSO-treated siControl sample. Results shown are of a representative experiment ± SE. Remaining cells were pooled and then lysed with 0.5% NP40 buffer. Expression of endogenous Spy1 was determined by SDS-PAGE and immunoblot.

Spy1 is a novel cell cycle regulatory gene (12). Overexpression of Spy1 protein in mammalian cells enhances cell growth and prematurely activates cdk2. In the Xenopus oocyte system, X-Spy1 activates cdk2 and promotes meiotic maturation, indicative of progression through the G2/M transition (11, 15). Thus, Spy1 appears to be a potent regulator of cell cycle progression. Furthermore, in S. pombe, X-Spy1 protein expression partially rescues a Rad1-deficient yeast strain after UV radiation (11). However, the possible involvement of Spy1 in mammalian DNA damage regulatory checkpoints has not been described previously.

To explore the role of Spy1 in cellular responses to DNA damage, we expressed the recently identified human Spy1 (12) in the mammalian 293T cell line (human embryonic kidney) with or without exposure to 0.25 mm CPT. CPT is a chemical DNA damage agent that interacts with topoisomerase I by stabilizing DNA-topoisomerase I covalent intermediate (16). This interaction inhibits topoisomerase I activity, as the enzyme is trapped in a DNA-protein complex, leading to arrest of DNA synthesis and cell division. Notably, the survival of cells expressing Myc-tagged Spy1 was substantially increased compared with mock cells, even after extended periods of CPT exposure (Fig. 1,A). A dosage curve of CPT including concentrations ranging from 0.1 mm to 1.0 mm (3-h exposure time) assessed that overexpression of Spy1 protein enhances cell viability substantially through 0.5 mm concentration (Fig. 1 B).

To determine whether other chemical DNA damage agents have an effect on cell viability in the presence of Spy1 protein, we assayed transfected 293T cells treated with cisplatin and HU. Cisplatin is a drug that is used widely in cancer chemotherapy. Its detailed mechanism of action has not been completely elucidated, but it involves covalent adducts with DNA resulting in DNA cross-links that interfere with replication (16). Cells expressing Spy1 protein demonstrate an enhanced cell survival response in the presence of various doses of cisplatin (Fig. 1,C). HU also inhibits DNA replication, because of the depletion of dNTP pools, which then leads to an inhibition of mitosis (17, 18, 19). Cells expressing Spy1 protein demonstrate an enhanced cell survival response with exposure to various doses of HU (Fig. 1 D). These results show that exogenously expressed human Spy1 substantially enhances survival in cells treated with CPT, cisplatin, or HU.

To determine whether Spy1 maintains a DNA damage response function in other mammalian cells, we assayed two additional cell lines for cell viability: COS-1 (SV40-transformed simian kidney) and NIH3T3 (mouse embryonic fibroblast). A dosage curve of CPT including concentrations ranging from 0.1 mm to 1.0 mm assessed that overexpression of Myc-Spy1 protein enhances cell viability substantially for both cell lines (Fig. 2, A and B). Spy1-enhanced NIH3T3 cell survival closely paralleled 293T cell survival (Fig. 1,B; Fig. 2 B), suggesting that embryonic cell lines are better suited to characterize Spy1 function. In addition, we demonstrated previously that Spy1 mRNA is present in both 293T and NIH3T3 cells, additionally indicating that these cell lines respond appropriately to the exogenous expression of Spy1 (Ref. 12; data not shown).

To confirm that the increase in cell viability is specific to Spy1 expression we used a GFP expression vector as a control. COS-1, NIH3T3, and 293T cells were transiently transfected by either Myc-Spy1 or GFP expression vectors and then exposed to 0.25 mm CPT 24 h after transfection (Fig. 2 C). Only Spy1-expressing cells had a substantial increase in cell viability. Mock-treated and GFP-expressing cells from each cell line did not show an enhancement of cell survival. These results demonstrate that the DNA damage response observed in each cell type is because of the specific expression of Spy1 protein.

To determine the long-term effects of Spy1 expression on cell survival, we performed clonogenic survival assays. As demonstrated in Fig. 3,A, CPT exposure over the dose range tested led to significant cytotoxicity in mock-expressing NIH3T3 cells. In contrast, Spy1-expressing cells showed an increase in survival 14 days after treatment. Myc-tagged Spy1 expression was examined in cell lysates by immunoblot analysis (Fig. 3,A, right). Furthermore, a time course study of cells treated with 10 μm CPT demonstrated that Spy1-expressing cells retain a significant increase in cell viability over their mock counterparts (Fig. 3,B). Immunoblot analysis demonstrated the presence of Myc-tagged Spy1 protein in cell lysates (Fig. 3 B, right). These data from clonogenic survival assays demonstrate that exogenous Spy1 protein in the presence of CPT significantly decreases the ability of this DNA damaging agent to cause cell death.

To assess if Spy1 contributes to protecting cellular DNA in the presence of CPT, we measured the induction of DNA fragmentation by exploiting the alkaline comet assay. This assay allows for the detection of both single- and double-stranded DNA breaks, and, therefore, is a highly sensitive method to directly examine the amount of DNA damage incurred in a single cell. NIH3T3 cells expressing either mock or Spy1 proteins were exposed to 10 μm of CPT and then analyzed for comet expression 4 days after treatment. In mock cells, >56% of the cells examined contained a comet tail, indicating severe DNA damage (Fig. 4,A, panel b; Fig. 4,B). In contrast, only 10% of Spy1-expressing cells had damaged DNA (Fig. 4,A, panel d; Fig. 4,B). Both mock and Spy1-expressing cells treated with DMSO alone resulted in <1% of their respective cells with comets (Fig. 4,A, panels a and c; and Fig. 4 B). These results demonstrate that Spy1-expressing cells exhibit less DNA damage than their mock counterparts after chemically induced DNA damage by CPT.

Considering that Spy1 has been shown to bind to and prematurely activate cdk2 in mammalian cells (12), we wished to examine whether exogenous Spy1 associates with cdk2 under CPT-induced DNA damage conditions. We transiently transfected 293T cells with Myc-Spy1 and used cdk2 antisera to immunoprecipitate endogenous cdk2. Immunoblot analysis for associated Spy1 demonstrated that endogenous cdk2 binds to human Spy1 (Fig. 5,A, top panel, Lane 4). Interestingly, with exposure to 0.1 mm CPT there is an enhanced association between cdk2 and Spy1 (Fig. 5,A, top panel, Lane 6). As a control for Spy1 and cdk2 expression, whole cell lysates are depicted in Lanes 1 and 2 (top and bottom panels, respectively). The bottom panel in Fig. 5 A demonstrates equal expression of cdk2 protein in cdk2 immunoprecipitates (Lanes 3–6). These results demonstrate that endogenous cdk2 binds to exogenously expressed human Spy1, and this interaction is enhanced with cellular exposure to CPT.

To determine whether cdk2 kinase activity is required for Spy1-induced cell survival, we transiently transfected 293T cells with Myc-Spy1 with or without α cdk2-DN. This cdk2 mutant effectively inhibits cdk2 kinase activity (14). As shown in Fig. 5,B and as shown previously (12), the expression of Spy1 protein enhances cell proliferation significantly. With the addition of cdk2-DN, Spy1-enhanced proliferation is blocked under conditions of normal cell growth (Fig. 5,B; Ref. 12). In the presence of CPT, Spy1-expressing cells fare better than their mock counterparts, as there is a significant increase in cell number. In comparison, in the presence of CPT with the addition of cdk2-DN, Spy1-induced cell viability is completely inhibited (Fig. 5,B). This result shows that Spy1 is no longer able to rescue CPT-treated cells in the presence of cdk2-DN and indicates that cdk2 is essential for Spy1-enhanced cell viability. This is not because of an alteration in the expression of Myc-Spy1 protein (Fig. 5 B, bottom panel).

We then wished to examine the kinase activity of cdk2 associated with Spy1 under conditions of genotoxic stress. Lysates from mock- or Myc-Spy1-transfected 293T cells were immunoprecipitated with antibodies against cdk2 and subjected to an in vitro kinase assay using histone H1 as a substrate. Spy1-expressing cells exhibited a dramatic increase in cdk2 kinase activity with cellular exposure to HU, CPT, or cisplatin (Fig. 5,C, top panel, Lanes 4, 6, or 8, respectively). As a control, cdk2 protein levels from cell lysates were examined and found to be equivalent (Fig. 5,C, middle panel). Cdk2 immunoprecipitates from untreated Spy1-expressing cells showed a significant decrease in activity compared with untreated mock cells (Fig. 5,C, top panel, compare Lane 2 to Lane 1). This result is because of an increase in recovery time after transfection (as described in “Materials and Methods;” data not shown). Under these experimental conditions a minimal amount of Spy1 protein binds to cdk2 (Fig. 5,C, bottom panel, Lane 2). Significantly, in the presence of the DNA damage agents, cdk2 immunoprecipitates demonstrate an increase in Spy1 association (Fig. 5 C, bottom panel, Lanes 4, 6, and 8, respectively). These data demonstrate that Spy1 enhances cdk2 kinase activity in the presence of DNA damage, which likely reflects the enhanced association of Spy1 with cdk2.

Using human Spy1 antisera we found that endogenous Spy1 protein levels in 293T cells are up-regulated with exposure to CPT (Fig. 6,A, top panel). In addition, with cellular exposure to cisplatin or HU, endogenous Spy1 is also up-regulated (Fig. 6 B, top panel). Similarly, several checkpoint proteins are up-regulated in response to genotoxic stresses, such as the transcription factors E2F-1 and p53, the growth arrest protein GADD45, and the cyclin-dependent kinase inhibitor p21 (20, 21, 22, 23). Thus, these results implicate Spy1 as a mediator of cellular responses to DNA damage.

To determine the physiological role of endogenous Spy1 in enhancing cell survival, siRNA techniques were used. Transient transfection of 293T cells with siRNA constructs designed against Spy1 (siSpy1) depletes the cells of Spy1 mRNA (12). As a control, siRNA directed against luc-GL2 (siControl), a bacterial reporter, was used. As demonstrated in Fig. 6,C, depleting Spy1 mRNA in 293T cells has a significant effect on cell survival. This difference in cell viability can be demonstrated throughout a range of CPT concentrations. In addition, endogenous Spy1 protein is detected in siControl samples, and Spy1 protein levels increase with exposure to CPT (Fig. 6 C, top panel). These results indicate that endogenous Spy1 is essential for cell survival during the DNA damage response.

One function of cell cycle checkpoints is to integrate cell cycle progression with DNA replication and repair. Therefore, the integrity of these checkpoints is considered essential in maintaining genetic stability. Mutations in checkpoint components may lead to aberrant cell cycle progression and, in the presence of DNA damage, may lead to subsequent genetic instability. DNA damage triggers a variety of cellular responses, including activation of DNA damage response pathways. In fission yeast, genetic evidence pointed to a model in which five checkpoint Rad proteins, Rad1, Rad9, Rad17, Rad26, and Hus1, sense DNA alterations and then cooperate to send a signal through Rad3 (1). Rad3 can also function in the absence of several of these Rad genes, suggesting that Rad3 may interact with other proteins involved in the DNA damage response (4).

In S. pombe the cell cycle checkpoint gene Rad1 is required to ensure that mitosis does not occur in the presence of DNA damage (8, 9). X-Spy1 was initially isolated from a DNA damage screen using a Rad1-deficient strain of S. pombe. Expression of the X-Spy1 gene product was found to enhance yeast viability under both UV and γ-radiation conditions (11), indicating that Spy1 complements a Rad1 deficiency. Here we describe a role for human Spy1 in the DNA damage response pathway in mammalian cells, which is consistent with X-Spy1 function in yeast. With CPT-induced DNA damage we have demonstrated that mammalian cell viability is enhanced with the overexpression of Spy1. In addition, Spy1 enhanced cell survival of cells treated with cisplatin and HU, both inhibitors of DNA replication events. Clonogenic studies also revealed that Spy1 expression enhanced long-term cell survival, and alkaline comet assays demonstrated that Spy1-expressing cells exhibit significantly less damaged DNA after CPT-induced DNA damage. These results indicate that Spy1 expression either prevents DNA damage from occurring or promotes repair of damaged DNA. Consistent with Spy1 having a role in the DNA damage response, endogenous Spy1 protein levels are up-regulated in response to DNA damage. Several checkpoint proteins, such as p53, are up-regulated in response to genotoxic stresses (24, 25). In addition, cells are overly sensitized to DNA damage when endogenous Spy1 protein is ablated, indicating that Spy1 is required for the proper response to DNA damage and cellular recovery. Altogether, these data indicate that Spy1 plays a physiological role in determining cell survival after treatment with genotoxic agents.

The DNA damage response is dependent on the activation of cdk2, a key effector in S phase progression. We had demonstrated previously that Spy1 binds to and prematurely activates cdk2 in undamaged cells (11, 12). In addition, Spy1 has been demonstrated recently to bind to cdk2 without a cyclin partner (26). These results led us to believe that Spy1 is a potent activator of cell cycle progression through interaction with cdk2. In this report we demonstrate that the Spy1-cdk2 interaction is enhanced in the presence of CPT-induced DNA damage, indicating that Spy1 complexes with active cdk2 throughout the DNA damage response. We also show that cdk2 kinase activity is substantially increased with the expression of Spy1 in cells with compromised DNA. Furthermore, expression of a DN form of cdk2 overrides Spy1 function in damaged cells, indicating that cdk2 activity is required for Spy1-enhanced cell survival.

The expression of Spy1 not only promotes cell proliferation under normal circumstances, but promotes cell survival under conditions of genotoxic stress. Our results demonstrate that Spy1 participates in cellular responses to DNA damage and cooperates with cdk2 in the positive regulation of cell proliferation and cellular recovery. Two possible roles for Spy1 can be envisioned that are consistent with our data. In one model, expression of Spy1 protein may override potential cell cycle checkpoints, thereby leading to increased proliferation, possibly even in the presence of DNA damage. Alternatively, Spy1 expression may result in increased cell viability by participating in cell cycle arrest and/or repair of the damaged DNA. We believe our results are consistent with the latter model and suggest a role for Spy1 in checkpoint responses. This is based on: (a) the endogenous up-regulation of Spy1 protein under conditions of DNA damage; (b) the increased clonogenic survival of cells expressing Spy1; and (c) the decreased DNA damage exhibited by Spy1-expressing cells after CPT-induced damage. It will be interesting to determine which other checkpoint proteins involved in DNA damage responses may associate with human Spy1. Likely candidates are the human homologues of the Rad proteins. These proteins serve as DNA sensors, and are activated during both DNA replication and DNA damage events. Therefore, Spy1 may function during the DNA damage response as a component of DNA replication and repair complexes.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grant RO1-CA34456 (to D. J. D.) and NIH/National Cancer Institute Grant T32-CA09523 (to E. A. B.). L. A. P. acknowledges support from the University of California, San Diego Pete Lopiccola Fellowship in Cancer Research.

3

The abbreviations used are: X-Spy1, Xenopus Speedy; CPT, camptothecin; HU, hydroxyurea; siRNA, small interference RNA; GFP, green fluorescent protein; TBS-T, TBS with 0.05% Tween 20; DN, dominant-negative.

Fig. 1.

Expression of Spy1 enhances cell survival under various conditions of DNA damage. A, 293T viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 0.25 mm CPT for 2, 4, 8, or 24 h. B, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 0.1, 0.5, or 1.0 mm CPT for 3 h. C, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 10, 20, or 40 μm cisplatin for 1 h. D, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 1, 2, or 3 mm HU for 16 h. Spy1-expressing cells were counted via indirect immunofluorescence. Results shown reflect the percentage of treated cells (relative to untreated cells). Each graph represents the average of three experiments; bars, ±SD.

Fig. 1.

Expression of Spy1 enhances cell survival under various conditions of DNA damage. A, 293T viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 0.25 mm CPT for 2, 4, 8, or 24 h. B, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 0.1, 0.5, or 1.0 mm CPT for 3 h. C, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 10, 20, or 40 μm cisplatin for 1 h. D, viability curves of mock 293T cells and Myc-Spy1-transfected 293T cells exposed to 1, 2, or 3 mm HU for 16 h. Spy1-expressing cells were counted via indirect immunofluorescence. Results shown reflect the percentage of treated cells (relative to untreated cells). Each graph represents the average of three experiments; bars, ±SD.

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Fig. 2.

Expression of Spy1 enhances cell survival with CPT-induced DNA damage in COS-1 and NIH3T3 cell lines. A, viability curves of mock COS-1 cells and Myc-Spy1-transfected COS-1 cells exposed to 0.1, 0.5, or 1.0 mm CPT for 18 h. B, viability curves of mock NIH3T3 cells and Myc-Spy1-transfected NIH3T3 cells exposed to 0.1, 0.5, or 1.0 mm CPT for 18 h. C, bar graph comparing cell viability of three cell lines exposed to 0.25 mm CPT. Mock, Myc-Spy1-transfected, and GFP-transfected cells were exposed to 0.25 mm CPT as described previously. Positive cells were counted via indirect immunofluorescence. Results shown reflect the percentage of treated cells (relative to untreated cells). Each graph represents the average of three experiments; bars, ±SD.

Fig. 2.

Expression of Spy1 enhances cell survival with CPT-induced DNA damage in COS-1 and NIH3T3 cell lines. A, viability curves of mock COS-1 cells and Myc-Spy1-transfected COS-1 cells exposed to 0.1, 0.5, or 1.0 mm CPT for 18 h. B, viability curves of mock NIH3T3 cells and Myc-Spy1-transfected NIH3T3 cells exposed to 0.1, 0.5, or 1.0 mm CPT for 18 h. C, bar graph comparing cell viability of three cell lines exposed to 0.25 mm CPT. Mock, Myc-Spy1-transfected, and GFP-transfected cells were exposed to 0.25 mm CPT as described previously. Positive cells were counted via indirect immunofluorescence. Results shown reflect the percentage of treated cells (relative to untreated cells). Each graph represents the average of three experiments; bars, ±SD.

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Fig. 3.

Survival response of Spy1-expressing cells after CPT treatment. A, clonogenic survival curves for NIH3T3 cells expressing Myc-tagged Spy1 or empty vector (mock) treated with various doses of CPT. Results shown are the average number of colonies (containing >50 cells) formed 14 days after treatment and represent two independent experiments with duplicate plates per experiment. Right panel, immunoblot of Myc-Spy1 expression in NIH3T3 cell lysates from a representative experiment at day 0 (Lanes 3 and 4). As a control, the right bottom panel depicts actin expression. B, clonogenic survival curves for NIH3T3 cells expressing Myc-tagged Spy1 or empty vector (mock) treated with 10 μm CPT (at day 0). Results shown are the mean of survival (days after treatment) determined in three independent experiments with duplicate plates per experiment; bars, ±SE. Right panel, immunoblot of Myc-Spy1 expression in NIH3T3 cell lysates from a representative experiment at day 0 (Lanes 3 and 4). As a control, the right bottom panel depicts actin expression.

Fig. 3.

Survival response of Spy1-expressing cells after CPT treatment. A, clonogenic survival curves for NIH3T3 cells expressing Myc-tagged Spy1 or empty vector (mock) treated with various doses of CPT. Results shown are the average number of colonies (containing >50 cells) formed 14 days after treatment and represent two independent experiments with duplicate plates per experiment. Right panel, immunoblot of Myc-Spy1 expression in NIH3T3 cell lysates from a representative experiment at day 0 (Lanes 3 and 4). As a control, the right bottom panel depicts actin expression. B, clonogenic survival curves for NIH3T3 cells expressing Myc-tagged Spy1 or empty vector (mock) treated with 10 μm CPT (at day 0). Results shown are the mean of survival (days after treatment) determined in three independent experiments with duplicate plates per experiment; bars, ±SE. Right panel, immunoblot of Myc-Spy1 expression in NIH3T3 cell lysates from a representative experiment at day 0 (Lanes 3 and 4). As a control, the right bottom panel depicts actin expression.

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Fig. 4.

Alkaline comets photographed to visualize 4′,6-diamidino-2-phenylindole-stained DNA. A, NIH3T3 cells expressing empty vector (mock) or Myc-tagged Spy1 were treated with 10 μm CPT and then analyzed for fragmented DNA 4 days after treatment. Panels are as listed: (a) mock cell, (b) mock cell treated with CPT, (c) Spy1-expressing cell, (d) Spy1-expressing cell treated with CPT. B, quantitation of damaged DNA in mock and Spy1-expressing cells exposed to CPT as measured by the alkaline comet assay. Results represent the average of two independent experiments with 100 cells (nuclei) analyzed per experiment; bars, ±SD.

Fig. 4.

Alkaline comets photographed to visualize 4′,6-diamidino-2-phenylindole-stained DNA. A, NIH3T3 cells expressing empty vector (mock) or Myc-tagged Spy1 were treated with 10 μm CPT and then analyzed for fragmented DNA 4 days after treatment. Panels are as listed: (a) mock cell, (b) mock cell treated with CPT, (c) Spy1-expressing cell, (d) Spy1-expressing cell treated with CPT. B, quantitation of damaged DNA in mock and Spy1-expressing cells exposed to CPT as measured by the alkaline comet assay. Results represent the average of two independent experiments with 100 cells (nuclei) analyzed per experiment; bars, ±SD.

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Fig. 5.

Spy1-cdk2 association is required for cell survival. A, increased association between Spy1 and cdk2 with CPT exposure. Myc-Spy1-transfected 293T cells were exposed with or without 0.1 mm CPT for 12 h, harvested, lysed, and then immunoprecipitated with cdk2 antisera to examine the Spy1-cdk2 association under conditions of genotoxic stress. Whole cell lysate (WCL) samples (Lanes 1 and 2) and cdk2 immunoprecipitates (Lanes 3–6) were analyzed by 10% SDS-PAGE followed by immunoblotting with Myc antisera to detect Myc-tagged Spy1 expression (top panel). As a control, the bottom panel demonstrates the equal expression of endogenous cdk2 protein. B, cdk2-DN ablates the DNA damage response function of Spy1. Myc-Spy1-transfected, cdk2-DN-transfected, and Myc-Spy1/cdk2-DN-cotransfected 293T cells were exposed with or without 0.01 mm CPT for 20 h. Cells were counted via trypan blue exclusion. A representative experiment is shown depicting the average total cell number of each sample; bars, ±SE. Lysates were analyzed by 10% SDS-PAGE followed by immunoblotting with Myc antisera to detect Myc-Spy1 expression. C, DNA damage enhances Spy1-associated cdk2 kinase activity. Mock-transfected (Lanes 1, 3, 5, and 7) and Myc-Spy1-transfected (Lanes 2, 4, 6, and 8) 293T cells were exposed to 0.1 mm HU, 0.025 mm CPT, or 20 μm cisplatin for 24 h, as indicated. Cells were harvested 48 h after transfection, immunoprecipitated with antibodies against cdk2, and then assayed for kinase activity toward histone H1 (top panel). As a control, the middle panel shows the presence of cdk2 protein in cell lysates. The bottom panel demonstrates associated Spy1 protein in cdk2 immunoprecipitates.

Fig. 5.

Spy1-cdk2 association is required for cell survival. A, increased association between Spy1 and cdk2 with CPT exposure. Myc-Spy1-transfected 293T cells were exposed with or without 0.1 mm CPT for 12 h, harvested, lysed, and then immunoprecipitated with cdk2 antisera to examine the Spy1-cdk2 association under conditions of genotoxic stress. Whole cell lysate (WCL) samples (Lanes 1 and 2) and cdk2 immunoprecipitates (Lanes 3–6) were analyzed by 10% SDS-PAGE followed by immunoblotting with Myc antisera to detect Myc-tagged Spy1 expression (top panel). As a control, the bottom panel demonstrates the equal expression of endogenous cdk2 protein. B, cdk2-DN ablates the DNA damage response function of Spy1. Myc-Spy1-transfected, cdk2-DN-transfected, and Myc-Spy1/cdk2-DN-cotransfected 293T cells were exposed with or without 0.01 mm CPT for 20 h. Cells were counted via trypan blue exclusion. A representative experiment is shown depicting the average total cell number of each sample; bars, ±SE. Lysates were analyzed by 10% SDS-PAGE followed by immunoblotting with Myc antisera to detect Myc-Spy1 expression. C, DNA damage enhances Spy1-associated cdk2 kinase activity. Mock-transfected (Lanes 1, 3, 5, and 7) and Myc-Spy1-transfected (Lanes 2, 4, 6, and 8) 293T cells were exposed to 0.1 mm HU, 0.025 mm CPT, or 20 μm cisplatin for 24 h, as indicated. Cells were harvested 48 h after transfection, immunoprecipitated with antibodies against cdk2, and then assayed for kinase activity toward histone H1 (top panel). As a control, the middle panel shows the presence of cdk2 protein in cell lysates. The bottom panel demonstrates associated Spy1 protein in cdk2 immunoprecipitates.

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Fig. 6.

Endogenous Spy1 is up-regulated in response to DNA damage and is essential for the DNA damage response. A, 293T cells up-regulate Spy1 protein in response to CPT exposure. Lysates from 293T cells exposed with or without 40 μm CPT for 12 h were analyzed by 10% SDS-PAGE and then immunoblotted with human Spy1 antisera for expression of endogenous Spy1 (top panel). As a control, the bottom panel depicts actin expression. B, 293T cells up-regulate Spy1 protein in response to cisplatin or HU exposure. Lysates from 293T cells exposed to 40 μm cisplatin or 1 mm HU for 6 h were analyzed by 10% SDS-PAGE, and then immunoblotted with human Spy1 antisera for expression of endogenous Spy1 (top panel). As a control, the bottom panel depicts actin expression. C, ablation of Spy1 protein sensitizes 293T cells to DNA damage. siRNA techniques were used to ablate Spy1 mRNA and Spy1 protein. Bar graph depicting the percentage of viable 293T cells exposed to 0, 1.7, 3.3, or 6.7 μm CPT concentrations for 12 h (percentages reflect the fraction of CPT-treated samples over the DMSO-treated siControl sample). Total cell populations were counted via trypan blue exclusion. Results shown are of a representative experiment; bars, ±SE. Lysates were analyzed by 10% SDS-PAGE followed by immunoblotting with Spy1 antisera to detect endogenous Spy1 expression (top panel). As a control, the bottom panel depicts actin expression.

Fig. 6.

Endogenous Spy1 is up-regulated in response to DNA damage and is essential for the DNA damage response. A, 293T cells up-regulate Spy1 protein in response to CPT exposure. Lysates from 293T cells exposed with or without 40 μm CPT for 12 h were analyzed by 10% SDS-PAGE and then immunoblotted with human Spy1 antisera for expression of endogenous Spy1 (top panel). As a control, the bottom panel depicts actin expression. B, 293T cells up-regulate Spy1 protein in response to cisplatin or HU exposure. Lysates from 293T cells exposed to 40 μm cisplatin or 1 mm HU for 6 h were analyzed by 10% SDS-PAGE, and then immunoblotted with human Spy1 antisera for expression of endogenous Spy1 (top panel). As a control, the bottom panel depicts actin expression. C, ablation of Spy1 protein sensitizes 293T cells to DNA damage. siRNA techniques were used to ablate Spy1 mRNA and Spy1 protein. Bar graph depicting the percentage of viable 293T cells exposed to 0, 1.7, 3.3, or 6.7 μm CPT concentrations for 12 h (percentages reflect the fraction of CPT-treated samples over the DMSO-treated siControl sample). Total cell populations were counted via trypan blue exclusion. Results shown are of a representative experiment; bars, ±SE. Lysates were analyzed by 10% SDS-PAGE followed by immunoblotting with Spy1 antisera to detect endogenous Spy1 expression (top panel). As a control, the bottom panel depicts actin expression.

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We thank Ed Harlow and Sander van den Heuvel for the generous gift of the hemagglutinin-tagged DN cdk2 vector (pCMVcdk2D145N). We thank April N. Meyer for critical review of the manuscript, Laura J. Castrejon for editorial assistance, and Marjan Haghnia for reagents.

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