CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cogswell, J. P.
Right arrow Articles by Neill, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cogswell, J. P.
Right arrow Articles by Neill, S. D.
Cell Growth & Differentiation Vol. 11, 615-623, December 2000
© 2000 American Association for Cancer Research


Articles

Dominant-Negative Polo-like Kinase 1 Induces Mitotic Catastrophe Independent of cdc25C Function

John P. Cogswell1, Chadwick E. Brown, John E. Bisi and Suzanne D. Neill

Department of Functional Genetics, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709

Abstract

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377–385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.

Introduction

Accurate transmission of genetic information during cell division is dependent on the fidelity of chromosome repair, replication, and segregation. Checkpoint controls monitor these processes for accuracy and completion and, when errors are detected, arrest the cell cycle to allow sufficient time for correction of the errors and completion of these events (1, 2, 3) . Deregulated expression of, or mutations in, cell cycle checkpoint genes can lead to genetic instability and have been associated with the development of cancer (4, 5, 6, 7) .

In mammalian cells, the G2-M checkpoint that responds to unreplicated or damaged DNA can be activated by phosphorylation of Thr-14 and Tyr-15 on cdk12 (also called cdc2; Refs. 8, 9, 10, 11, 12 ). Removal of these phosphorylations by the dual-specificity phosphatase cdc25C is a key event in the prophase-to-metaphase transition of the cell cycle (13) . cdc25C itself undergoes NH2-terminal phosphorylation to become fully active (14, 15, 16, 17) . Early on, cdk1 was identified as a kinase capable of activating cdc25C, which suggested that these two partners could participate in an autoamplification loop (18 , 19) . However, the specific kinase that triggers cdc25C activation was not known (20) . In 1996, Kumagai and Dunphy (21) identified a cdc25 phosphorylating kinase, Plx1, from frog oocytes that turned out to be a homologue of the conserved Drosophila gene called polo (see below). Both biochemical and cell-cycle-timing experiments have been used to make a strong case for Plx1 in activation of cdc25 function (21, 22, 23) .

The polo gene was first identified in mutant screens of Drosophila as causing multiple types of mitotic abnormalities (24) . Homologues have been identified in frogs (Plx1), yeast (Cdc5 in Saccharomyces cerevisiae and Plo1 in Schizosaccharomyces pombe), and mammals (Plk1) that likewise have critical roles in mitosis (25 , 26) . All of the PLK1 homologues share a conserved 30-amino-acid sequence called the polo-box as well as additional conserved sequences in the NH2-terminal kinase domain (25 , 26) . Other polo family members [e.g., proliferation related kinase/fibroblast growth factor inducible kinase and serum inducible kinase (PRK/FNK and SNK)] have been identified through sequence homology, although their functional roles may not be exclusive to mitosis (27, 28, 29, 30, 31) . Interestingly, human PRK has been shown to phosphorylate cdc25C in vitro (30) . Whether PLK family members are the sole activating kinases for cdc25C or can play other roles in activating cdk function is not known at this time (25 , 26 , 32) .

The change in localization of PLK1 during mitosis suggests that PLK1 substrates could be located at multiple sites including centrosomes in prophase, spindle pole bodies in metaphase, and cytokinetic bridges in telophase (33 , 34) . This pattern parallels that seen with a mAb MPM-2, which recognizes mitotic phosphorylations on at least 50 proteins (35, 36, 37, 38) , including cdc25C (21 , 39) . Moreover, the Drosophila polo protein appears to be required for MPM-2 reactivity in vivo (40) . Consistent with its changing localization, PLK1 has been implicated in functions as diverse as centrosome maturation, microtubule dynamics, and cytokinesis (34 , 41, 42, 43) . The substrates identified to date, including tubulins and mitotic kinesin-like proteins as well as anaphase promoting complex subunits, are consistent with the multiple roles of PLK1 in the G2-to-M transition and mitotic exit (34 , 41 , 44 , 45) . PLK1 is also necessary for the destruction of mitotic substrates at the end of mitosis (46 , 47) . In view of its multiple targets and functions, PLK1 appears to be a pleiotropic regulator of mitosis.

Abrogation of PLK1 function through antibody microinjection was shown to block a centrosome maturation step in both normal H68 human fibroblasts and HeLa cervical carcinoma cells (42) , but only the latter entered into mitosis and cell death. Because microsurgical dissection of centrosomes has been shown to block all aspects of mitosis (48) , Lane and Nigg (42) hypothesized that PLK1 may regulate a centrosome maturation checkpoint that is defective in HeLa cells. They further suggested that the HeLa cell defect resulted from a loss of dependence for PLK1 in cdc25C activation. To more closely study the role of PLK1 in tumor-specific apoptosis and checkpoint function, we used adenovirus to deliver and overexpress dnPLK1 in multiple cell types. Our studies demonstrate that inhibition of PLK1 function is proapoptotic in most tumor lines compared with normal cell lines and that this antitumor activity is independent of the role of PLK1 in activating cdc25C phosphatase.

Results

dnPLK1 Is Proapoptotic in Most Tumor Cells Relative to Normal Cell Counterparts.
Previous studies showed that microinjection of PLK1 antibodies induced mitotic catastrophe in a HeLa cervical carcinoma cell line, whereas Hs68 normal human diploid fibroblasts arrested in the G2 phase of the cell cycle (42) . Likewise, transient transfection of a dnPLK1 plasmid has been shown to elicit mitotic delay and cytokinetic abnormalities in HeLa cells, although the same comparison to normal cells was not reported (43) . The mitotic catastrophe in HeLa cells injected with PLK1 antibodies was attributed to a defect in a centrosome maturation checkpoint, perhaps related to a loss of PLK1-dependent cdc25C activation, although the results were not extrapolated to other tumors, nor was an assessment of cdc25C function performed (42) .

To address whether PLK1 inhibition caused tumor selective apoptosis in a range of tumor types, a dnPLK1 mutant (K82M) was cloned behind a cytomegalovirus promoter in an E1-defective adenoviral vector, and the gene product delivered via infection into 10 tumor lines and 3 normal lines. The 10 tumors were selected based on their diverse origins and included breast carcinomas (MDA MB 231, MDA MB 435S, HS578T, MCF-10A, and MCF7), osteosarcomas (SAOS-2 and U-2OS), an ovarian carcinoma (SKOV3), a colon carcinoma (SW480), and a lung carcinoma (NCI H596). They were also selected based on their relative inability to complement E1 function in the adenovirus, thus circumventing the complicating cytopathic effects that result from viral DNA replication seen in HeLa and other tumor cell types (49) . The tumors were compared with two normal cell lines of epithelial origin—HMECs and NHKs—as well as a normal human diploid fibroblast cell line (IMR90). Expression of dominant negative protein was accurately quantitated relative to the endogenous protein (see "Materials and Methods") at the 24-h time point largely to avoid any effects of cell death on the quantitation. A high multiplicity of infection, 500 MOI (equivalent to 500 pfu per cell) was used to ensure a sufficient expression of dominant negative protein to abrogate PLK1 function. The percentage and fold increases in Sub-2N DNA content relative to control adenovirus infection were quantified at several time points postinfection by flow cytometric analysis of propidium iodide-stained cells.

The results in Fig. 1Citation clearly reveal that expression of the dnPLK1 protein caused significant cell death by 3 days in 6 of 10 tumor lines, SAOS-2, U-2OS, SK-OV-3, MCF10A, NCI-H596, and SW480. Cell death was considered significant if dnPLK induced at least a 20% increase in Sub-2N DNA content and at least a 2-fold increase in fold apoptosis. In many tumor lines, significant apoptosis was achieved at high ratios of dominant-negative:endogenous PLK. Some cell lines (e.g., SK-OV-3 and NCI H596) showed significant apoptosis at much lower dominant-negative:endogenous PLK1 expression ratios. Three tumor lines were classified as "insensitive" to dnPLK1 because two (MDA 435S and MCF7) were relatively unaffected, and a third (HS578T) was only slightly affected despite equivalent or greater overexpression of the dominant-negative protein compared with the "sensitive" tumors.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 1. dnPLK1 induces selective apoptosis in tumor cells versus normal cells. Cells were infected at 500 MOI of dnPLK1 and processed for FACS analysis at different time points. Percentage increase in Sub-2N is calculated from % dnPLK Ad - % control Ad. Fold > Sub-2N is calculated from % dnPLK ÷ % control Ad. DN/WT, the ratio of protein expression determined from the dilution of dnPLK1-expressing lysate that gave equivalent expression to control virus-infected samples 24 h postinfection.

 
dnPLK1 induced minimal apoptosis in normal epthelial cells, HMECs and NHKs, although the proliferation of these cells was considerably slowed. HMECs showed only an 8% increase in Sub-2N DNA content even out to 4 days postinfection and despite the much greater relative expression of the dominant negative protein compared with the tumor lines (Fig. 1)Citation . Even tumors that grew as slowly as HMECs (e.g., SAOS-2 and H596) showed more rapid and profound cell death, which suggested that apoptosis can be independent of the rate of cell proliferation.

Normal human diploid fibroblast IMR90 cells demonstrated significant apoptosis in response to dnPLK1 (Fig. 1)Citation . Because the dnPLK1:endogenous PLK1 ratio was much higher in IMR90 cells than in most tumor lines, we suspected that the dn-PLK1-induced apoptotic response might still be tumor-selective. To examine this question, we quantitated the dnPLK1:endogenous protein ratio in SAOS-2 and IMR90 cells over a range of MOI at a time when the apoptotic responses of both cell lines were similar. The results clearly show that the level of apoptosis is directly correlated with the expression of dnPLK1 in both of the cell lines and that significantly more dnPLK1 is required to elicit an equivalent apoptotic response in IMR90 compared with SAOS-2 cells (Fig. 2)Citation .



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. IMR90 cells require a higher dnPLK1:endogenous PLK1 ratio to elicit the equivalent apoptotic response to SAOS-2. Both of the cell types were infected with dnPLK1 at 62.5, 125, 250, and 500 MOI. Percentage increase in Sub-2N DNA content was determined by flow cytometry. One in four dilutions of dnPLK1 Ad cell lysates were compared with control virus lysates to determine fold overexpression of dnPLK1 protein. For each 2-fold increase in MOI, SAOS-2 cells showed a 1.5-fold increase in dnPLK1 level, whereas IMR90 cells showed a 2-fold increase.

 
Flow cytometric analysis of the cell lines demonstrated that the induction of a G2-M cell cycle arrest was strong (>30% increase) in U-20S and SAOS-2, moderate or weak (10–30% increase) in most of the rest, or nonexistent in a few (e.g., MCF-10A and IMR90). Because dnPLK1 induced a rounding up of the IMR90 fibroblasts, we explored whether PLK might also affect substrate adherence. Immunofluorescent staining for actin and tubulin demonstrated highly disrupted cytoskeletal networks in virtually all IMR90 cells by 48 h postinfection (data not shown). This effect was seen in dnPLK1 Ad-infected cells but not with control virus-infected cells, which suggested that high-level expression of dnPLK1 in fibroblasts can induce a loss of substrate adherence and apoptosis. The phenotype elicited in IMR90 cells mimics the cell-cycle-independent apoptosis that results when normal cells are deprived of substrate adherence (50 , 51) . Unlike IMR90, only about 8% of HMECs showed obvious disruption of their cytoskeletal networks under the same conditions, which suggested an epithelial cell difference in the response to dnPLK1 (data not shown). Because it is possible that the loss of substrate adherence also contributes to the apoptotic phenotype in tumors with a weak G2-M arrest, we wanted to eliminate this effect from our functional analysis of dnPLK1 on mitosis and apoptosis. Therefore, we focused our attention on cell lines like SAOS-2 and U-2OS that exhibited clear-cut G2-M arrests without any detectable adherence effects.

dnPLK1 Induces Mitotic Catastrophe in SAOS-2 and U-2OS Tumor Cells but Spares Normal HMECs.
To determine whether the G2-M arrest and apoptosis attributable to dnPLK1 expression were linked or separate events, we used flow cytometry of propidium iodide-stained SAOS-2 and U-2OS cells to examine how the percentage 4N DNA content and Sub-2N DNA content changed with respect to time and virus dose. At 24 h, expression of the dnPLK1 caused dramatic increases in 4N DNA content in both of the cell types (Fig. 3, A and B)Citation . The time-dependent loss in 4N cells corresponded with an increase in cells with Sub-2N DNA content. The cell-death rate was dependent on the MOI and, thus, the amount of dominant-negative protein expressed. These results suggested that ablation of PLK1 function through expression of a dnPLK1 protein could cause SAOS-2 and U-20S cells to arrest in G2 or mitosis before progressing to apoptosis.



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 3. G2-M arrest precedes apoptosis in SAOS-2 and U-2OS osteosarcomas. Graphs of 4N DNA content versus Sub-2N DNA content in SAOS-2 (A) and U-20S (B). Tumor cells were infected at 50, 100, 200, and 500 MOI of dnPLK Ad and processed for FACS analysis at 24, 48, and 72 h to determine DNA content. Apoptosis was calculated from percentage of Sub-2N DNA content. 4N DNA contents in control adenovirus populations were 34% for SAOS-2 and 38% for U-20S (single point {blacksquare}).

 
We then used immunostaining to look for defects in mitotic spindle formation in SAOS-2, U-2OS, and HMECs. We coimmunostained cells with antibodies directed against {alpha}-tubulin to see spindle components and {gamma}-tubulin to stain centrosomes, and included DAPI to visualize chromatin condensation. With these markers we could quantify the percentage mitotic figures (Table 1)Citation and assess the integrity of the mitotic apparatus (Fig. 4)Citation . At 24 h postinfection with 200 MOI of virus, 44% of dnPLK1 Ad-infected SAOS-2 cells were in mitosis compared with 4% in control Ad-infected cells. More strikingly, 63% of the dnPLK mitoses were abnormal compared with 8% in the control (Table 1)Citation . Similar results were seen in U-20S cells in which dnPLK1 induced a >5-fold increase in abnormal mitoses. Abnormal mitoses in SAOS-2 and U-20S cells showed evidence of disrupted spindle formation, {gamma}-tubulin staining that was abnormally reduced compared with interphase cells, and nonuniform distribution of condensed chromatin (Fig. 4, A–D)Citation . These results suggest that apoptosis in SAOS-2 and U-2OS cells was a consequence of catastrophic mitoses.


View this table:
[in this window]
[in a new window]
 
Table 1 Quantification of abnormal mitoses induced by dnPLK1 in SAOS-2 and U-2OS tumors and normal HMEC cells

Percentages total mitoses were calculated from at least 1000 cells. Percentages of mitoses that were abnormal, unipolar, or abnormal unipolar were determined by examining at least 100 mitoses.

 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4. DN-PLK1 induces abnormal mitoses in SAOS-2 and U-20S but not in HMECs. Cells were infected at 200 MOI of dnPLK1 and processed for immunohistochemistry at different time points. SAOS-2 cells immunostained 24 h postinfection for {alpha}-tubulin (A), {alpha}-tubulin and DAPI (B), or gamma-tubulin (C). Arrows, abnormal prophase mitoses with disrupted mitotic spindles and poor centrosomal {gamma}-tubulin staining. U-2OS cells immunostained 48 h postinfection for {alpha}-tubulin (D). Arrows, abnormal prophase and metaphase cells. HMECs were immunostained 48 h postinfection with {alpha}-tubulin and DAPI (E) or {gamma}-tubulin (F). Arrow, prophase cell with normal monoastral spindle. Metaphase cell is just below.

 
dnPLK1 Ad infection of HMECs had entirely different consequences even at a later 48-h time point and despite a much higher overall expression (see Fig. 1Citation ). dnPLK1 induced only a 3-fold increase in mitotic cells, which suggested that fewer cells were being delayed (Table 1)Citation . Fully formed spindles and a normal distribution of chromosomes characterized HMEC mitoses whether the cells were in prophase or metaphase (Fig. 4, E and F)Citation . Perhaps the biggest distinction from SAOS-2 cells was that the {gamma}-tubulin immunostaining in the mitotic cells was unchanged relative to interphase cells (Fig. 4, E and F)Citation or control Ad-treated HMECs (data not shown). Because proportionally more mitotic cells were arrested at the prophase stage of mitosis in HMECs, we wanted to determine whether or not mitotic catastrophe was specific to the later-stage mitotic arrests prevalent in SAOS-2 cells. We addressed this question by quantifying the fraction of monoastral mitoses in each cell type that were abnormal (Table 1)Citation . In SAOS-2 cells, dnPLK1 Ad induced a >2-fold increase in monoastral mitoses, of which 41% had defects in centrosomal {gamma}-tubulin staining and spindle formation. In contrast, of the almost 4-fold increase in monoastral mitoses in HMECs, only 2% were morphologically abnormal. These results suggest that dnPLK1 induces a defective prophase in SAOS-2 tumor cells, whereas HMECs have a normal prophase. These results confirm that normal HMECs show an intrinsic difference in their response to dnPLK1.

Tumor-specific Apoptosis Is Independent of cdc25C Function.
In Xenopus, xPLK1 is essential for the mitotic phosphorylation and activation of cdc25C on its NH2 terminus (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 21, 22, 23) . In turn, activated cdc25C is essential for entry into, and progression through, mitosis through its activation of cdk1 in frog and mammalian systems (8, 9, 10, 11, 12) . Lane and Nigg (42) hypothesized that microinjection of PLK1 antibodies blocked this cdc25C-regulated checkpoint pathway in Hs68 cells but not in HeLa cervical carcinoma cells, which allowed the tumor cells to enter mitosis prematurely. Because adenovirus can infect 100% of the cells in a population, we could make the first assessment of the effect of PLK1 ablation on mitotic phosphorylation of cdc25C in mammalian cells. To measure the effect of dnPLK1, we infected G1-synchronized SAOS-2 and U-2OS cells and then treated with the microtubule inhibitor nocodazole to arrest cells in mitosis. As expected, nocodazole induced a broad band of lower-mobility cdc25C protein (Fig. 5ACitation , open arrow) that represented the mitotically phosphorylated form because treatment with {lambda} phosphatase increased its mobility to that seen with interphasic cdc25C (compare Lanes 2 and 4, Fig. 5ACitation ). Mitotically phosphorylated cdc25C was seen in control virus-infected cells treated with nocodazole but was completely blocked at 100 MOI and partially blocked at 30 MOI of dnPLK1 Ad in both cell lines (Fig. 5B)Citation . Similar results were seen in normal HMECs (Fig. 5C)Citation at the same MOI as SAOS-2 and U-2OS tumor cells, although the degree of mitotic phosphorylation was less evident attributable in part to a less efficient nocodazole-induced mitotic arrest. Mitotic phosphorylation of cdc25C in an insensitive MCF7 tumor (Fig. 5D)Citation was also blocked by dnPLK1, although higher MOI (650 and 300) were required because dnPLK1 was not as effectively overexpressed in the mitotic population compared with the interphase population of cells (data not shown). Concordant with the effects on phosphorylation of cdc25C, 650 MOI were sufficient to considerably ablate cdc25C-dependent activation of cdk1 (see Table 2Citation ) and had no greater effect on apoptosis than that observed at 500 MOI (Fig. 1Citation , data not shown).



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 5. Cell death is independent of cdc25C function. A, cycling and nocodazole-treated U-20S cells were treated with {lambda} phosphatase, and cdc25C mobility was determined by Western blotting. I, interphase cdc25C. M, mitotic cdc25C. B, SAOS-2 and U-20S cells were synchronized in G1, infected at 100 and 30 MOI of dnPLK or control virus, treated with nocodazole, and then harvested 24 h later for Western blotting with cdc25C antibody. C, HMECs harvested after 48 h. D, MCF-7 cells infected with 650 and 300 MOI and harvested after 24 h. Open arrow, mitotic form of cdc25C. Solid arrow, interphase form of cdc25C.

 

View this table:
[in this window]
[in a new window]
 
Table 2 Effect of dnPLK1 on cdk1 kinase activity

Cyclin B1 antibody was used to immunoprecipitate cdk1 from control and from dnPLK1 Ad-infected cells prepared as above for cdc25C phosphorylation status. cdk1 activity against Histone H1 substrate was determined.

 
To confirm that the lack of mitotic phosphorylation corresponded to a loss of cdc25C function, we infected G1-synchronized cells with high doses of dnPLK1 or control virus and released the cells into nocodazole. We immunoprecipitated cdk1 through its partner cyclin B1 and performed histone H1 kinase assays (Table 2)Citation . These experiments showed that phosphorylation of the histone H1 substrate in vitro was most inhibited in HMECs (98%), although it required 48 h in nocodazole to obtain a good mitotic arrest with nocodazole. cdk1 kinase activity was equally inhibited in U-20S cells and MCF7 cells at 24 h postinfection (88 and 85%, respectively), although MCF7 cells were insensitive to the dnPLK1 virus. These data argue that the mitotic catastrophe observed in the U-20S tumor line is not attributable solely to a lack of dependence for PLK1 in activating cdc25C, and that loss of cdc25C activation is not sufficient to induce apoptosis in MCF7 cells.

Discussion

Overexpression of PLK1 could have important consequences in tumor formation. PLK1 overexpression has been shown to be transforming in NIH3T3 cells (52) and has been correlated with poor prognosis in small cell lung (53) and squamous cell carcinomas of the head and neck (54) . Previous work (42) suggested PLK1 could have a different function in HeLa tumor cells versus normal Hs68 fibroblasts. Microinjection of PLK1 antibodies caused mitotic catastrophe in HeLa cells, whereas the same treatment of nonimmortalized Hs68 cells elicited a G2 arrest (42) . Transient transfection of a dnPLK1 gene into HeLa cells had somewhat different results from antibody microinjection studies in that most of the mitotic arrests were bipolar and cytokinesis appeared disrupted (43) . To study PLK1 function in more detail, we used an adenoviral vector to deliver a dnPLK1 gene to 10 tumor lines and 3 normal cell lines. Here we demonstrated that 6 of 10 tumor lines were at least 2-fold and up to 11-fold more sensitive to inhibition of PLK1 function than normal epithelial cells (Fig. 1)Citation . Even the one normal cell line, IMR90, which demonstrated a significant increase in Sub-2N DNA content response, required more dnPLK1 expression to elicit the same apoptotic response as that in the SAOS-2 tumor line (Fig. 2)Citation . In the sensitive tumor lines, SAOS-2 and U-20S, we showed that dnPLK1 induced a G2-M cell cycle arrest within 24 h but that these arrested cells declined coincident with the onset of apoptosis (Fig. 3)Citation . These data extend previous observations made with dnPLK1 in HeLa cells to other tumor lines and further show that these sensitive tumors show a selective apoptotic response versus normal epithelial counterparts.

Previous experiments unequivocally demonstrated that microinjected PLK1 antibodies blocked centrosome maturation in both HeLa tumor cells and normal Hs68 fibroblast cells as assessed by size of the centrosomes and lack of {gamma}-tubulin staining (42) . The effects of overexpression of dnPLK1 protein on centrosome maturation were not reported (43) . To further understand why tumor cells respond differently to a loss of PLK1 function, we used immunostaining to examine mitotic structures in two sensitive tumor lines, SAOS-2 and U-20S, versus unaffected normal human mammary epithelial cells (Fig. 4)Citation . To ensure that the phenotypic differences were not attributable to proliferation rates, we focused on the effects of dnPLK at 48 h in HMECs versus 24 h in the slow growing SAOS-2 cell line. It was obvious that dnPLK1 Ad caused a mitotic arrest in all cell types. SAOS-2 (and U-20S) cells were arrested with bipolar anaphase-like spindles or as prophase-like mitoses with monoastral arrays emanating from a single organizing center (Fig. 4, A–D)Citation . Most of the monoastral and bipolar spindles were morphologically abnormal (Table 1)Citation . The weak immunostaining of the centrosomes using the {gamma}-tubulin antibody is consistent with the failure of centrosome maturation noted by others (42) . The mitotic prophase cells in HMECs had fully formed, morphologically normal monoastral spindles (Fig. 4, E and F)Citation . The diminished {gamma}-tubulin staining seen in SAOS-2 cells was clearly not evident in HMECs, although a 3-fold increase in these prophase-like cells suggested that they, too, were arrested (Table 1)Citation . We suggest that dnPLK1 is arresting these cells by blocking the separation of the centrosomes. This effect on the HMECs appears to differ from the loss of centrosome maturation that occurs in Hs68 cells after microinjection of PLK1 antibodies (42) . These differences may reflect the type of functional ablation technique or the type of normal cell studied (42 , 43) .

Centrosome maturation is essential for the nucleation of mitotic microtubules. To confirm that abnormal centrosome maturation in SAOS-2 cells could, in part, account for the ensuing mitotic catastrophe, we quantitated the normal and abnormal monoastral mitoses in each cell type. Twenty-four % of all of the abnormal mitoses in control virus-infected SAOS-2 cells (7.7% of total cells) were of the monoastral type. The number of abnormal monoastral spindles increased from 24 to 41% in dnPLK1 Ad-infected SAOS-2 cells. Unlike SAOS-2 cells, the number of abnormal monoastral mitoses increased only from 1 to 2% in HMECs (Table 1)Citation . These data suggest that the loss of PLK1 function exacerbates existing mitotic defects in these tumor lines. Our observation that mitotic prophase is normal in HMECs suggests that HMECs escape mitotic catastrophe because their centrosome maturation is less affected by a loss of PLK1 function. Because PLK1 is important for the activation of the anaphase-promoting complex necessary for exit from mitosis, we also hyopthesize that apoptosis results when the sensitive tumor lines such as SAOS-2 or U-2OS cannot exit mitosis.

We also wanted to investigate the role of cdc25C in the differential apoptotic response between normal and tumor cells. cdc25C is required for the prophase-to-metaphase transition through the removal of inhibitory Thr-14 and Tyr-15 phosphorylations on cdk1 (9 , 10 , 13) . Lane and Nigg (42) suggested that HeLa cells bypassed a centrosome maturation checkpoint whereas normal cells did not, because PLK1 was not required for cdc25C activation in the former. However, an alternative model might be that PLK1 has independent roles in cdc25C activation and centrosome maturation. Our results clearly demonstrate not only that PLK1 is necessary for mitotic hyperphosphorylation of cdc25C in mammalian cells but that dnPLK1 blocked mitotic hyperphosphorylation of cdc25C (Fig. 5)Citation and activation of cdk1 (Table 2)Citation regardless of whether the cell line was sensitive or insensitive to dnPLK1. These data suggest that the inhibition of cdc25C inhibition by dnPLK1 is not sufficient to induce apoptosis in MCF7 cells. These data also argue that the mitotic catastrophe observed in the sensitive U-20S tumor line is not attributable to a lack of dependence for PLK1 in activating cdc25C. Although they do not exclude a role for cdc25C in the apoptotic response in sensitive tumor lines, they do indicate that the loss of cdc25C function itself is not sufficient.

Our data suggest that centrosome maturation can be a distinguishing feature in the response to loss of PLK1 function. Clearly, a prophase arrest prevents the HMECs from entering mitosis and catastrophe, presumably because bipolar movement of the centrosomes cannot occur without active cdk1. Improper nucleation of the mitotic spindle by the centrosomes likely leads to mitotic catastrophe in tumor lines like SAOS-2. Considering that PLK1 can directly interact with {alpha}- and {gamma}-tubulins (44) we hypothesize that tubulins may be key PLK1 substrates in tumor cell lines. We think it is likely that the loss of substrate adhesion and cell death in IMR90 cells mimics anoikis and may contribute to cell death in some tumor lines. It would be interesting to know whether disrupting PLK1 function through other techniques (e.g., antisense or small molecule inhibitor) causes the same loss of substrate adhesion because anoikis is being exploited as an antimetastatic and antiangiogenic strategy for cancer (55) .

This intrinsic determinant of sensitivity to the loss of PLK1 function might reflect how PLK1 interacts with, or is activated in, essential mitotic complexes. In view of the fact that PLK1 has multiple roles in mitotic progression, it is perhaps not surprising that there is cell-to-cell variability in the dependence of each function on PLK1. Although we see effects of dnPLK1 on phosphorylation of mitotic substrates such as cdc25C and other MPM-2 substrates (data not shown), the predominant effect of overexpressing a high amount of dnPLK1 protein may be disruption of essential mitotic complexes. In fact, overexpression of wild-type PLK1 was reported to block centrosome maturation and induce aberrant mitotic structures in HeLa cells (43) . Preliminary experiments in SAOS-2 and U-2OS cells using a wild-type PLK1 adenovirus suggest that hyperphosphorylation of cdc25C is revealed at lower MOI whereas higher MOI can block cdc25C phosphorylation and cause many of the mitotic defects seen with the dominant-negative (data not shown). Future studies will be aimed at comparing the consequences of expressing wild-type- versus dnPLK1 in these cells.

Why then do the tumor and normal cell lines used in this study show such a wide range of "sensitivity" to expression of the dominant negative protein when PLK1 should be essential for all cells? Perhaps overexpression of PLK1 protein in the sensitive tumor lines makes these cells more dependent on PLK1 for formation of essential mitotic complexes and consequent mitotic phosphorylations. Normal cells that have very low levels of PLK1 protein could use different mitotic complexes that are not as easily disrupted by dnPLK1 and could use PLK1 only to phosphorylate a few essential mitotic substrates. Perhaps overexpression of other mitotic kinases results in the formation of novel complexes or causes overlapping mitotic phosphorylation of essential substrates that make these cells insensitive to a loss of PLK1 function. Future work on the roles of PLK1 and other mitotic kinases in mitotic progression will help to answer these questions.

Materials and Methods

Cell lines, Maintenance, and Cell Cycle Synchronization.
The U-20S osteosarcoma cell line was obtained from the Lineberger Comprehensive Cancer Center Tissue Culture Facility (University of North Carolina at Chapel Hill, NC). All of the other tumor lines were obtained from the American Type Culture Collection (Rockville, MD). MCF-10A cells were maintained in DMEM/F12 supplemented with 10 µg/ml bovine insulin, 20 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, and 5% horse serum. All of the other tumor lines were maintained in DMEM-H (high glucose) with 10% fetal bovine serum from HyClone (Logan, UT). HMECs were purchased from Clonetics (San Diego, CA) and used at or before passage 9. The G1 cell cycle synchronization protocol consisted of treatment with 1 mM hydroxyurea for 16 h, release for 10 h, and retreatment with 1 mM hydroxyurea overnight. For HMECs, only one treatment with hydroxyurea was sufficient. The G2 cell synchronization included G1 synchronization, followed by adenovirus infection, then the addition of 0.2 µg/ml nocodazole 8 h postinfection, and finally harvest 24 h postinfection in tumor lines and 48 h postinfection in HMECs.

dnPLK1 Adenovirus and Infections.
The full-length human PLK1 gene was obtained from Incyte as clone 3180142. PCR mutagenesis was used to change Lys-82 to Met (AAG to ATG). Sequence was confirmed by double-stranded dideoxy sequencing. The cytomegalovirus promoter-driven, dnPLK1-expressing, E1(-), E3(-) Ad5 virus was constructed by transposon 7 transposition in Escherichia coli, as described previously (56) . The virus was titered and purified as described previously (57) , except that cesium chloride was removed by dialysis against 10% glycerol, 10 mM Tris-Cl (pH 8.0), 10 mM MgCl2, and 150 mM NaCl. The purified virus was stored at 20°C. Infections were done in 0.2 ml of DMEM-H per 2 x 105 cells containing 2% FCS at 37°C for 1 h with continuous rocking. For lipid-enhanced delivery, 0.1 ml each of adenovirus and lipofectamine diluted in serum-free growth media (6 x 106 pfu of adenovirus per 1 µg of lipofectamine) were mixed and incubated for 15 min at room temperature before addition to cells. Lipid delivery was essential for HS578T and MDAMB435S and was used to enhance dnPLK1 expression in MDAMB231 and MCF10A cells. For Western blots and immunofluorescence, cell lines were plated at 105 cells per 9.6-cm2 well. Twenty-four h after plating, the cells were infected with dnPLK Ad in DMEM-H plus 2% FCS (for tumor lines) or serum-free growth media (for HMECs or NHKs) with rocking for 1 h at 37°C. For kinase assays, cells were plated at 8 x 105 per 100-mm dish and infections were scaled up by 8-fold.

Western Blotting and Kinase Assays.
For Western blots, cells were lysed in RIPA buffer containing protease inhibitor cocktail (Boehringer Mannheim), 50 mM sodium fluoride, 2 mM sodium vanadate, and 10 µg/ml microcystin LR (Sigma) at 4°C. Protein concentrations were determined by the bicinchoninic acid assay (Pierce) and equal concentrations loaded per well. Ten % nonfat dry milk was used for blocking nitrocellulose and for antibody incubations. Western blotting antibodies were used at 0.5 µg/ml and included mouse mAb anti-PLK1 (No. 33–170; Zymed) and anti-cdc25C (#sc-327, Santa Cruz Biotechnology). Quantitation of dnPLK1 overexpression was performed making serial 1:4 dilutions of dnPLK1-infected lysates and determining the dilution that was equivalent to control virus infected lysate. Immunoprecipitations used 100 µg of cell lysate with 10 µg of cyclin B1 mAb (No. 14541C; PharMingen) and protein G Sepharose (Pharmacia Biotech). Precipitates were washed twice in RIPA, twice in RIPA plus 1 M NaCl, then twice in 50 mM HEPES (pH 7.5), and 150 mM NaCl. Beads were resuspended in histone H1 assay mix containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mg/ml histone H1, 1 mM DTT, 2 mM EGTA, 50 mM ATP, and 50 µCi/ml [{gamma}-32P]ATP and were incubated for 15 min at 30°C. Reactions were stopped with an equal volume of Laemmli sample buffer, heat-denatured, and electrophoresed on 12% SDS-PAGE mini-gels (Novex). Kinase activity was quantitated after a 4-h exposure on a PhosphoImager (Molecular Dynamics).

FACS Analysis.
FACS analysis for cell cycle and apoptosis was performed with a Becton Dickinson FACS Calibur. After infection, cells were washed in PBS without calcium and magnesium salts and then fixed in 70% methanol. Fixed cells were washed once with a solution containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 4% FCS, 0.5% Tween, and 0.1% sodium azide. Cell pellets were resuspended in PBS (Ca+2, Mg+2-free) with 0.1% Triton X-100, 100 µg/ml RNase A, and 40 µg/ml propidium iodide for staining of DNA. Cell cycle analysis was performed using Cell Quest software.

Immunocytochemistry.
SAOS-2, U-20S, and HMECs were plated at 105 per well of a 2-well glass chamber slide (Nalge; Nunc International) and, 24 h after plating, were infected with 200 pfu of virus per cell. Fixation and staining were done as described previously (58) using 1 h for each antibody-binding step. Primary antibodies were rat anti-{alpha}-tubulin (MCP77; Serotec) and mouse anti-{gamma}-tubulin (GTU-88; Sigma), each at 1:100. Secondary antibodies were fluorescein-conjugated donkey antirat IgG and rhodamine-conjugated donkey antimouse IgG (Jackson Immunoresearch) at 1:1000. Vectastain plus DAPI was added to stain DNA.

Microscopy.
Sections at x200 and cells at x40 were examined on a Leitz DMRX microscope and captured with Adobe Photoshop using a Hamamatsu chilled 3CCD camera, model C5810.

Acknowledgments

PLK1 clone 3180142 was obtained from Incyte Pharmaceuticals Inc. We thank Jui-Lan Su for antibody optimizations and Cathy Finlay, Gordon McIntyre, Sally Kornbluth, Bob Abraham, and Tony Means for their scientific input.

Footnotes

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 To whom requests for reprints should be addressed, at Department of Functional Genetics, Glaxo Wellcome Inc., 5 Moore Drive, Research Triangle Park, NC 27709. Phone: (919) 483-3449; E-mail: jpc30240{at}glaxowellcome.com Back

2 The abbreviations used are: cdk, cyclin-dependent kinase; PLK, polo-like kinase; dnPLK, dominant-negative PLK; dnPLK1 Ad, adenovirus expressing dnPLK1; MPM-2, mitotic protein monoclonal 2; FACS, fluorescence-activated cell sorting; MOI, multiplicity/multiplicities of infection; NHK, normal human keratinocyte; HMEC, human mammary epithelial cell; DAPI, 4',6-diamidino-2-phenylindole; RIPA, radioimmunoprecipitation assay; pfu, plaque-forming unit(s); mAb, monoclonal antibody. Back

Received for publication 6/22/00. Revision received 10/10/00. Accepted for publication 10/25/00.

References

  1. Elledge S. J. Cell cycle checkpoints: preventing an identity crisis. Science (Washington DC), 274: 1664-1672, 1996.[Abstract/Free Full Text]
  2. Murray, A. W. The genetics of cell cycle checkpoints. Curr. Opin. Genet. Dev., 5: 5-l l, 1995.
  3. Paulovich A. G., Toczyski D. P., Hartwell L. H. When checkpoints fail. Cell, 88: 315-321, 1997.[Medline]
  4. Lengauer C., Kinzler K. W., Vogelstein B. Genetic abnormalities in human cancers. Nature (Lond.), 396: 643-649, 1998.[Medline]
  5. Marx J. How cells cycle towards cancer. Science (Washington DC), 263: 319-321, 1994.[Free Full Text]
  6. Orr-Weaver, T. L, and Weinberg, R. A. The difficulty in separating sisters. Nature (Lond.), 392: 223–224, 1998.
  7. Sherr C. J. Cancer cell cycles. Science (Washington DC), 274: 1672-1677, 1996.[Abstract/Free Full Text]
  8. Atherton-Fessler S., Liu F., Gabrielli B., Lee M. S., Peng C-Y., Piwnica-Worms H. Cell cycle regulation of the p34cdc2 inhibitory kinases. Mol. Biol. Cell, 5: 989-1001, 1994.[Abstract/Free Full Text]
  9. Gabrielli B. G., Lee M. S., Walker D. H., Piwnica-Worms H., Maller J. L. cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex. J. Biol. Chem., 267: 18040-18046, 1992.[Abstract/Free Full Text]
  10. Gu Y., Rosenblatt J., Morgan D. O. Cell cycle regulation of cdk2 activity by phosphorylation of Thr160 and Tyr15. EMBO J., 11: 3995-4005, 1992.[Medline]
  11. Heald R., McLoughlin M., McKeon F. Human Wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated cdc2 kinase. Cell, 74: 463-474, 1993.[Medline]
  12. Smythe C., Newport J. W. Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2. Cell, 68: 787-797, 1992.[Medline]
  13. Borgne A., Meijer L. Sequential dephosphorylation of p34cdc2 on Thr-14 and Tyr-15 at the prophase/metaphase transition. J. Biol. Chem., 271: 27847-27854, 1996.[Abstract/Free Full Text]
  14. Hoffman I., Clarke P. R., Jessus-Marcote M., Karsenti E., Draetta G. Phosphorylation and activation of human cdc25-C by Cdc2-cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J., 12: 53-63, 1993.[Medline]
  15. Izumi T., Walker D., Maller J. L. Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. Mol. Biol. Cell, 3: 927-939, 1992.[Abstract/Free Full Text]
  16. Jessus C., Beach D. Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclinB. Cell, 68: 323-332, 1992.[Medline]
  17. Kumagai, A., and Dunphy, W. G. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell 70: 139–151, 1992.
  18. Izumi T., Maller J. L. Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase block initiation of M-phase. Mol. Biol. Cell, 4: 1337-1350, 1993.[Abstract/Free Full Text]
  19. Strausfeld U., Fernandez A., Capony J-P., Girard F., Lautredou N., Derancourt J., Labbe J-C., Lamb N. J. C. Activation of p34(cdc2) protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34 (cdc2) on sites phosphorylated at mitosis. J. Biol. Chem., 269: 5989-6000, 1994.[Abstract/Free Full Text]
  20. Izumi T., Maller J. L. Phosphorylation and activation of the Xenopus cdc25 phosphatase in the absence of cdc2 and cdk2 kinase activity. Mol. Biol. Cell, 6: 215-226, 1995.[Abstract/Free Full Text]
  21. Kumagai A., Dunphy W. G. Purification and molecular cloning of Plx1, a cdc25-regulatory kinase from Xenopus egg extracts. Science (Washington DC), 273: 1377-1380, 1996.[Abstract]
  22. Abrieu A., Brassac T., Galas S., Fisher D., Labbe J-C., Doree M. The polo-like kinase plx1 is a component of the MPF amplification loop at the G2/M phase transition of the cell cycle in Xenopus eggs. J. Cell Sci., 111: 1751-1757, 1998.[Abstract/Free Full Text]
  23. Qian Y-W., Erikson E., Li C., Maller J. L. Activated polo-like kinase plx1 is required at multiple points during mitosis in Xenopus laevis. Mol. Cell Biol., 18: 4262-4271, 1998.[Abstract/Free Full Text]
  24. Sunkel, C. E., and Glover, D. M. Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J. Cell Sci. 89: 25–38, 1988.
  25. Glover D. M., Hagan I. M., Tavares A. A. M. Polo-like kinases: a team that plays throughout mitosis. Genes and Development, 12: 3777-3787, 1998.[Free Full Text]
  26. Nigg E. A. Polo-like kinases: positive regulators of cell division from start to finish. Curr. Opin. Cell Biol., 10: 776-783, 1999.
  27. Chase D., Feng Y., Hanshew B., Winkles J. A., Longo D. L., Ferris D. K. Expression and phophorylation of fibroblast-growth-factor-inducible kinase (Fnk) during cell-cycle progression. Biochem. J., 333: 655-660, 1998.
  28. Donohue P. J., Alberts G. F., Guo Y., Winkles J. A. Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase. J. Biol. Chem., 270: 10351-10357, 1995.[Abstract/Free Full Text]
  29. Li B., Ouyang B., Pan H., Reissmann P. T., Slamon D. J., Arceci R., Lu L., Dai W. PRK, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas. J. Biol. Chem., 271: 19402-19408, 1996.[Abstract/Free Full Text]
  30. Ouyang B., Pan H., Lu L., Li J., Stambrook P., Li B., Dai W. The physical association and phosphorylation of cdc25C protein phosphatase by Prk. J. Biol. Chem., 272: 28646-28651, 1997.[Abstract/Free Full Text]
  31. Simmons D. L., Neel B. G., Steven R., Evett G., Erikson R. L. Identification of an early-growth-response gene encoding a novel protein kinase. Mol. Cell. Biol., 12: 4164-4169, 1992.[Abstract/Free Full Text]
  32. Patel R., Holt M., Philipova R., Moss S., Schulman H., Hidaka H., Whitaker M. Calcium/calmodulin-dependent phosphorylation and activation of cdc25-C at the G2/M phase transition in HeLa cells. J. Biol. Chem., 274: 7958-7968, 1999.[Abstract/Free Full Text]
  33. Golsteyn R. M., Mundt K. E., Fry A. M., Nigg E. A. Cell cycle regulation of the activity and subcellular localization of PLK1, a human protein kinase implicated in mitotic spindle function. J. Cell Biol., 129: 1617-1628, 1995.[Abstract/Free Full Text]
  34. Lee, K. S., Yuan, Y-L. O., Kuriyama, R., and Erikson, R. L. PLK is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1. Mol. Cell. Biol., 15: 7143–7151, 1995.
  35. Davis F. M., Tsao T. Y., Fowler S. K., Rao P. N. Monoclonal antibodies to mitotic cells. Proc. Natl. Acad. Sci. USA, 80: 2926-2930, 1983.[Abstract/Free Full Text]
  36. Taagepera S., Rao P. N., Drake F. H., Gorbsky G. J. DNA topoisomerase II{alpha} is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2. Proc. Natl. Acad. Sci. USA, 90: 8407-8411, 1993.[Abstract/Free Full Text]
  37. Taagepera S., Campbell M. S., Gorbsky G. J. Cell-cycle regulated localization of tyrosine and threonine phosphoepitopes at the kinetochores of mitotic chromosomes. Exp. Cell. Res., 221: 249-260, 1995.[Medline]
  38. Vandre D. D., Davis F. M., Rao P. N., Borisy G. G. Phosphoproteins are components of mitotic microtubule organizing centers. Proc. Natl. Acad. Sci. USA, 81: 4439-4443, 1984.[Abstract/Free Full Text]
  39. Kuang J., Ashorn C. L., Gonzalez-Kuyvenhoven M., Penkala J. E. cdc25 is one of the MPM-2 antigens involved in the activation of maturation promoting factor. Mol. Biol. Cell, 5: 135-145, 1994.[Abstract/Free Full Text]
  40. Logarinho E., Sunkel C. E. The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. J. Cell Sci., 111: 2897-2909, 1998.[Abstract/Free Full Text]
  41. Adams R. R., Tavares A. A. M., Salzberg A., Bellen H. J., Glover D. M. Pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes and Development, 12: 1483-1494, 1998.[Abstract/Free Full Text]
  42. Lane H. A., Nigg E. A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (plk1) in the functional maturation of mitotic centrosomes. J. Cell Biol., 135: 1701-1713, 1996.[Abstract/Free Full Text]
  43. Mundt K. E., Golsteyn R. M., Lane H. A., Nigg E. A. On the regulation and function of human polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression. Biochem. Biophys Res. Comm., 239: 377-385, 1997.[Medline]
  44. Feng Y., Hodge D. R., Palrnieri G., Chase D. L., Longo D. L., Ferris D. K. Association of polo-like kinase with {alpha}-, ß-, and {gamma}-tubulins in a stable complex. Biochem. J., 339: 435-442, 1999.
  45. Kotani S., Tegendrich S., Fujii M., Jorgenson P-M., Watanabe N., Hoog C., Hieter P., Todokoro K. PKA and MPF-activated polo-like kinase regulate anaphase-promoting complex activity and mitosis progression. Molecular Cell, 1: 371-380, 1998.[Medline]
  46. Descombes P., Nigg E. A. The polo-like kinase plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J., 17: 1328-1335, 1988.[Abstract]
  47. Shirayama M., Zachariae W., Ciosk R., Nasmyth K. The polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J., 17: 1336-1349, 1988.[Abstract]
  48. Maniotis A., Schliwa M. Microsurgical removal of centrosomes block cell reproduction and centriole generation in BSC-1 cells. Cell, 67: 495-504, 1991.[Medline]
  49. Dedieu, J-F., Vigne, E., Torrent, C., Jullien, C., Mahfouz, I., Caillaud, J-M., Aubailly, N., Orsini, C,, Guillame, J-M., Opolon, P., Delaire, P., Perricaudet, M., and Yeh, P. Long term gene delivery into the livers of immunocompetant mice with E1/E4-defective adenoviruses. J. Virol., 71: 4626–4637, 1997.
  50. Frisch S. M., Ruoslahti E. Integrins and anoikis. Curr. Opin. Cell Biol., 9: 701-706, 1997.[Medline]
  51. Krestkow J. K., Rak J., Filmus J., Kerbel R. S. Functional dissociation of anoikis-like cell death and activity of stress-activated protein kinase. Biochem. Biophys. Res. Comm., 260: 48-53, 1999.[Medline]
  52. Smith M. R., Wilson M. L., Hamanaka R., Chase D., Kung H-F., Longo D. L., Ferris D. K. Malignant transformation of mammalian cells initiated by constitutive expression of the polo-like kinase. Biochem. Biophys. Res. Comm., 234: 397-405, 1997.[Medline]
  53. Wolf G., Elez R., Doermer A., Holtrich U., Ackermann H., Stutte H. J., Altmannsberger H-M., Rubsamen-Waigmann H. R., Strebhardt K. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer. Oncogene, 14: 543-549, 1997.[Medline]
  54. Knecht R., Elez R., Oechler M., Solbach C., von Ilberg C., Strebhardt K. Prognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neck. Cancer Res., 59: 2794-2797, 1999.[Abstract/Free Full Text]
  55. Ruoslahti E. Fibronectin and its integrin receptors in cancer. Adv. Cancer Res., 76: 1-20, 1999.[Medline]
  56. Richards C. A., Brown C. E., Cogswell J. P., Weiner M. P. The Admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli. Biotechniques, 29: 146-154, 2000.[Medline]
  57. Ghersa, P., Pescini-Gobert, R, Sattonnet-Roche, P., Richards, C. A., Merlo Pick, E., and Hooft van Huijsduijnen, R. Highly controlled gene expression using combinations of a tissue-specific promoter, recombinant adenovirus and a tetracycline-regulatable transcription factor. Gene Ther., 5: 1213–1220, 1998.
  58. Su, J-L, Kilpatrick, K. E., Champion, B. R., Morris, D. C., Lehmann, J. M., and Kost, T. A. Fluorescent microtiter screening assay of immunocytochemically reactive antibodies. Biotechniques, 22: 320–324, 1997.



This article has been cited by other articles:


Home page
Cancer Res.Home page
J. Triscott, C. Lee, C. Foster, B. Manoranjan, M. R. Pambid, R. Berns, A. Fotovati, C. Venugopal, K. O'Halloran, A. Narendran, et al.
Personalizing the Treatment of Pediatric Medulloblastoma: Polo-like Kinase 1 as a Molecular Target in High-Risk Children
Cancer Res., November 15, 2013; 73(22): 6734 - 6744.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
D.-C. Lin, Y. Zhang, Q.-J. Pan, H. Yang, Z.-Z. Shi, Z.-H. Xie, B.-S. Wang, J.-J. Hao, T.-T. Zhang, X. Xu, et al.
PLK1 Is Transcriptionally Activated by NF-{kappa}B during Cell Detachment and Enhances Anoikis Resistance through Inhibiting {beta}-Catenin Degradation in Esophageal Squamous Cell Carcinoma
Clin. Cancer Res., July 1, 2011; 17(13): 4285 - 4295.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
Y. Degenhardt and T. Lampkin
Targeting Polo-like Kinase in Cancer Therapy
Clin. Cancer Res., January 15, 2010; 16(2): 384 - 389.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
A. G. Gilmartin, M. R. Bleam, M. C. Richter, S. G. Erskine, R. G. Kruger, L. Madden, D. F. Hassler, G. K. Smith, R. R. Gontarek, M. P. Courtney, et al.
Distinct Concentration-Dependent Effects of the Polo-like Kinase 1-Specific Inhibitor GSK461364A, Including Differential Effect on Apoptosis
Cancer Res., September 1, 2009; 69(17): 6969 - 6977.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
H. Yim and R. L. Erikson
Polo-Like Kinase 1 Depletion Induces DNA Damage in Early S Prior to Caspase Activation
Mol. Cell. Biol., May 15, 2009; 29(10): 2609 - 2621.
[Abstract] [Full Text] [PDF]


Home page
Mol. Biol. CellHome page
V. Sherwood, R. Manbodh, C. Sheppard, and A. D. Chalmers
RASSF7 Is a Member of a New Family of RAS Association Domain-containing Proteins and Is Required for Completing Mitosis
Mol. Biol. Cell, April 1, 2008; 19(4): 1772 - 1782.
[Abstract] [Full Text] [PDF]


Home page
Clin. Cancer Res.Home page
L. M. Leoni, B. Bailey, J. Reifert, H. H. Bendall, R. W. Zeller, J. Corbeil, G. Elliott, and C. C. Niemeyer
Bendamustine (Treanda) Displays a Distinct Pattern of Cytotoxicity and Unique Mechanistic Features Compared with Other Alkylating Agents
Clin. Cancer Res., January 1, 2008; 14(1): 309 - 317.
[Abstract] [Full Text] [PDF]


Home page
Proc. Natl. Acad. Sci. USAHome page
F. Stegmeier, M. E. Sowa, G. Nalepa, S. P. Gygi, J. W. Harper, and S. J. Elledge
The tumor suppressor CYLD regulates entry into mitosis
PNAS, May 22, 2007; 104(21): 8869 - 8874.
[Abstract] [Full Text] [PDF]


Home page
Molecular Cancer TherapeuticsHome page
T. J. Lansing, R. T. McConnell, D. R. Duckett, G. M. Spehar, V. B. Knick, D. F. Hassler, N. Noro, M. Furuta, K. A. Emmitte, T. M. Gilmer, et al.
In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1
Mol. Cancer Ther., February 1, 2007; 6(2): 450 - 459.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
B. Spankuch, S. Heim, E. Kurunci-Csacsko, C. Lindenau, J. Yuan, M. Kaufmann, and K. Strebhardt
Down-regulation of Polo-like Kinase 1 Elevates Drug Sensitivity of Breast Cancer Cells In vitro and In vivo
Cancer Res., June 1, 2006; 66(11): 5836 - 5846.
[Abstract] [Full Text] [PDF]


Home page
FASEB J.Home page
W. Malorni and C. Fiorentini
Is the Rac GTPase-activating toxin CNF1 a smart hijacker of host cell fate?
FASEB J, April 1, 2006; 20(6): 606 - 609.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
E. Y. Chuang, X. Chen, M.-H. Tsai, H. Yan, C.-Y. Li, J. B. Mitchell, H. Nagasawa, P. F. Wilson, Y. Peng, M. M. Fitzek, et al.
Abnormal Gene Expression Profiles in Unaffected Parents of Patients with Hereditary-Type Retinoblastoma
Cancer Res., April 1, 2006; 66(7): 3428 - 3433.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
M. R. Bhonde, M.-L. Hanski, J. Budczies, M. Cao, B. Gillissen, D. Moorthy, F. Simonetta, H. Scherubl, M. Truss, C. Hagemeier, et al.
DNA Damage-induced Expression of p53 Suppresses Mitotic Checkpoint Kinase hMps1: THE LACK OF THIS SUPPRESSION IN p53MUT CELLS CONTRIBUTES TO APOPTOSIS
J. Biol. Chem., March 31, 2006; 281(13): 8675 - 8685.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
F. Eckerdt, J. Yuan, K. Saxena, B. Martin, S. Kappel, C. Lindenau, A. Kramer, S. Naumann, S. Daum, G. Fischer, et al.
Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells
J. Biol. Chem., November 4, 2005; 280(44): 36575 - 36583.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
R. Guan, P. Tapang, J. D. Leverson, D. Albert, V. L. Giranda, and Y. Luo
Small Interfering RNA-Mediated Polo-Like Kinase 1 Depletion Preferentially Reduces the Survival of p53-Defective, Oncogenic Transformed Cells and Inhibits Tumor Growth in Animals
Cancer Res., April 1, 2005; 65(7): 2698 - 2704.
[Abstract] [Full Text] [PDF]


Home page
aacredbookHome page
R. A. Mook Jr.
The Importance and Complexities of Target Class Selectivity in Drug Discovery
Am. Assoc. Cancer Res. Educ. Book, April 1, 2005; 2005(1): 223 - 226.
[Full Text] [PDF]


Home page
J Biol ChemHome page
L. Tsvetkov and D. F. Stern
Interaction of Chromatin-associated Plk1 and Mcm7
J. Biol. Chem., March 25, 2005; 280(12): 11943 - 11947.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
R. W. Gunawardena, H. Siddiqui, D. A. Solomon, C. N. Mayhew, J. Held, S. P. Angus, and E. S. Knudsen
Hierarchical Requirement of SWI/SNF in Retinoblastoma Tumor Suppressor-mediated Repression of Plk1
J. Biol. Chem., July 9, 2004; 279(28): 29278 - 29285.
[Abstract] [Full Text] [PDF]


Home page
Molecular Cancer TherapeuticsHome page
P. J. Gray Jr, D. J. Bearss, H. Han, R. Nagle, M.-S. Tsao, N. Dean, and D. D. Von Hoff
Identification of human polo-like kinase 1 as a potential therapeutic target in pancreatic cancer
Mol. Cancer Ther., May 1, 2004; 3(5): 641 - 646.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
Y.-S. Seong, K. Kamijo, J.-S. Lee, E. Fernandez, R. Kuriyama, T. Miki, and K. S. Lee
A Spindle Checkpoint Arrest and a Cytokinesis Failure by the Dominant-negative Polo-box Domain of Plk1 in U-2 OS Cells
J. Biol. Chem., August 30, 2002; 277(35): 32282 - 32293.
[Abstract] [Full Text] [PDF]


Home page
Cancer Res.Home page
J. Yuan, A. Kramer, F. Eckerdt, M. Kaufmann, and K. Strebhardt
Efficient Internalization of the Polo-Box of Polo-like Kinase 1 Fused to an Antennapedia Peptide Results in Inhibition of Cancer Cell Proliferation
Cancer Res., August 1, 2002; 62(15): 4186 - 4190.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cogswell, J. P.
Right arrow Articles by Neill, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cogswell, J. P.
Right arrow Articles by Neill, S. D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation