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Cancer Research 67, 9150, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-0025
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Cell Division Cycle 25B Phosphatase Is Essential for Benzo(a)Pyrene-7,8-Diol-9,10-Epoxide–Induced Neoplastic Transformation

Sanjay K. Srivastava1,3, Pallavi Bansal1,2, Tetsuya Oguri1, John S. Lazo1,2,3 and Shivendra V. Singh1,3

1 Department of Pharmacology, 2 Drug Discovery Institute, and 3 University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Requests for reprints: Shivendra V. Singh, Hillman Cancer Center Research Pavilion, Suite 2.32A, 5117 Center Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-7828; E-mail: singhs{at}upmc.edu or John S. Lazo, Department of Pharmacology, Drug Discovery Institute, University of Pittsburgh, Biomedical Science Tower 3, Suite 10040, 3501 Fifth Avenue, Pittsburgh, PA 15260. Phone: 412-648-9200; Fax 412-648-9009; E-mail: lazo{at}pitt.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell division cycle 25B (Cdc25B) phosphatase controls entry into mitosis and regulates recovery from G2-M checkpoint-induced arrest. In the present study, we show that exposure of diploid mouse embryonic fibroblasts (MEF) to the ultimate carcinogen anti-benzo(a)pyrene (BP)-7,8-diol-9,10-epoxide (anti-BPDE) resulted in a concentration- and time-dependent increase in Cdc25B protein levels. Chronic exposure of wild-type (Cdc25B+/+) MEFs to anti-BPDE (0.1 µmol/L) caused neoplastic transformation characterized by colony formation in culture and tumor production in nude mice. In contrast, the Cdc25B null MEFs were resistant to anti-BPDE–induced transformation. Furthermore, a carcinogenic dose of the parent hydrocarbon (BP) increased Cdc25B protein levels in the target organ, lung. The biological importance of elevated Cdc25B levels was documented by the early reentry into mitosis of cells overexpressing ectopic Cdc25B levels even in the presence of DNA damage following anti-BPDE exposure, whereas control cells resumed only after DNA damage was repaired. We conclude that Cdc25B has an essential role in anti-BPDE–induced neoplastic transformation, including regulation of cell cycle resumption in the presence of DNA damage. [Cancer Res 2007;67(19):9150–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Benzo(a)pyrene (BP) is the prototype of the polycyclic aromatic hydrocarbon family of environmental pollutants that are tumorigenic in experimental animals and suspected human carcinogens (1). The tumorigenic activity of BP has been attributed to its metabolite BP-7,8-diol-9,10-epoxide (anti-BPDE), which is formed via epoxidation and hydration reactions catalyzed by cytochrome P450–dependent monooxygenases and epoxide hydrolase, respectively (2–5). Anti-BPDE is highly reactive toward cellular nucleophiles, including DNA and glutathione (6, 7). Covalent interaction of the epoxide functional group of anti-BPDE with exocyclic amino groups of the purine bases in DNA is considered a critical reaction in the initiation of anti-BPDE–induced cancers (7).

Cell division cycle 25B (Cdc25B) is a dual-specificity phosphatase that plays pivotal role in controlling cell cycle progression by catalyzing removal of covalently attached contiguous phosphates and the subsequent activation of cyclin-dependent kinase 1 (Cdk1; ref. 8). Among the Cdc25 family members, Cdc25B has been proposed to regulate reentry into mitosis after DNA damage (8–10). Importantly, Cdc25B is oncogenic (11) and its overexpression has been documented in a variety of human cancers, including head and neck, colon, and non–small cell lung cancer (12–14). Cdc25B also interacts with the steroid receptors (15), suggesting that some Cdk-independent sites might contribute to the oncogenic potential of Cdc25B. In addition, transgenic mice that overexpress Cdc25B in mammary epithelium exhibit mammary gland hyperplasia and increased susceptibility to 9,10-dimethyl-1,2-benzanthracene–induced mammary tumorigenesis (16, 17).

We showed previously that anti-BPDE exposure of lung cancer cells and terminal squamous differentiated human bronchial epithelial cells, but not undifferentiated bronchial epithelial cells, resulted in induction of Cdc25B protein expression (18). In addition, the growth rate of lung cancer cells increased significantly compared with untreated cells following chronic exposure to anti-BPDE (18). In the present study, we markedly extended these findings by showing that Cdc25B was essential for anti-BPDE–induced neoplastic transformation using diploid mouse embryonic fibroblasts (MEF) from wild-type (WT; Cdc25B+/+) and Cdc25B null mice (Cdc25B–/–). We also provide evidence that Cdc25B could promote tumorigenesis by accelerating the kinetics of cell cycle resumption even in the presence of DNA damage following anti-BPDE treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Diploid MEFs derived from WT and Cdc25B null mice were generously provided by Dr. Peter J. Donovan (University of California, Irvine, CA; ref. 19). Normal male diploid CCL-202 human lung fibroblasts were obtained from American Type Culture Collection. BP and its metabolites were obtained from the National Cancer Institute, Chemical Carcinogen Reference Standard Repository. Gö6976 was purchased from EMD Biosciences. The antibody against Cdc25B, which has been extensively validated by us (20), was from Transduction Laboratories. Other antibodies used were as follows: anti–Tyr15 Cdk1 and anti–checkpoint kinase 1 (Chk1; Sigma-Aldrich Co.); anti-actin, p53, and p21 (Santa Cruz Biotechnology); anti–histone H3 (Ser10; Upstate Biotechnology); and anti-Chk1 (Ser317), p53 (Ser15), and {gamma}H2AX (Cell Signaling Technology). Six- to 8-week-old female athymic mice were purchased from Harlan Sprague-Dawley, whereas 6- to 8-week-old female A/J mice were procured from the National Cancer Institute (Frederick, MD).

Cell culture and immunoblotting methods. WT diploid MEF and Cdc25B null MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, and antibiotics at 37°C in a humidified atmosphere of 5% CO2 and 95% air. CCL-202 cells were maintained in Eagle's minimum essential medium supplemented with 10% FBS and 2 mmol/L L-glutamine. The MEFs were exposed to the desired concentration of anti-BPDE, BP, BP-7,8-diol, or BP-7,8,9,10-tetrol for the indicated times. Stock solutions of BP and its metabolites were prepared in DMSO, and an equal volume of DMSO was added to the controls. Treated and control MEFs were harvested using EDTA-trypsin, washed with ice-cold PBS, and lysed with a solution containing 50 mmol/L Tris, 1% Triton X-100, 0.1% SDS, 150 mmol/L NaCl, 2 mmol/L Na3VO4, 2 mmol/L EGTA, 12 mmol/L ß-glycerol phosphate, 10 mmol/L NaF, 16 µg/mL benzamidine-HCl, 10 µg/mL phenanthroline, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and 1 nmol/L phenylmethylsulfonyl fluoride. After a 30-min incubation on ice, the cell lysate was cleared by centrifugation at 14,000 rpm for 30 min. Equal amounts of lysate proteins were subjected to SDS-PAGE followed by immunoblotting as described by us previously (18).

Transformation assay. WT as well as Cdc25B null MEFs (~2 x 104) were seeded in T25 flasks and allowed to attach overnight. The medium was replaced with fresh complete medium containing 0.1 µmol/L anti-BPDE or an equal volume of DMSO. Three separate cultures of both types of MEFs were independently exposed to anti-BPDE or DMSO. After a 48-h incubation at 37°C, the medium was replaced with fresh complete medium without anti-BPDE or DMSO and the fibroblasts were allowed to attain 70% to 80% confluency. The MEFs were harvested by trypsin treatment, reseeded, and exposed again to anti-BPDE or DMSO as described above. This treatment protocol was repeated four times over a period of 4 weeks. After the fourth exposure, a change in morphology was noticed for anti-BPDE–exposed WT MEFs. At this stage, anti-BPDE and DMSO treatments were stopped.

Colony formation assay. The anchorage-independent growth of MEFs was determined by colony formation efficiency in soft agar. Approximately 3,000 DMSO- or BPDE-treated MEFs were mixed at 37°C with 2 mL of 0.33% (w/v) soft agar (Sigma-Aldrich) and then poured onto a layer of previously set 2 mL of 0.9% soft agar (w/v) in six-well tissue culture plates. The soft agar suspensions were prepared in the complete DMEM. Cells were incubated for 2 weeks and then the growth of colonies was observed under an inverted microscope (Zeiss Thornwood) at x10 magnification.

Cell cycle resumption and DNA damage studies. U2OS cells expressing HA-Cdc25B3 under a tetracycline (Tet)–regulated promoter (a gift from Prof. Bernard Ducommun, Université Paul Sabatier, Toulouse, France) were maintained in DMEM constituted with 10% FBS, 100 µg/mL G418, 1% penicillin-streptomycin, and 2 µg/mL Tet as described previously (10). Cells were plated 24 h before inducing Cdc25B for 16 h by removing Tet. Control cells were cultured in the presence of Tet to suppress ectopic Cdc25B expression. We then examined mitotic reentry of asynchronous cell populations treated with 100 nmol/L anti-BPDE. Cells were either harvested 0 to 24 h after anti-BPDE treatment in cold lysis buffer for immunoblotting, as described above, or fixed in ice-cold 70% ethanol for flow cytometry. Induction of DNA damage in the presence of elevated levels of Cdc25B was examined by immunoblotting for phosphorylated {gamma}H2AX, p53, and Chk1. For flow cytometry analysis, cells were fixed overnight, washed with PBS containing 1% bovine serum albumin, and permeabilized with PBS containing 0.25% Triton X-100 for 8 min on ice. Following washing, cells were then incubated for 2 h with 1 µg anti–phospho-Ser10-histone H3, a well-accepted marker of mitosis. Cells were washed an additional time before incubating with antirabbit IgG-Alexa 488 (1:75) for 40 min in the dark. Finally, cells were washed again and resuspended in 500 µL propidium iodide for 15 min before flow cytometry analysis.

Animal experiments. For tumor xenograft assay, anti-BPDE– or DMSO-exposed WT or Cdc25B null MEFs (106 cells) were s.c. injected into the right flank of athymic mice (four mice per group). Animals of each group were monitored thrice weekly for the appearance of tumors. Single tumors were formed and the tumor volume was calculated as described previously (21). To determine in vivo effect of BP administration on pulmonary and hepatic Cdc25B protein level, we fed female A/J mice an AIN-76 semipurified (ICN) diet for 1 week before treatment. Mice were divided into two groups of 12 mice per group. The experimental group of mice was p.o. gavaged with 2 mg BP in 0.1 mL corn oil, whereas the control animals received an equal volume of corn oil alone. Four mice from both groups were sacrificed at 6, 12, and or 24 h after BP or corn oil administration. Lung and liver tissues were collected, washed with ice-cold PBS, and stored at –80°C until processed for immunoblotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-BPDE increases Cdc25B protein levels. To determine whether anti-BPDE altered Cdc25B levels in MEFs, we treated WT MEFs with various concentrations of anti-BPDE. As shown in Fig. 1A , we noted a concentration-dependent induction of Cdc25B with a 4-fold increase at 5 and 10 µmol/L anti-BPDE. In time course studies using 10 µmol/L anti-BPDE (Fig. 1B), the induction of Cdc25B protein level was evident as early as 4 h after treatment and persisted for the duration of the experiment (24 h after treatment). We saw no significant cytotoxicity when MEFs were treated for 24 h with 10 µmol/L anti-BPDE consistent with our previous observations with 128-88T and A549 cells (18). In agreement with the known function of Cdc25B in catalyzing dephosphorylation of Thr14 and Tyr15 of Cdk1 (8), anti-BPDE–mediated induction of Cdc25B protein was accompanied by a sharp decline in the level of Tyr15 Cdk1 (Fig. 1B) similar to terminal squamous differentiated bronchial epithelial cells and lung cancer cells (18). Next, we addressed whether induction of Cdc25B protein was unique to anti-BPDE by determining the effects of parent hydrocarbon (BP), BP-7,8-diol (precursor of anti-BPDE), and BP-7,8,9,10-tetrol (hydrolysis product of anti-BPDE) on Cdc25B protein level by immunoblotting (Fig. 2 ). Unlike anti-BPDE, Cdc25B levels were marginally increased by treatment of MEFs with BP-7,8-diol or seemed to be decreased by either BP or BP-7,8,9,10-tetrol (Fig. 2). The decrease in Cdc25B was unexpected and requires additional studies to explain. Nonetheless, these results suggested that the epoxide functional group of anti-BPDE was required for inducing Cdc25B protein levels.


Figure 1
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  		Figure 1. Anti-BPDE increases Cdc25B protein levels in MEFs. A, immunoblotting for Cdc25B protein using lysates from WT MEFs treated for 24 h with the indicated concentrations of anti-BPDE. The membrane was stripped and reprobed with anti-actin antibody to ensure equal protein loading. Data are from a representative experiment that was repeated thrice using independently prepared lysates with similar results. B, immunoblotting for Cdc25B and Tyr15 Cdk1 using lysates from WT MEFs treated with 10 µmol/L anti-BPDE for the indicated times. The membrane was stripped and reprobed with anti-actin antibody to ensure equal protein loading. Data are from a representative experiment that was repeated twice with similar results. The numbers above are the numerical representation of the mean densitometry values normalized to actin. p-Cdk1, phosphorylated Cdk1.

 

Figure 2
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  		Figure 2. Increase in Cdc25B levels is dependent on anti-BPDE. Immunoblotting for Cdc25B protein using lysates from WT MEFs treated for 24 h with the indicated concentrations of BP (A), BP-7,8-diol (B), or BP-7,8,9,10-tetrol (C). The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Immunoblotting was done at least twice using independently prepared lysates, and the results were comparable. The numbers above are the numerical representation of the mean densitometry values normalized to actin.

 
Cdc25B is essential for anti-BPDE–induced tumorigenesis. We next determined the potential functional significance of Cdc25B induction after anti-BPDE treatment. We postulated that if Cdc25B induction was important in anti-BPDE–induced neoplasia, it might affect anti-BPDE–induced neoplastic transformation. Using previously described MEFs from WT and Cdc25B null mice (19), we observed that the WT MEFs began to form colonies of rounded cells after repeated exposures to 0.1 µmol/L anti-BPDE over a period of 4 weeks (Fig. 3A , III). Similar results were seen in three separate cultures of the WT MEFs that were exposed to anti-BPDE. In contrast, anti-BPDE treatment of Cdc25B null MEFs did not cause a visible change in their morphology (Fig. 3A, IV). The fibroblast-like morphology was also maintained when either the WT or the Cdc25B null MEFs were chronically exposed to DMSO (Fig. 3A, compare I and II). To confirm the role of Cdc25B in anti-BPDE–induced transformation, we plated DMSO- or anti-BPDE–treated WT and Cdc25B null MEFs in soft agar and observed colony formation 2 weeks later. As shown in the Supplementary Fig. S1 (I–IV), only anti-BPDE–treated WT MEFs formed colonies. These results substantiate that Cdc25B has an important role in anti-BPDE–induced transformation.


Figure 3
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  		Figure 3. Cdc25B is essential for anti-BPDE–induced tumorigenesis. A, light microscopy of crystal violet–stained cultures of DMSO-exposed (I and II) or anti-BPDE–exposed (III and IV) MEFs derived from WT mice (I and III) or Cdc25B null mice (II and IV). Note that only anti-BPDE–exposed WT MEFs (III) acquired the ability to form colonies. B, tumorigenic potential of anti-BPDE–exposed WT MEFs in female athymic mice. Compare II of B for a representative mouse of this group. C, growth curve for tumors on s.c. injection of 106 anti-BPDE–exposed WT MEFs. Tumor volume data. Points, mean (n = 4); bars, SD.

 
To confirm the transformed phenotype in vivo, we s.c. injected athymic mice with the anti-BPDE–exposed WT MEFs. As seen in Fig. 3B (II), the anti-BPDE–exposed WT MEFs produced tumors after s.c. injection in nude mice. Tumors were observed in each of the four mice that were injected with anti-BPDE–exposed WT MEFs. Figure 3C depicts the growth curve for tumors resulting from s.c. injection of anti-BPDE–exposed WT MEFs. In contrast, the anti-BPDE–exposed Cdc25B null MEFs failed to produce tumors in nude mice (compare IV of Fig. 3B for a representative mouse of this group). The DMSO-exposed MEFs of both types (WT and Cdc25B null) were also nontumorigenic in nude mice (Fig. 3B, I and III, respectively).

BP administration increases pulmonary Cdc25B in vivo. To further test the hypothesis that the anti-BPDE induction of Cdc25B protein was biologically relevant, we examined the in vivo effect of BP administration to mice on Cdc25B protein content in the lung, which is a target organ for BP-induced cancer in mice. Female A/J mice, a strain sensitive to BP-induced pulmonary tumorigenesis (22), were p.o. treated with a carcinogenic dose of BP or an equal volume of the corn oil (vehicle for BP). Four mice from both groups were sacrificed at different time intervals, and the lungs and livers were collected and processed for Cdc25B immunoblotting (Fig. 4 ). Densitometric scanning of the Cdc25B immunoreactive bands followed by normalization to loading control (actin) revealed that 24 h after BP administration, there was a significant increase in pulmonary Cdc25B, which was accompanied by decrease in the level of Tyr15 Cdk1 at 24 h after BP (Fig. 4A). In contrast, hepatic Cdc25B protein level was unaffected by BP administration (data not shown), as would be predicted by the lack of BP-induced hepatocarcinogenesis in this mouse model. As shown in Fig. 4B, Cdc25B was also induced in normal diploid human lung fibroblasts (CCL-202) when treated with anti-BPDE (10 µmol/L) in a time-dependent manner.


Figure 4
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  		Figure 4. BP administration increases pulmonary Cdc25B protein level in vivo and in diploid lung fibroblasts. A, the effect of BP treatment on pulmonary Cdc25B and p-Cdk1 protein level was determined by immunoblotting using 14,000 x g supernatant fractions from the lungs of control (corn oil treated) and BP-treated mice. Columns, mean after normalizing with actin loading control (n = 3); bars, SE. *, P < 0.05, control versus treated groups by Student's t test. B, CCL-202 cells were plated 24 h before exposure to anti-BPDE. Cells were treated with anti-BPDE and harvested at the indicated time points. CCL-202 cells were pretreated with Gö6976 (1 µmol/L) for 30 min or DMSO before anti-BPDE (1 µmol/L) or DMSO treatment for 1 h. Immunoblotting was done as indicated. Representative of two independent experiments done in duplicate.

 
Cdc25B induction involves the Chk1 pathway. Recently, we have observed that UV irradiation increases Cdc25B protein levels by activating ATR/Chk1 pathway potentially via post-transcriptional mechanism (20). Similarly, in recent studies, it was shown that replication fork stalled by anti-BPDE–adducted DNA activates ATR/Chk1 (23, 24). To test the hypothesis that anti-BPDE increases Cdc25B by activating Chk1, we first confirmed Chk1 activation in CCL-202 cells following anti-BPDE treatment (data not shown). To block Chk1 activation, we pretreated cells with Gö6976 (1 µmol/L) for 30 min followed by anti-BPDE treatment (1 µmol/L) for an additional 1 h. As shown in Fig. 4B, pretreatment with Chk1 inhibitor Gö6976 (25) blocked the increase in Cdc25B protein levels.

Cdc25B regulates recovery in the presence of DNA damage. Cdc25B has been identified recently to have a crucial role in the cell cycle resumption following DNA damage (9, 10) and this process might enable cells to survive genotoxic stress. A disruption in the presumptive protective pathway could contribute to tumor development by permitting cell division without completion of DNA repair. We therefore postulated that BPDE-induced transformation might be linked to Cdc25B induction and the subsequent promotion of cell cycle resumption in the presence of DNA damage. To test this hypothesis, we first examined mitotic reentry in asynchronous cell populations with differing levels of Cdc25B (22) after exposure to anti-BPDE (100 nmol/L). Tet was removed from U2OS cells for 16 h to induce Cdc25B expression before anti-BPDE treatment. Using histone H3 Ser10 phosphorylation as a marker of mitotic reentry, we found that both control and Cdc25B-overexpressing U2OS cells initially activate a G2-M checkpoint as shown by the reduction in the percentage of mitotic cells at 2 h after treatment. The relative mitotic ratio of anti-BPDE–treated control cells was 0.77 ± 0.06%, which was similar to the anti-BPDE–treated Cdc25B-overexpressing cells (0.71 ± 0.11%; Fig. 5A and B ). In contrast, 12 h after anti-BPDE treatment, there was a pronounced inhibition of mitotic reentry in cells with suppressed ectopic Cdc25B (+Tet; 0.19 ± 0.01%), whereas Cdc25B-overexpressing U2SO cells (–Tet) had a higher mitotic ratio (0.46 ± 0.08%). Twenty-four hours after treatment, Cdc25B-overexpressing cells (–Tet) were fully recovered as indicated by percentage of mitotic cells of 1.16 ± 0.06%, whereas control cells populations (+Tet) continued to restrict cellular entry into mitosis (0.53 ± 0.04%). Furthermore, based on the DNA profile of cells at 12 h compared with 0 and 2 h, DMSO-treated cell progressed through cell cycle as indicated by increase in the number of cells in G2 phase. Cell cycle progression of anti-BPDE–treated cells was delayed with a majority of cells in S phase (Supplementary Fig. S2). Additionally, 24 h after anti-BPDE treatment, 47.9 ± 3.6% of control cells and 40.5 ± 1.8% of Cdc25B-overexpressing cells were in G2-M phase, indicating cell progression from S phase at 12 h. In contrast, DMSO-treated cells potentially divided between 12 and 24 h as indicated by increased percentage of cells in G1 (29.6 ± 2.0% of control cells and 19.7 ± 0.7% of Cdc25B-overexpressing cells), suggesting that anti-BPDE–treated cells were delayed in cell cycle compared with DMSO-treated cells. As shown in Fig. 5C, treatment with anti-BPDE caused DNA damage within 2 h after treatment indicated by {gamma}H2AX phosphorylation. The intensity of {gamma}H2AX phosphorylation increased with time (4 and 8 h) in BPDE-treated samples. Cell populations that maintained high ectopic Cdc25B levels displayed prolonged {gamma}H2AX irrespective of exposure to anti-BPDE. It is noteworthy that Cdc25B-overexpressing cells treated with anti-BPDE resumed cell cycle progression despite this apparent DNA damage.


Figure 5
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  		Figure 5. Cdc25B regulates recovery in the presence of DNA damage. A, U2OS cells were cultured for 16 h in the presence (WT Cdc25B) or absence of Tet (elevated Cdc25B). The two populations were then treated with either DMSO or anti-BPDE (100 nmol/L). Cells were fixed at the indicated time points and stained for phospho-histone H3. B, percentage of mitotic cells following anti-BPDE treatment normalized to respective DMSO control fixed at the same time as anti-BPDE–treated cells. Columns, mean (n = 4); bars, SE. *, P < 0.05; ***, P < 0.0001, determined by two-tailed unpaired t test. C, immunoblotting of cell lysates for {gamma}H2AX from Cdc25B-induced or WT U2OS cells treated with DMSO or anti-BPDE. Cells were harvested at different time points as indicated. The experiment was repeated twice using independently prepared lysates with similar results.

 
Cdc25B induction deregulates checkpoints. Recent studies have shown that in early precursor lesions, activated oncogenes, such as Cdc25A, cyclin E, Cdc6, and E2F, could aberrantly regulate cell cycle progression, thus causing replication stress and DNA damage, which potentially activates checkpoint response, including phosphorylated ATM, Chk2, phosphorylated {gamma}H2AX, and p53 (26, 27). Activation of checkpoints thus leads to cell death or senescence, thereby preventing tumor progression, but the development of defects in the checkpoint response could lead to genetic instability. The exact mechanism by which the defects in the checkpoint response are accumulated is not completely understood but one mechanism may be selection pressure induced by activated oncogenes. Because overexpression of Cdc25B alone caused phosphorylation of {gamma}H2AX (Fig. 5C), we tested whether Cdc25B also functions similarly to Cdc25A where overexpression of Cdc25A in U20S cells was shown to activate checkpoints. Thus, we examined the phosphorylation status of {gamma}H2AX, Ser15 on p53, and Ser317 on Chk1 after chronic treatment with BPDE. As shown in Fig. 6A , hyperphosphorylation of these DNA damage–sensing proteins was sustained for at least 5 days after elevation of ectopic Cdc25B. UV-treated cell lysate was used as a positive control for the phosphorylation of {gamma}H2AX, activation of checkpoints. These results are consistent with the generation of a stressful environment after elevation of Cdc25B, which could be ideal for selection of cells with compromised checkpoints. Interestingly, Cdc25B overexpression for 5 days did not affect the cell growth as was reported previously (10). Cdc25B overexpression can reduce clonogenic survival, however (10). Chronic treatment of WT MEF with anti-BPDE resulted in the loss of p53 and p21, also consistent with compromised checkpoints. This loss seemed to at least partially require the presence of Cdc25B as loss of p53 was attenuated in the Cdc25B null MEFs (Fig. 6B and C). Surprisingly, p21 levels increased in Cdc25B null MEFs after anti-BPDE treatment.


Figure 6
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  		Figure 6. Cdc25B induction could deregulate checkpoints. A, overexpression of Cdc25B induces DNA damage. U2OS cells were harvested 1 to 5 d after Tet removal and DNA damage was assessed by immunoblotting for phosphorylated p53 or Chk1 or expression of {gamma}H2AX. Overexpression of Cdc25B was confirmed using a HA antibody. Representative of two independent experiments done in duplicate with similar results. B, immunoblotting for p53 and p21 proteins using lysates from WT (Cdc25B+/+) and Cdc25B null (Cdc25B–/–) MEFs chronically exposed to 0.1 µmol/L anti-BPDE (T) or DMSO (C) over a period of 4 wks. The experiment was repeated twice using independently prepared lysates, and the results were similar. C, densitometric scanning data for p53 and p21 immunoblots after normalization to actin loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Cdc25 phosphatases are vital controllers of cell entry and progression into mitosis. Our understanding of the relative importance of each of the family members in the regulation of Cdk-cyclin complexes after DNA damage–induced cell cycle arrest continues to evolve. Although initial studies using mice lacking functional Cdc25B suggested a relatively normal phenotype, more recent data from several groups indicate that Cdc25B is required for an efficient exit from G2 arrest (9, 10, 20). Proteins, such as polo-like kinase-1 (Plk1), which controls the half-life of the Cdc25-inactivating kinase Wee1, are also required for this process (9). These observations have led to the hypothesis that an increase in endogenous Cdc25B could be part of the normal response to recover from DNA damage. The data presented herein indicate that induction of Cdc25B protein could be an important event in anti-BPDE–induced neoplasia. Consistent with previous studies (16, 17), treatment of WT MEFs with anti-BPDE resulted in a transformed phenotype that was characterized by colony formation in culture, soft agar, and tumor production in nude mice on s.c. injection. In contrast, MEFs derived from Cdc25B null mice failed to form colonies or to produce tumors in athymic mice (Fig. 3). Treatment of WT MEFs with the parent hydrocarbon failed to increase Cdc25B protein level, which was probably attributable to poor bioactivation of BP in MEFs. In contrast to cells treated with anti-BPDE, cells treated with either BP or BP-tetrol, which is a nontumorigenic metabolite of anti-BPDE, seemed to have reduced levels of Cdc25B (Fig. 2). We have no explanation at this time for the apparent decrease seen with BP or BP-tetrol but this is worthy of further study. Collectively, however, these data suggest that the epoxide functional group, which is essential for DNA damage, has an important role in anti-BPDE–mediated induction of Cdc25B protein.

The induction of a Cdc25 family member after carcinogen-mediated DNA damage would seem at first rather surprising as this could facilitate Cdk activation and cell cycle progression rather than checkpoint activation. Indeed, with most DNA-damaging agents, Cdc25A is phosphorylated and rapidly degraded (28–31). Recent studies indicate that anti-BPDE can induce Chk1- and Chk1-dependent degradation of Cdc25A (24, 32). Our results showing that cells activate G2-M checkpoint immediately following anti-BPDE irrespective of Cdc25B levels (Fig. 5) is consistent with the model that initial cell cycle arrest following DNA damage might be more dependent on the loss of Cdc25A. If, however, there is an increase in basal level of Cdc25B due to chronic exposure to DNA-damaging anti-BPDE, then resumption could occur in the presence of DNA damage (Fig. 6). Such an event could explain why transgenic mice overexpressing Cdc25B in mammary epithelium have increased susceptibility to 9,10-dimethyl-1,2-benzanthracene–induced mammary tumorigenesis (17). These results are analogous to a recent study showing that Chk1 and Plk1 regulate checkpoint adaptation/premature recovery in human cells following ionizing radiation (33). These findings suggest that human cells, like their yeast counterpart, can divide in the presence of DNA damage, which could increase the risk in diploid cells for development of genetically unstable cells that may progress toward cancer. Interestingly, positive regulators (Plk1 and Cdc25B) of premature recovery/checkpoint adaptation are overexpressed in many human tumors and negative regulator (Chk1) is mutated in several malignancies (30, 34–37). Taken together, these findings indicate that Cdc25B could promote premature recovery from checkpoints without completion of DNA repair.

It is interesting to note that the increased expression of Cdc25B even in the absence of an overt DNA-damaging agent also resulted in DNA damage and activation of checkpoints (Fig. 6). These results are consistent with previous reports describing activation of checkpoints in response to replication stress induced by overexpression of Cdc25A, cyclin E, and E2F (26, 27). Based on these results, it was proposed that in precancerous lesions, oncogenic stress could regulate selection of cells with compromised checkpoint signaling, thus leading to genetic instability and tumor progression. Hence, markedly reduced level of p53 in the chronically anti-BPDE–exposed WT MEFs with a transformed phenotype is consistent with a role for Cdc25B in checkpoint deregulation. Surprisingly, p21 levels increased in anti-BPDE–treated Cdc25B null MEFs. These results are very similar to Wani et al. (38), who found increased p21 levels in response to anti-BPDE treatment irrespective of p53 levels.

Finally, induction of Cdc25B may not be restricted to the carcinogen anti-BPDE. Recently, Cdc25B was found to be elevated in cells chronically exposed to ionizing radiation. Repeated exposure to ionizing radiation leads to tumorigenic phenotype in these cells. These results suggest that increase in Cdc25B levels could potentially play an important role in tumorigenesis induced by several different DNA-damaging agents (39).


   Acknowledgments
 
Grant support: National Cancer Institute USPHS grants CA076348, CA078039, CA 106953, and CA052995; National Institute of Environmental Health Sciences grant ES009140; the Fiske Drug Discovery Fund; and the Lung Cancer Specialized Programs in Research Excellence CA 90440. Department of Pharmacology Predoctoral Fellowships (P. Bansal).

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.

We thank Profs. Bernard Ducommun and Peter Donovan for providing U2OS Tet-regulated Cdc25B cells and WT and Cdc25B null MEFs and Yan Shi for technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

S.K. Srivastava and P. Bansal contributed equally to this work.

Received 1/ 3/07. Revised 7/ 2/07. Accepted 7/31/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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