Nasopharyngeal carcinoma is a cancer with its highest prevalence among the southern Chinese and is rare elsewhere in the world. The main treatment modalities include chemotherapy and radiotherapy. However, tumor chemoresistance often limits the efficacy of nasopharyngeal carcinoma treatment and reduces survival rates. Thus, identifying new selective chemotherapeutic drugs for nasopharyngeal carcinoma treatment is needed. In this current study, the antitumor efficacy of a polo-like kinase inhibitor, Ro5203280, was investigated. Ro5203280 induces tumor suppression both in vitro and in vivo. An inhibitory effect was observed with the highly proliferating cancer cell lines tested, but not with the nontumorigenic cell line. Real-time cell proliferation and fluorescence-activated cell sorting (FACS) analysis, together with immunohistochemical (IHC), immunofluorescence, and Annexin V staining assays, were used to evaluate the impact of drug treatment on cell cycle and apoptosis. Ro5203280 induces G2–M cell-cycle arrest and apoptosis. Western blotting shows it inhibits PLK1 phosphorylation and downregulates the downstream signaling molecule, Cdc25c, and upregulates two important mitosis regulators, Wee1 and Securin, as well as the DNA damage-related factor Chk2 in vitro and in vivo. In vivo tumorigenicity assays with Ro5203280 intravenous injection showed its potent ability to inhibit tumor growth in mice, with no observable signs of toxicity. These findings suggest the potential usefulness of Ro5203280 as a chemotherapeutic targeting drug for nasopharyngeal carcinoma treatment. Mol Cancer Ther; 12(8); 1393–401. ©2013 AACR.

Nasopharyngeal carcinoma is a unique malignancy with an exceptionally high incidence in Southeast Asia, but it is rare elsewhere in the world. There are several cofactors that are believed to contribute to nasopharyngeal carcinoma development, including host genetics, Epstein–Barr virus (EBV) infection, and environmental factors. The loss of function of tumor suppressor genes (TSG) and loss of control of cell-cycle–regulating genes such as cyclin D1 (1) are important for nasopharyngeal carcinoma development. The nasopharynx is located deep inside the skull, making surgical excision a difficult first option line of therapy. Therefore, chemotherapy and radiotherapy are commonly used to treat patients with nasopharyngeal carcinoma. At present, the choices for chemotherapy in nasopharyngeal carcinoma are limited and chemoresistance often contributes to treatment failures. Therefore, there is a real need to identify alternative drugs for selectively targeting nasopharyngeal carcinoma.

In this current study, the drug Ro5203280 was tested and confirmed to inhibit nasopharyngeal carcinoma tumor cell growth. This drug mainly targets the polo-like kinase 1 (PLK1) protein. PLK1 is one of the well-studied members of the PLK family, which is an important regulator of different signaling pathways that are responsible for cell-cycle progression and regulation of mitosis (2). PLK1 expression is commonly detected in the highly proliferating adult tissues, including the spleen and testis (3), and it serves as a marker of cellular proliferation (4). Overexpression of PLK1 is observed in many different malignancies including nasopharyngeal carcinoma, non–small cell lung cancer (NSCLC), breast, endometrium, colon, esophagus, ovary, pancreas, stomach, prostate, glioblastoma, leukemia, melanoma, and also other head and neck cancers (3, 5–8). The overexpression of PLK1 was reported to associate with the high mitotic rate found in cancer (9).

The PLK1 protein plays various functional roles in G2–M cell-cycle control. It regulates the entry of cells into mitosis by phosphorylating the Wee1 protein and facilitating its degradation (10), and thereby activating the cyclin B/Cdk1 complex activity. Cdc25c regulates Cdk1 protein activation by removing the inhibitory phosphorylation of the Cdk1 protein (11). PLK1 also plays a role in the G2–M checkpoint. It is inhibited as a response to DNA damage-responsive ATM and ATR proteins (12). Moreover, PLK1 takes part in regulating the mitotic spindle formation by controlling the recruitment of γ-tubulin to centrosomes (13). PLK1 can also activate the anaphase-promoting complex (APC) to promote DNA segregation (14). This suggests that PLK1 plays an important role in cancer.

On the basis of the unique and important function of PLK1 in cancer, PLK1 has been molecularly targeted for cancer treatment. Interestingly, injection of a PLK1-specific antibody to transformed cells can induce mitotic arrest (13). Several PLK1 inhibitors, including BI2536, BI6727, Volasertib, GSK461364, and HMN-214, were used in clinical trials for colorectal cancer, NSCLC, and other type of solid tumors (15–17). Phase II clinical studies of BI2536 have now been completed. In a recent study, an orally available PLK1-specific inhibitor was reported to induce cell-cycle arrest and apoptosis in cancer cell lines and xenograft tumor models (18). These earlier studies all suggest an important potential role of chemotherapeutic PLK1 inhibitors for targeted cancer treatment. Ro5203280 is a new PLK1 inhibitor that is now undergoing preclinical drug studies. Its structure is a close analog to BI2536, but has improved activity to PLK1. Therefore, more studies are warranted to investigate and confirm the functions of this new drug in inhibiting tumor growth.

In nasopharyngeal carcinoma, the function of PLK1 is still unclear and there are no reports to date on investigating the usefulness of PLK1 inhibitors in nasopharyngeal carcinoma chemotherapy. In this study, the function of the novel PLK1 inhibitor Ro5203280 was investigated. Results showed its efficacy in inhibiting nasopharyngeal carcinoma tumor cell growth in vitro and tumor formation in vivo by inducing cell-cycle arrest and apoptosis.

Cell lines

Three nasopharyngeal carcinoma cell lines (HONE1, HK1, and C666-1) and an immortalized nasopharyngeal epithelial cell line (NP460) were used in this current study. HONE1 and C666-1 were established from poorly differentiated nasopharyngeal carcinoma tumors, whereas HK1 was from a well-differentiated nasopharyngeal carcinoma. C666-1 is the only EBV-positive nasopharyngeal carcinoma cell line available for studies. NP460 is a normal nasopharyngeal epithelial cell line, which was immortalized by human telomerase. Culture conditions for these cell lines were as described previously (19–22). All cell lines used in the current study were tested and confirmed to be mycoplasma negative. They were obtained from the Hong Kong NPC AoE Cell Line Repository and have been authenticated using the AmpFℓSTR Identifier PCR Amplification kit (Life Technologies).

Drug preparation and Ambit selectivity screen

The Ro5203280 drug was provided by Roche Pharma Research and Early Development (23). The nocodazole was purchased from Sigma-Aldrich. For in vitro studies, Ro5203280 and nocodozole were dissolved in dimethyl sulfoxide (DMSO) solution. For the in vivo studies, the Ro5203280 was dissolved in a vehicle consisting of PEG400/acetate buffer (pH 5.0). The Ambit selectivity screen was conducted according to the manufacturer's protocol (Ambit Biosciences).

Real-time cell proliferation assay

The real-time cell proliferation assay was conducted using the E-plate and the xCelligence System (Roche) according to the manufacturer's instruction. In brief, a total of 1 × 104 HONE1, 1 × 104 HK1, 2 × 104 C666-1, and 2 × 104 NP460 cells were seeded onto an E-plate. Drug treatment was begun 24 hours after cell seeding. The cell proliferation index was automatically recorded by the xCelligence system computer program. All cell lines were treated with varying concentrations of Ro5203280 from 25, 50, 100, 200, 500, and 1,000 nmol/L or with DMSO, which is the in vitro vehicle control. The percentage of cell proliferation inhibition was calculated using the formula: % inhibition = 100 − (cell index of the treated well/cell index of untreated well) × 100% (24). All experiments were carried out in duplicate at least twice.

In vivo nude mouse tumorigenicity assay

The in vivo nude mouse tumorigenicity assay was conducted as described previously (25). All mice were kept in the Laboratory Animal Unit in the University of Hong Kong [Hong Kong (SAR), People's Republic of China] according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) international guidelines. In brief, a total of 1 × 107 cells of the tumorigenic nasopharyngeal carcinoma cell lines, HONE1 and C666-1, were injected subcutaneously into 2 groups of athymic BALB/c Nu/Nu 6 to 8-week old nude mice. A total of 44 and 27 mice were used for the in vivo nude mouse tumorigenicity assay for HONE1 and C666-1 cell lines, respectively. Tumor sizes were measured weekly. After the tumors reached a size of 100 mm3 or more, the drug was then injected into the mice intravenously. Drug concentrations of 15 mg/kg body weight (low dose) and 30 mg/kg body weight (high dose) were used for HONE1 treatment, whereas only the lower dose was used for C666-1. The drug was injected into the mice following a drug-dosing cycle regimen of 2 days on and 5 days off (2+/5−).

Cell-cycle and Annexin V apoptosis analysis

The cell-cycle distribution of the samples and Annexin V apoptosis assay were analyzed by FACSCantoII (BD Bioscience) flow cytometry, as previously described (26). For the cell-cycle distribution of samples, in brief, a total of 1 × 106 cells was seeded on a T25 flask before treatment. The cell density of the untreated control reached more than 80% confluence before being subjected to the flow cytometric analysis. The cells were treated for 24 hours. A 200 nmol/L drug concentration was used for HONE1, HK1, and C666-1. A total of 10,000 stained cells were analyzed by fluorescence-activated cell sorting (FACS). The Annexin V apoptosis assay was conducted by using the Annexin V, Alexa Fluor 647 Conjugate assay (Life Technologies) and nucleic acid staining dye, 7-aminoactinomycin D (BD Bioscience), according to the manufacturer's instructions.

Immunofluorescence and immunohistochemical staining

The immunofluorescence and immunohistochemical (IHC) staining was conducted as previously described (27, 28). Antibodies specific for phosphorylated-Histone H3 and γ-tubulin were used for IHC and immunofluorescence staining, respectively (Table 1). The stained cells were observed under fluorescence microscopy (Nikon).

Table 1.

Antibody information

AntibodyHostDilutionCompanyCatalog no.
p-PLK1-T210 Rabbit 1:1000 Cell Signaling 5472 
PLK1 Rabbit 1:1000 Cell Signaling 4513 
WEE1 Rabbit 1:1000 GeneTex GTX111392 
Securin Rabbit 1:1000 GeneTex GTX62173 
Cdc25c Rabbit 1:1000 GeneTex GTX102809 
Cyclin B Rabbit 1:200 GeneTex GTX100911 
Cdk1 Rabbit 1:1000 GeneTex GTX108120 
p-Chk2(T68) Rabbit 1:1000 Cell Signaling 2661 
α-Tubulin Rabbit 1:1000 GeneTex GTX108784 
p-Histone H3(S10) Rabbit 1:200 Santa Cruz Sc8656 
γ-Tubulin Mouse 1:500 Sigma-Aldrich T5326 
AntibodyHostDilutionCompanyCatalog no.
p-PLK1-T210 Rabbit 1:1000 Cell Signaling 5472 
PLK1 Rabbit 1:1000 Cell Signaling 4513 
WEE1 Rabbit 1:1000 GeneTex GTX111392 
Securin Rabbit 1:1000 GeneTex GTX62173 
Cdc25c Rabbit 1:1000 GeneTex GTX102809 
Cyclin B Rabbit 1:200 GeneTex GTX100911 
Cdk1 Rabbit 1:1000 GeneTex GTX108120 
p-Chk2(T68) Rabbit 1:1000 Cell Signaling 2661 
α-Tubulin Rabbit 1:1000 GeneTex GTX108784 
p-Histone H3(S10) Rabbit 1:200 Santa Cruz Sc8656 
γ-Tubulin Mouse 1:500 Sigma-Aldrich T5326 

Western blot analysis

Western blot analyses of the PLK, phosphorylated PLK, and different signalling targets were conducted as previously described (26, 29). The protein cell lysates from the cell lines and from mouse tumors were extracted as previously described (24, 29). In brief, 1 × 106 cells were seeded on a T25 flask before treatment. The cell density of the untreated control reached more than 80% confluence. The cells were treated for 24 hours at a 200 nmol/L drug concentration. The protein was then extracted by radioimmunoprecipitation assay solution supplemented with proteinase inhibitors. The antibodies and conditions used in this study are listed in Table 1. The α-tubulin was used as an internal loading control.

Statistical analysis

The real-time cell proliferation assay results represent the arithmetic mean ± range of experimental data. The SEM was used to calculate the SE of the in vivo assay. The Student t test was used to determine the confidence levels for group comparisons. A P < 0.05 was considered as statistically significant.

Ro5203280 is a selective inhibitor for PLK1

Ro5203280 is a small-molecular PLK1 inhibitor that is structurally similar to BI2536 (30), but with an improved activity (Fig. 1A; ref. 23). In the Ambit protein kinase selectivity screen, a panel of 315 wild-type and mutant forms of kinases was investigated as described previously (31). R05203280 exhibited a high selectivity and showed a very low Kd (0.09 nmol/L) to PLK1 when compared with the 0.2 nmol/L Kd of BI2536. Although Ro5203280 does not bind to most of the protein kinases tested (Supplementary Table S1), for 8 protein kinases, it exhibits a Kd ranging from 70 to 2,000 nmol/L in the Ambit selectivity screen. The specificity of the PLK1 inhibitor Ro5203280 is summarized in Fig. 1B. Like BI2536, Ro5203280 also exhibits potent and broad activity in suppressing a large panel of 30 cell lines with IC50 as low as 6 nmol/L (data not shown).

Figure 1.

Inhibitory effect of Ro5203280 on NPC cell lines. A, chemical structure of Ro5203280. B, ambit selectivity screen specificity of PLK1 inhibitor Ro5203280. C, in vitro growth inhibitory effect of Ro5203280 in 3 nasopharyngeal carcinoma cell lines (HONE1, HK1, and C666-1) and the immortalized nasopharyngeal cell line NP460. The relative inhibition rate was compared with their corresponding untreated control. The cells were treated with different concentrations of Ro5203280 (25, 50, 100, 200, 500, and 1,000 nmol/L) for 24 hours.

Figure 1.

Inhibitory effect of Ro5203280 on NPC cell lines. A, chemical structure of Ro5203280. B, ambit selectivity screen specificity of PLK1 inhibitor Ro5203280. C, in vitro growth inhibitory effect of Ro5203280 in 3 nasopharyngeal carcinoma cell lines (HONE1, HK1, and C666-1) and the immortalized nasopharyngeal cell line NP460. The relative inhibition rate was compared with their corresponding untreated control. The cells were treated with different concentrations of Ro5203280 (25, 50, 100, 200, 500, and 1,000 nmol/L) for 24 hours.

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Ro5203280 inhibits in vitro cell and in vivo tumor growth

The efficacy of Ro5203280 in inhibiting in vitro cell growth was first investigated by the real-time cell proliferation assay. Comparisons of in vitro cell growth of nasopharyngeal carcinoma cell lines with the immortalized nontumorigenic nasopharyngeal epithelial cell line, NP460, showed EC50 values for Ro5203280 of 78 nmol/L for HONE1, 193 nmol/L for HK1, and 358 nmol/L for C666-1, in contrast to 950 nmol/L for NP460 (Fig. 1C). The inhibitory effect decreases dramatically at drug concentrations less than 200 nmol/L. This shows that Ro5203280 can inhibit in vitro nasopharyngeal carcinoma cell growth.

The Ro5203280 is also able to inhibit in vivo tumor growth. In a previous Roche in-house study, different dosage levels and dosing regimes of Ro5203280 were tested to identify an effective in vivo dosage and schedule for Ro5203280 for different cancer cell lines, including the colorectal adenocarcinoma and small cell lung cancer (data not shown). Results suggested that the effective dosage is around 30 to 40 mg/kg with a 2+/5− dosing regimen by intravenous injection. Both HONE1 and C666-1 cell lines are highly tumorigenic in nude mice (Fig. 2A and Table 2). For the HONE1 cell line, significant tumor growth inhibition can be observed with Ro5203280 treatment (P = 0.002) in both the low-dosage (15 mg/kg) and high-dosage (30 mg/kg) groups when compared with the vehicle control group, which was treated with PEG400/acetate buffer (pH 5.0). For C666-1, because a low dosage of Ro5203280 was found to induce significant tumor suppression in the in vivo tumorigenicity assay (P < 0.0005), the higher dose was not tested. The Ro5203280 does not show any acute toxicity in the mice, as assessed by mouse behavior and body conditions including hydration and changes of body weight (Fig. 2B) and gross necropsy. The effectiveness of the Ro5203280 in in vivo levels was further confirmed by IHC staining with p-Histone H3 antibody. P-Histone H3 is a pharmacodynamic biomarker of PLK1 inhibition. Ro5203280-treated HONE1 and C666-1 tumors showed elevated p-Histone H3 levels (Supplementary Fig. S1). This finding further supports the potent role of Ro5203280 in nasopharyngeal carcinoma tumor inhibition.

Figure 2.

In vivo tumor growth inhibition studies in nasopharyngeal carcinoma. A, in vivo growth inhibitory effect of Ro5203280 on nasopharyngeal carcinoma cell lines HONE1 and C666-1. The curves represent an average tumor volume of all injected animals (16 mice for HONE1/vehicle, 15 mice for HONE1/15 mg/kg i.v. 2+/5−, 13 mice for HONE1/30 mg/kg i.v. 2+/5−, 10 mice for C666-1/Vehicle, and 17 mice for C666-1/15 mg/kg i.v. 2+/5−). Drug treatments were initiated when the average tumor size reached 100 mm3. The vehicle control forms large tumors; Ro5203280 treatment resulted in reduced tumor sizes. B, changes in body weight of the experimental animals. No significant changes in the body weights were observed in the experimental animals, indicating low toxicity of the administered drug.

Figure 2.

In vivo tumor growth inhibition studies in nasopharyngeal carcinoma. A, in vivo growth inhibitory effect of Ro5203280 on nasopharyngeal carcinoma cell lines HONE1 and C666-1. The curves represent an average tumor volume of all injected animals (16 mice for HONE1/vehicle, 15 mice for HONE1/15 mg/kg i.v. 2+/5−, 13 mice for HONE1/30 mg/kg i.v. 2+/5−, 10 mice for C666-1/Vehicle, and 17 mice for C666-1/15 mg/kg i.v. 2+/5−). Drug treatments were initiated when the average tumor size reached 100 mm3. The vehicle control forms large tumors; Ro5203280 treatment resulted in reduced tumor sizes. B, changes in body weight of the experimental animals. No significant changes in the body weights were observed in the experimental animals, indicating low toxicity of the administered drug.

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Table 2.

Summary of Ro5203280 in vivo tumorigenicity assays

Cell lines/treatment descriptionNumber of residual tumors after drug treatment/number of injection sitesPa
HONE1/Vehicle 16/16 – 
HONE1/15 mg/kg i.v. 2+/5− 10/15 0.0019 
HONE1/30 mg/kg i.v. 2+/5− 5/13 0.0018 
C666-1/Vehicle 10/10 – 
C666-1/15 mg/kg i.v. 2+/5− 2/17 <0.0005 
Cell lines/treatment descriptionNumber of residual tumors after drug treatment/number of injection sitesPa
HONE1/Vehicle 16/16 – 
HONE1/15 mg/kg i.v. 2+/5− 10/15 0.0019 
HONE1/30 mg/kg i.v. 2+/5− 5/13 0.0018 
C666-1/Vehicle 10/10 – 
C666-1/15 mg/kg i.v. 2+/5− 2/17 <0.0005 

aP value obtained by comparison with vehicle control.

Ro5203280 induces cell-cycle arrest at G2–M phase by inhibiting mitotic spindle formation

The tumor inhibitory mechanism for Ro5203280 was investigated. FACS analysis confirmed that 24 hours treatment of cells with 200 nmol/L Ro5203280 could induce cell-cycle G2–M arrest, as illustrated for HONE1 and HK1 (Fig. 3A). The percentage of cells in G2–M in HONE1, HK1, and C666-1 increased from 21.9% to 52.9%, 13.7% to 39.5%, and 15.4% to 19.6%, respectively, when compared with the vehicle control, whereas for NP460, no significant increase in the percentage of G2–M cells before and after treatment was observed. These results suggested that Ro5203280 has the ability to inhibit cell proliferation through G2–M arrest in HONE1 and HK1 cells. The Ro5203280-treated HONE1 and HK1 cells formed multinucleated cells (Fig. 3A and Supplementary Fig. S2).

Figure 3.

Cell cycle and cell death studies of the Ro5203280-treated nasopharyngeal carcinoma and nasopharyngeal cell lines. A, representative images of FACS analysis in Ro5203280-treated nasopharyngeal carcinoma cell lines HONE1, HK1, and C666-1 and the nasopharyngeal cell line NP460. The cells were untreated, vehicle treated, and drug treated. Treatment was with 200 nmol/L Ro5203280 for 24 hours. B, representative images of the Annexin V staining assay of untreated, vehicle, and drug-treated nasopharyngeal carcinoma HONE1 and NP460 cell lines. The Ro5203280-treated HONE1 cells showed a higher level of cell death when compared with the Ro5203280-treated NP460.

Figure 3.

Cell cycle and cell death studies of the Ro5203280-treated nasopharyngeal carcinoma and nasopharyngeal cell lines. A, representative images of FACS analysis in Ro5203280-treated nasopharyngeal carcinoma cell lines HONE1, HK1, and C666-1 and the nasopharyngeal cell line NP460. The cells were untreated, vehicle treated, and drug treated. Treatment was with 200 nmol/L Ro5203280 for 24 hours. B, representative images of the Annexin V staining assay of untreated, vehicle, and drug-treated nasopharyngeal carcinoma HONE1 and NP460 cell lines. The Ro5203280-treated HONE1 cells showed a higher level of cell death when compared with the Ro5203280-treated NP460.

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Ro5203280 induced cell-cycle arrest through inhibition of PLK1. The ability of treatment for 24 hours of 200 nmol/L Ro5203280 to induce mitotic spindle formation was investigated by 4′, 6-diamidino-2-phenylindole (DAPI) and immunofluorescence staining. After DAPI staining, abnormal mitoses were observed in the Ro5203280-treated cells, providing evidence of nonuniform distributions of condensed chromatin (Supplementary Fig. S3A). All 3 nasopharyngeal carcinoma cell lines showed significantly increased populations of cells with abnormal mitoses (Supplementary Fig. S3B); this was not the case for NP460. This is consistent with the FACS analysis. Immunofluorescence staining results showed that the Ro5203280-treated HONE1 cells could selectively inhibit mitotic spindle formation when compared with the untreated and vehicle controls (Supplementary Fig. S2). A mitotic arrest inducer, nocodazole, was used as a mitotic arrest positive control. The Ro5203280-treated cells showed similar mitotic spindle inhibition effects as observed with nocodazole. These results indicate the role of Ro5203280 in inhibition of the mitotic spindle formation.

Ro5203280 induces apoptosis in nasopharyngeal carcinoma cells

The ability of Ro5203280 to induce apoptosis was quantified by Annexin V staining with FACS analysis. After 24 hours of 200 nmol/L Ro5203280 treatment, HONE1 cells showed a higher proportion of early apoptotic cells (20.8%), as compared with the untreated (3.77%) and vehicle controls (7.71%; Fig. 3B). In NP460, no significant increase in apoptotic cells could be observed in the Ro5203280-treated cells. This further supports the antiapoptotic ability of Ro52035280 in cancer cells.

Ro5203280 inhibits PLK1 and its downstream target activities to inhibit tumor growth

Ro5203280 is a PLK1 inhibitor. The expression levels of PLK1 in the nasopharyngeal carcinoma cell lines were investigated. The PLK1 protein is upregulated in all 3 nasopharyngeal carcinoma cell lines when compared with NP460 (Fig. 4A). Phosphorylation levels of PLK1 were decreased in HONE1, HK1, and C666-1 cell lines treated with 200 nmol/L Ro5203280 (Fig. 4B). Postdrug treatment, levels of downstream proteins were determined. Wee1 and Securin, which are important molecules negatively regulating mitosis, were upregulated in the nasopharyngeal carcinoma cell lines treated with 200 nmol/L Ro5203280 (Fig. 4B). After treatment with 200 nmol/L Ro5203280, there was downregulation of the mitosis promoter, Cdc25c, in the nasopharyngeal carcinoma cell lines, HONE1 and HK1, but there was no significant change in the Cdc25c levels in C666-1 (Fig. 4B). The cyclin B protein level dropped after treatment with 200 nmol/L Ro5203280 in HONE1 and HK1 cell lines, but this was not observed at the other Ro5203280 treatment concentrations. The protein levels of total Cdk1 did not show any significant differences after Ro5203280 treatment. Interestingly, one of the DNA damage checkpoint proteins, Chk2, seemed to have a higher phosphorylation level in nasopharyngeal carcinoma cells when treated with 200 nmol/L Ro5203280. This is suggestive that Ro5203280 may also be involved in regulating this signaling pathway.

Figure 4.

Western blot analysis of changes in molecular signaling after Ro5203280 treatment. A, PLK1 expression levels in nasopharyngeal carcinoma cell lines HONE1, HK1, and C666-1 and nasopharyngeal cell line NP460. The expression levels were normalized to the internal control α-tubulin. B, PLK, cell-cycle control, and DNA damage-related signaling pathways analysis of the Ro5203280-treated nasopharyngeal carcinoma cells. The expression levels were normalized to the internal control α-tubulin. The cells were untreated (U), vehicle (V), and drug treated (T), as indicated. The phosphorylation status of p-PLK1 in Ro5203280-treated cells with different concentrations of Ro5203280 is shown. Downregulation of cyclin B and Cdc25c and upregulation of Wee1, securin, and pChk2 is observed in the drug-treated nasopharyngeal carcinoma cell lines. There is no significant change in the levels of Cdk1 protein in the Ro5203280-treated cells. The expression levels were normalized to the internal control α-tubulin.

Figure 4.

Western blot analysis of changes in molecular signaling after Ro5203280 treatment. A, PLK1 expression levels in nasopharyngeal carcinoma cell lines HONE1, HK1, and C666-1 and nasopharyngeal cell line NP460. The expression levels were normalized to the internal control α-tubulin. B, PLK, cell-cycle control, and DNA damage-related signaling pathways analysis of the Ro5203280-treated nasopharyngeal carcinoma cells. The expression levels were normalized to the internal control α-tubulin. The cells were untreated (U), vehicle (V), and drug treated (T), as indicated. The phosphorylation status of p-PLK1 in Ro5203280-treated cells with different concentrations of Ro5203280 is shown. Downregulation of cyclin B and Cdc25c and upregulation of Wee1, securin, and pChk2 is observed in the drug-treated nasopharyngeal carcinoma cell lines. There is no significant change in the levels of Cdk1 protein in the Ro5203280-treated cells. The expression levels were normalized to the internal control α-tubulin.

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After Ro5203280 treatment in vivo, tumor proteins were analyzed by Western blot analysis. Interestingly, phosphorylation of PLK1 was reduced in both HONE1 and C666-1 cell lines. Wee1, Securin, and phosphorylated Chk2 show upregulation in the Ro5203280-treated HONE1 and C666-1 tumors, whereas the Cdc25c and Cyclin B show downregulation (Supplementary Fig. S4). This further confirms the important role of Ro5203280 in nasopharyngeal carcinoma growth inhibition.

Nasopharyngeal carcinoma is a deadly cancer that is usually diagnosed only at a late stage of cancer progression. Current treatment practice uses only limited standard anticancer drugs. With the development of drug resistance, curative treatment for patients with late-stage nasopharyngeal carcinoma remains a clinical challenge, and improved anticancer drugs are needed.

PLK1 overexpression is commonly observed in many different cancers (3, 5–7). PLK1 is the best characterized member in the PLK family, and increasing evidence suggests that PLK1 serves as a good candidate for cancer drug targeting in several malignancies; various PLK1 inhibitors are efficacious as anticancer drugs (18, 32, 33). A previous study showed that inhibition of PLK1 function suppresses cell growth and increases radiation sensitivity in medulloblastoma cell lines (34). In a diffuse large B-cell lymphoma study, a small-molecule PLK1 inhibitor, MLN0905, was confirmed to have antitumor effects (35). MLN0905 induces mitotic arrest and inhibits in vivo tumorigenicity (35). In recent clinical trial studies, a PLK1 inhibitor, BI2536, was used in a randomized phase II clinical trial of pancreatic exocrine adenocarcinoma (36). Furthermore, another PLK1 inhibitor, BI6727, was found to maintain broad antitumor activities in several solid tumors (37). However, BI2536 failed to show sufficient efficacy in phase II clinical trials; there was a low response rate and no significant difference in patient outcomes (36, 38). Ro5203280 is a close analog to BI2536, but with an improved activity against PLK1. These findings provide the rationale for evaluating the function of this novel PLK1 inhibitor in nasopharyngeal carcinoma. Ro5203280 holds promise as a novel molecularly targeted treatment drug for nasopharyngeal carcinoma.

In this current study, the effectiveness of Ro5203280 in selectively suppressing nasopharyngeal carcinoma growth, while sparing inhibition of the nontumorigenic NP460 cell line, shows its potential to serve as a chemotherapy drug. The 3 nasopharyngeal carcinoma cell lines displayed different EC50 values, reflective of the levels of expression of PLK1, the molecular target for Ro5203280. For NP460, which only expresses low levels of PLK1, Ro5203280 has little effect on its growth. On the other hand, PLK1 is expressed in the highly proliferative cancer cells and is regarded as a cell proliferation marker (3, 4). These findings further support the potential efficacy of using this drug for treatment of nasopharyngeal carcinoma. The Ro5203280 compound does not induce any noticeable acute toxicity in the mice. However, due to inherent limitations in the use of mouse models, the primary hematologic toxicity seen in patients treated with other PLK1 inhibitors could not be studied but remains a potential side effect that warrants further future scrutiny.

PLK1 is involved in the cell-cycle G2–M transition control. The G2–M checkpoint is one of the important checkpoints for DNA damage repair and maintenance of genomic integrity (39). Normal cells suffering DNA damage do not undergo mitosis. If the damaged cells are unable to be repaired, they are destined to undergo apoptosis. On the other hand, inhibition of the mitosis can result in multinucleated cells. The multinucleated cells will finally undergo apoptosis. Therefore, the G2–M checkpoint is a good target for treating cancer (39). In cancer, the abnormal PLK1 activity can interfere with the normal G2–M checkpoint and thus, damaged cells will escape from the DNA repair mechanism and also cell-cycle control. Ro5203280 inhibits PLK1 kinase activity in cancer cells and induces G2–M arrest in the nasopharyngeal carcinoma cell lines. This finding helps to explain the low toxicity of Ro5203280 in the mice as the nontumor cells are spared.

PLK1 is a key regulator modulating different signaling pathways to control the G2–M transition. PLK1 induces Wee1 phosphorylation and thus, facilitates its degradation through proteasome-dependent degradation after ubiquitination by the E3 ubiquitin ligase (11). Wee1 is a crucial kinase in regulating the cell-cycle G2–M transition. It was confirmed to regulate DNA replication in human cells (40). It can phosphorylate Cdk1 and inhibit its function to drive cell-cycle progression (41). It can also induce apoptosis as observed during early xenopus embryonic development (42). Cdc25c removes the inhibitory phosphorylation of the Cdk1 protein and activates its functional role in mitosis (11). Early studies show that cyclin B was upregulated in the PLK1 knockdown nasopharyngeal carcinoma cell lines (8). However, in our current study, the cyclin B protein level decreased in the 200 nmol/L Ro5203280-treated cells; this may be related to cell apoptosis. At other Ro5203280 treatment concentrations, cyclin B protein was maintained at a high level.

In this current study, securin was found to be upegulated in Ro5203280-treated nasopharyngeal carcinoma cells. Securin is another key regulator for monitoring chromosome segregation, which is controlled by PLK1. PLK1 activates the APC function by degradation of APC inhibitors such as Emi1 (43). The activated APC then degrades securin and releases the separase to allow chromosome segregation (44). The upregulation of securin by Ro5203280 also indicates its potential ability to cause G2–M arrest.

Furthermore, PLK1 plays a vital role for the G2–M recovery at the DNA damage checkpoint. Upregulation of phosphorylated Chk2 protein, which is crucial for the DNA damage response and induces cell-cycle G2–M arrest to allow DNA repair, was observed after Ro5203280 treatment. If the cell fails to repair the damage, it will undergo apoptosis (45). The DNA damage-induced cell-cycle arrest can inactivate PLK1 function. However, in cancer cells, PLK1 malfunction can override the G2–M arrest induced by DNA damage (46). PLK1 inactivates Chk2 kinase activity and thus, arrests the DNA damage checkpoint signaling (47). The inhibition of PLK1 function by Ro5203280 helps to maintain Chk2 function, allows the cell to undergo cell-cycle arrest, and prevents cell proliferation. Our results are further supported by a previous PLK1 study in nasopharyngeal carcinoma. Knockdown of PLK1 by siRNA in C666-1 and HK1 cell lines resulted in cell-cycle G2–M arrest, apoptosis, downregulation of Cdc25c, and suppression of in vivo tumorigenesis (8). This further supports our findings of the potential usefulness of the PLK1 inhibitor, Ro5203280, to serve as a candidate chemotherapeutic drug for nasopharyngeal carcinoma.

Interestingly, our data show that C666-1 is more sensitive to Ro5203280 in vivo than in vitro. One possible explanation is that C666-1 grows relatively slowly in vitro but comparatively fast in the nude mouse. The changes in the molecular signaling pathways can help to explain differences observed in the efficacy of Ro52032820. In the Western blot analysis of C666-1 mouse tumors, downregulation of the PLK1 downstream signaling target, CDC25C, is more obvious in Ro5203280-treated mouse tumors than in the in vitro study. Also the levels of upregulation of the Wee1 and Securin were much higher in the C666-1 mouse tumors than observed in vitro. This further supports our results that the Ro5203280 has a higher efficacy on C666-1 in vivo than in vitro.

In conclusion, the PLK1 inhibitor Ro5203280 shows a high efficacy for suppressing nasopharyngeal carcinoma growth with low toxicity in mouse studies. It suppresses nasopharyngeal carcinoma growth by inducing G2–M arrest and mitotic arrest and finally induces apoptosis through inhibiting PLK1 phosphorylation. It restricts the PLK1 function, then activates the signaling molecules that are negatively regulated by PLK1, and inactivates the molecules that are positively regulated by PLK1. Thus, this novel PLK1 inhibitor targeting multiple pathways is a promising anticancer drug for nasopharyngeal carcinoma treatment.

S.W. Tsao has commercial research grant Roche Pharma Research and Early Development. M.L. Lung has commercial research grant from Roche Pharma Research and Early Development. No potential conflicts of interest were disclosed by the other authors.

Conception and design: A.K.L. Cheung, J.C.Y. Ip, H.L. Lung, J.Z. Wu, S.W. Tsao, M.L. Lung

Development of methodology: A.K.L. Cheung, J.C.Y. Ip

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.K.L. Cheung, J.C.Y. Ip, M.L. Lung

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.K.L. Cheung, J.C.Y. Ip, H.L. Lung, J.Z. Wu, M.L. Lung

Writing, review, and/or revision of the manuscript: A.K.L. Cheung, J.C.Y. Ip, H.L. Lung, J.Z. Wu, M.L. Lung

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.K.L. Cheung, J.C.Y. Ip, J.Z. Wu

Study supervision: M.L. Lung

The authors thank the use of the Faculty Core Facility of the Li Ka Shing Faculty of Medicine, HKU for the flow cytometry equipment, and Dr. Daniel Lee (Roche) for support.

This work was supported by funding from Roche Pharma Research and Early Development, China.

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.
Hui
AB
,
Or
YY
,
Takano
H
,
Tsang
RK
,
To
KF
,
Guan
XY
, et al
Array-based comparative genomic hybridization analysis identified cyclin D1 as a target oncogene at 11q13.3 in nasopharyngeal carcinoma
.
Cancer Res
2005
;
65
:
8125
33
.
2.
Andrysik
Z
,
Bernstein
WZ
,
Deng
L
,
Myer
DL
,
Li
YQ
,
Tischfield
JA
, et al
The novel mouse polo-like kinase 5 responds to DNA damage and localizes in the nucleolus
.
Nucleic Acids Res
2010
;
38
:
2931
43
.
3.
Strebhardt
K
,
Ullrich
A
. 
Targeting polo-like kinase 1 for cancer therapy
.
Nat Rev Cancer
2006
;
6
:
321
30
.
4.
Yuan
J
,
Horlin
A
,
Hock
B
,
Stutte
HJ
,
Rubsamen-Waigmann
H
,
Strebhardt
K
. 
Polo-like kinase, a novel marker for cellular proliferation
.
Am J Pathol
1997
;
150
:
1165
72
.
5.
Renner
AG
,
Dos Santos
C
,
Recher
C
,
Bailly
C
,
Creancier
L
,
Kruczynski
A
, et al
Polo-like kinase 1 is overexpressed in acute myeloid leukemia and its inhibition preferentially targets the proliferation of leukemic cells
.
Blood
2009
;
114
:
659
62
.
6.
Eckerdt
F
,
Yuan
J
,
Strebhardt
K
. 
Polo-like kinases and oncogenesis
.
Oncogene
2005
;
24
:
267
76
.
7.
Takai
N
,
Hamanaka
R
,
Yoshimatsu
J
,
Miyakawa
I
. 
Polo-like kinases (Plks) and cancer
.
Oncogene
2005
;
24
:
287
91
.
8.
Shi
W
,
Alajez
NM
,
Bastianutto
C
,
Hui
AB
,
Mocanu
JD
,
Ito
E
, et al
Significance of Plk1 regulation by miR-100 in human nasopharyngeal cancer
.
Int J Cancer
2010
;
126
:
2036
48
.
9.
Kneisel
L
,
Strebhardt
K
,
Bernd
A
,
Wolter
M
,
Binder
A
,
Kaufmann
R
. 
Expression of polo-like kinase (PLK1) in thin melanomas: a novel marker of metastatic disease
.
J Cutan Pathol
2002
;
29
:
354
8
.
10.
Watanabe
N
,
Arai
H
,
Nishihara
Y
,
Taniguchi
M
,
Hunter
T
,
Osada
H
. 
M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP
.
Proc Natl Acad Sci U S A
2004
;
101
:
4419
24
.
11.
Tominaga
Y
,
Wang
A
,
Wang
RH
,
Wang
X
,
Cao
L
,
Deng
CX
. 
Genistein inhibits Brca1 mutant tumor growth through activation of DNA damage checkpoints, cell cycle arrest, and mitotic catastrophe
.
Cell Death Differ
2007
;
14
:
472
9
.
12.
van Vugt
MA
,
Smits
VA
,
Klompmaker
R
,
Medema
RH
. 
Inhibition of Polo-like kinase-1 by DNA damage occurs in an ATM- or ATR-dependent fashion
.
J Biol Chem
2001
;
276
:
41656
60
.
13.
Lane
HA
,
Nigg
EA
. 
Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes
.
J Cell Biol
1996
;
135
:
1701
13
.
14.
Kotani
S
,
Tugendreich
S
,
Fujii
M
,
Jorgensen
PM
,
Watanabe
N
,
Hoog
C
, et al
PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression
.
Mol Cell
1998
;
1
:
371
80
.
15.
Olmos
D
,
Barker
D
,
Sharma
R
,
Brunetto
AT
,
Yap
TA
,
Taegtmeyer
AB
, et al
Phase I study of GSK461364, a specific and competitive Polo-like kinase 1 inhibitor, in patients with advanced solid malignancies
.
Clin Cancer Res
2011
;
17
:
3420
30
.
16.
Jimeno
A
,
Li
J
,
Messersmith
WA
,
Laheru
D
,
Rudek
MA
,
Maniar
M
, et al
Phase I study of ON 01910.Na, a novel modulator of the Polo-like kinase 1 pathway, in adult patients with solid tumors
.
J Clin Oncol
2008
;
26
:
5504
10
.
17.
Mross
K
,
Frost
A
,
Steinbild
S
,
Hedbom
S
,
Rentschler
J
,
Kaiser
R
, et al
Phase I dose escalation and pharmacokinetic study of BI 2536, a novel Polo-like kinase 1 inhibitor, in patients with advanced solid tumors
.
J Clin Oncol
2008
;
26
:
5511
7
.
18.
Valsasina
B
,
Beria
I
,
Alli
C
,
Alzani
R
,
Avanzi
N
,
Ballinari
D
, et al
NMS-P937, an orally available, specific, small molecule Polo-Like Kinase 1 inhibitor with antitumor activity in solid and haematological malignancies
.
Mol Cancer Ther
2012
;
11
:
1006
16
.
19.
Cheung
ST
,
Huang
DP
,
Hui
AB
,
Lo
KW
,
Ko
CW
,
Tsang
YS
, et al
Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus
.
Int J Cancer
1999
;
83
:
121
6
.
20.
Glaser
R
,
Zhang
HY
,
Yao
KT
,
Zhu
HC
,
Wang
FX
,
Li
GY
, et al
Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas
.
Proc Natl Acad Sci U S A
1989
;
86
:
9524
8
.
21.
Huang
DP
,
Ho
JH
,
Poon
YF
,
Chew
EC
,
Saw
D
,
Lui
M
, et al
Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx
.
Int J Cancer
1980
;
26
:
127
32
.
22.
Yip
YL
,
Tsang
CM
,
Deng
W
,
Cheung
PY
,
Jin
Y
,
Cheung
AL
, et al
Expression of Epstein-Barr virus-encoded LMP1 and hTERT extends the life span and immortalizes primary cultures of nasopharyngeal epithelial cells
.
J Med Virol
2010
;
82
:
1711
23
.
23.
Cao
SX
,
Feher
V
,
Ichikawa
T
,
Jones
B
,
Kaldor
S
,
Kiryanov
A
, et al
inventors
. 
Takeda Pharmaceutical Company Limited, assignee
.
WO2009/042711
. 
2009 Apr 2
.
24.
Law
EW
,
Cheung
AK
,
Kashuba
VI
,
Pavlova
TV
,
Zabarovsky
ER
,
Lung
HL
, et al
Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma
.
Oncogene
2012
;
31
:
728
38
.
25.
Cheng
Y
,
Poulos
NE
,
Lung
ML
,
Hampton
G
,
Ou
B
,
Lerman
MI
, et al
Functional evidence for a nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome 3p21.3
.
Proc Natl Acad Sci U S A
1998
;
95
:
3042
7
.
26.
Lung
HL
,
Cheung
AKL
,
Xie
D
,
Cheng
Y
,
Kwong
FM
,
Murakami
Y
, et al
TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma
.
Cancer Res
2006
;
66
:
9385
92
.
27.
Cheung
AK
,
Ko
JM
,
Lung
HL
,
Chan
KW
,
Stanbridge
EJ
,
Zabarovsky
E
, et al
Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis
.
Proc Natl Acad Sci U S A
2011
;
108
:
8390
5
.
28.
Cheung
AK
,
Lung
HL
,
Ko
JM
,
Cheng
Y
,
Stanbridge
EJ
,
Zabarovsky
ER
, et al
Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma
.
Proc Natl Acad Sci U S A
2009
;
106
:
14478
83
.
29.
Cheung
AK
,
Lung
HL
,
Hung
SC
,
Law
EW
,
Cheng
Y
,
Yau
WL
, et al
Functional analysis of a cell cycle-associated, tumor-suppressive gene, protein tyrosine phosphatase receptor type G, in nasopharyngeal carcinoma
.
Cancer Res
2008
;
68
:
8137
45
.
30.
DePinto
W
,
Chu
XJ
,
Yin
X
,
Smith
M
,
Packman
K
,
Goelzer
P
, et al
In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials
.
Mol Cancer Ther
2006
;
5
:
2644
58
.
31.
Steegmaier
M
,
Hoffmann
M
,
Baum
A
,
Lenart
P
,
Petronczki
M
,
Krssak
M
, et al
BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo
.
Curr Biol
2007
;
17
:
316
22
.
32.
Frost
A
,
Mross
K
,
Steinbild
S
,
Hedbom
S
,
Unger
C
,
Kaiser
R
, et al
Phase i study of the Plk1 inhibitor BI 2536 administered intravenously on three consecutive days in advanced solid tumours
.
Curr Oncol
2012
;
19
:
e28
35
.
33.
Su
Y
,
Vilgelm
AE
,
Kelley
MC
,
Hawkins
O
,
Liu
Y
,
Boyd
KL
, et al
RAF265 inhibits the growth of advanced human melanoma tumors
.
Clin Cancer Res
2012
;
18
:
2184
98
.
34.
Harris
PS
,
Venkataraman
S
,
Alimova
I
,
Birks
DK
,
Donson
AM
,
Knipstein
J
, et al
Polo-like kinase 1 (PLK1) inhibition suppresses cell growth and enhances radiation sensitivity in medulloblastoma cells
.
BMC Cancer
2012
;
12
:
80
.
35.
Shi
JQ
,
Lasky
K
,
Shinde
V
,
Stringer
B
,
Qian
MG
,
Liao
D
, et al
MLN0905, a small-molecule PLK1 inhibitor, induces antitumor responses in human models of diffuse large B-cell lymphoma
.
Mol Cancer Ther
2012
;
11
:
2045
53
.
36.
Mross
K
,
Dittrich
C
,
Aulitzky
WE
,
Strumberg
D
,
Schutte
J
,
Schmid
RM
, et al
A randomised phase II trial of the Polo-like kinase inhibitor BI 2536 in chemo-naive patients with unresectable exocrine adenocarcinoma of the pancreas - a study within the Central European Society Anticancer Drug Research (CESAR) collaborative network
.
Br J Cancer
2012
;
107
:
280
6
.
37.
Rudolph
D
,
Steegmaier
M
,
Hoffmann
M
,
Grauert
M
,
Baum
A
,
Quant
J
, et al
BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity
.
Clin Cancer Res
2009
;
15
:
3094
102
.
38.
Sebastian
M
,
Reck
M
,
Waller
CF
,
Kortsik
C
,
Frickhofen
N
,
Schuler
M
, et al
The efficacy and safety of BI 2536, a novel Plk-1 inhibitor, in patients with stage IIIB/IV non-small cell lung cancer who had relapsed after, or failed, chemotherapy: results from an open-label, randomized phase II clinical trial
.
J Thorac Oncol
2010
;
5
:
1060
7
.
39.
Chen
T
,
Stephens
PA
,
Middleton
FK
,
Curtin
NJ
. 
Targeting the S and G2 checkpoint to treat cancer. Drug Discov Today
2012
;
17
:
194
202
.
40.
Dominguez-Kelly
R
,
Martin
Y
,
Koundrioukoff
S
,
Tanenbaum
ME
,
Smits
VA
,
Medema
RH
, et al
Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease
.
J Cell Biol
2011
;
194
:
567
79
.
41.
Wu
CL
,
Kirley
SD
,
Xiao
H
,
Chuang
Y
,
Chung
DC
,
Zukerberg
LR
. 
Cables enhances cdk2 tyrosine 15 phosphorylation by Wee1, inhibits cell growth, and is lost in many human colon and squamous cancers
.
Cancer Res
2001
;
61
:
7325
32
.
42.
Wroble
BN
,
Finkielstein
CV
,
Sible
JC
. 
Wee1 kinase alters cyclin E/Cdk2 and promotes apoptosis during the early embryonic development of Xenopus laevis
.
BMC Dev Biol
2007
;
7
:
119
.
43.
Hansen
DV
,
Loktev
AV
,
Ban
KH
,
Jackson
PK
. 
Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1
.
Mol Biol Cell
2004
;
15
:
5623
34
.
44.
Eckerdt
F
,
Strebhardt
K
. 
Polo-like kinase 1: target and regulator of anaphase-promoting complex/cyclosome-dependent proteolysis
.
Cancer Res
2006
;
66
:
6895
8
.
45.
Taylor
WR
,
Stark
GR
. 
Regulation of the G2/M transition by p53
.
Oncogene
2001
;
20
:
1803
15
.
46.
Smits
VA
,
Klompmaker
R
,
Arnaud
L
,
Rijksen
G
,
Nigg
EA
,
Medema
RH
. 
Polo-like kinase-1 is a target of the DNA damage checkpoint
.
Nat Cell Biol
2000
;
2
:
672
6
.
47.
Bahassi el
M
. 
Polo-like kinases and DNA damage checkpoint: beyond the traditional mitotic functions
.
Exp Biol Med (Maywood)
2011
;
236
:
648
57
.