In order to characterize the function of Plk1 in vivo, mice heterozygous for Plk1 were generated by using gene trap ES cell line RRR358. In this ES cell line, one allele of Plk1 was disrupted by a gene trap vector inserted at exon 9, which abolished proper transcription of Plk1 (Fig. ). Exon 9 encodes part of the polo-box domain of Plk1, which is essential for its substrate binding (7
) and is required for targeting the kinase activity of Plk1 to various subcellular localizations (15
). Since the gene trap is on exon 9, a truncated mutant of Plk1 fused with β-geo should be translated. However, we could detect neither the fusion protein, using a β-galactosidase antibody, nor the truncated Plk1 mutant, using a Plk1 N-terminal antibody, by Western blotting using tissues from heterozygous mice (data not shown), suggesting that the fusion protein or truncated Plk1 could not be correctly translated or was extremely unstable. Thus, this Plk1 gene trap virtually abolishes Plk1's expression.
FIG. 1. Presence of Plk1 homozygous mutant embryos at E3.5. (a) Gene structure of WT and mutant (KO) allele of Plk1 locus. β-geo represents the gene trap vector. (b) Typical genotyping results for E3.5 embryos from Plk1 heterozygous mutant intercrossed (more ...)
Mice heterozygous for Plk1 were obtained by injecting ES cells into blastocysts and breeding chimeras with C57BL/6 mice. After several intercrosses of heterozygous mice, no homozygous null Plk1 mice were obtained (Table ), indicating that the complete deletion of Plk1 was embryonic lethal. To investigate at what stage Plk1-null embryos underwent cell death, we isolated embryos from matings of Plk1 heterozygous mice. The embryonic lethality occurred in the early embryonic stages, as no surviving Plk1 homozygous null embryos were obtained at E10.5 (Table ). We then harvested E3.5 embryos and discovered a population of abnormal embryos with four or eight cells that were consistently identified together with normal blastocysts. PCR genotyping suggested that all of these developmentally delayed embryos, but none of the normally developed blastocysts, were homozygous null for Plk1 (Fig. ). Analysis by reverse transcription-PCR also confirmed that these abnormal embryos did not contain any WT Plk1 transcripts (Fig. ). Embryos were then cultured in vitro for 4 days. None of the abnormal embryos showed further growth, and all underwent apoptosis (Fig. ), while normal blastocysts displayed proper hatching and outgrowth. These data suggest that Plk1 is essential for early embryonic development.
Summary of genotypes of offspring of Plk1 heterozygous mutant intercrossed mice
FIG. 2. Plk1 homozygous mutant embryos fail to survive after the eight-cell stage. Embryos harvested at E3.5 from heterozygous mutant intercrossed mice were cultured and imaged for 4 consecutive days. Pictures of typical abnormal and normal embryos are shown. (more ...)
Plk1 has multiple functions during the cell cycle and is required particularly for centrosome maturation, spindle assembly, and mitotic entry. To test if the embryonic lethality of these Plk1-null embryos was due to the inability to maintain a normal cell cycle, we performed immunostaining with an antibody against phosphorylated histone H3 serine 10, which is a marker for mitotic cells. All normal embryos displayed strong, concentrated staining in a portion of their cells, which were in mitosis. However, none of the abnormal embryos showed any positive staining (Fig. ). Although the number of total cells in the abnormal embryos was less than that in normal embryos, consistent observations of all the embryos suggested that this lower number of cells could not be the cause for the absence of phospho-H3S10. Further analysis of spindle assembly using an antibody against α-tubulin revealed that none of these abnormal embryos contained any assembled spindles, while normal spindle assembly was observed in normal embryos (Fig. ). These observations together indicate that Plk1 is indispensable for normal cell cycle progression in four-cell- or eight-cell-stage embryos.
FIG. 3. Plk1 homozygous mutant embryos failed to enter mitosis. Pictures of stained, typical abnormal and normal embryos are shown. (a) Embryos harvested at E3.5 from heterozygous mutant intercrossed mice were stained with an antibody against phosphorylated histone (more ...)
Since Plk1 was also reported to play a role in DNA replication (34
), we tested whether Plk1 is involved in DNA synthesis by using BrdU incorporation as a readout. Both normal and abnormal embryos incorporated BrdU (Fig. ), indicating that the initiation of DNA replication can occur in the absence of Plk1, although we were unable to conclude whether replication occurs normally or completely in Plk1-null cells.
From our in vitro studies using Plk1 embryos, we concluded that Plk1 deficiency leads to early embryonic lethality. However, Plk1+/− mice are born healthy and fertile, with no obvious effects except a slight decrease in Plk1 levels compared to those for Plk1 WT mice (Fig. ). Given the requirement of Plk1 for early embryogenesis and normal cell cycle progression, we hypothesized that loss of one Plk1 allele might cause problems with cell cycle control. Such defects may lead to chromosomal instability and promote tumorigenesis in these Plk1+/− mice. To test whether this is the case, we established a cohort of Plk1+/+ and Plk1+/− mice. We euthanized animals ranging from 50 to 70 weeks of age and performed necroscopies to search for tumorigenesis in these mice. The average age at euthanasia for both Plk1+/+ and Plk1+/− mice was 57 weeks (see Fig. S1 in the supplemental material). Interestingly and surprisingly, Plk1+/− mice developed tumors in various organs at a frequency of 27.5% (11 out of 40), compared with only 9% (3 out of 34) for Plk1+/+ mice. This increased incidence of tumors is highly significant by chi-square analysis (P < 0.001) (Fig. ). A significant portion of these tumors appeared to be lymphomas that invaded the lung and liver. Shown in Fig. is a lymphoma that invaded the liver, with a corresponding view of an H&E-stained section of the tumor. The rest of the tumors were lung carcinomas, except for one squamous cell carcinoma and one ovarian sarcoma (Fig. ). The increased incidence of tumors could potentially be caused by chromosomal instability, since Plk1 is important for mitotic transitions. To test this possibility, we harvested spleens from 6-month-old Plk1 WT and heterozygous mice and prepared chromosome spreads to determine whether Plk1 heterozygosity leads to aneuploidy, which may account for the subsequent tumorigenesis. We found that the heterozygous splenocytes contained a higher percentage of aneuploidies, suggesting that chromosomal instability is indeed present in somatic cells (Fig. ), which may eventually result in tumor formation in these Plk1+/− mice.
FIG. 4. Plk1 heterozygotes develop spontaneous tumors. (a) Plk1 levels are decreased in the livers of Plk1 heterozygous mice compared to those in the WT. IB, immunoblot. (b) Plk1+/+ and Plk1+/− mice were euthanized between the (more ...)
We also crossed Plk1 heterozygous mice onto a p53−/− background and determined whether the loss of p53 would rescue the embryonic lethality observed in Plk1−/− mice. Loss of p53 did not rescue the embryonic lethality of the Plk1 deletion, as only Plk1 heterozygotes were obtained from p53−/− Plk1+/− crossings (data not shown). The Plk1+/− p53−/− mice developed tumors at a higher frequency than p53−/− mice, although the tumor spectrum between the mice remained similar. All eight Plk1+/−p53−/− mice developed tumors, mostly lymphomas and sarcomas (Fig. ). In comparison, four out of eight Plk1+/+p53−/− mice developed tumors, mainly lymphomas and sarcomas. While the number of animals used in these experiments was limited, this finding is significant according to chi-square analysis (P < 0.05).
In conclusion, our results suggest that Plk1 is critical for maintaining the normal cell cycle. The absence of Plk1 leads to early embryonic lethality, and Plk1 heterozygous mice develop spontaneous tumors, suggesting that a normal level of Plk1 is critical for maintaining chromosomal stability. Future studies using conditional KO or hypomorphic Plk1 mice will allow for further analysis of the role of Plk1 as a putative tumor suppressor.
Our observations are consistent with previous reports of KO models of Plk1 homologs in other organisms, all of which support a critical role for Plk1 in cell cycle regulation. In Drosophila
, the polo2
mutant was lethal at the larval stage, probably due to a defect at the onset of mitosis. Although the polo1
mutant was viable, embryos from homozygous females showed a defect in spindle formation (36
). In budding yeast, the cdc5
mutant was lethal and displayed a dumbbell-shaped morphology and the consistent presence of mitotic spindles, indicating a defect in mitotic exit (16
). In fission yeast, the plo1
mutant was also lethal and displayed two distinct phenotypes, one with monopolar spindle formation and another with failed septation (29
), suggesting multiple roles for Plo1 during mitosis. The differences in phenotypes among various KO models in these organisms could reflect multiple roles of Plk1 in mitosis as well as the degree that each organism could tolerate the absence of Plks.
There are four members of the Plk family in mouse and human, all of which function in controlling cell cycle progression. All four members of the Plk family contain a serine/threonine kinase domain and a polo-box domain. Among them, Plk1, Plk2, and Plk3 have a tandem polo-box repeat, while Plk4 has a single polo box. The similar domain architecture could result in functional redundancy among Plks, which may explain why Plk2 KO mice are viable, albeit 20% smaller at birth. However, Plk4-null mice are embryonic lethal and die around E7.5, with increased mitotic cells in mutant embryos, suggesting a delay in progression through anaphase and the blockage of cell division. In this study, we showed that Plk1-null embryos had a perturbed progression of the cell cycle and were arrested at the eight-cell stage. These embryos might be arrested in the G2
phase and fail to enter mitosis, which would be consistent with previous reports of a role for Plk1 at mitotic entry (14
). It was also documented that Plk1 is required for recovery from G2
DNA damage-induced arrest (43
). However, recent studies suggest that Plk1 may not be absolutely required for mitotic entry; instead, cells without Plk1 activity showed long delays in late prophase before entering mitosis (12
). Moreover, Plk1 clearly has a critical role in cytokinesis (5
). Therefore, it is possible that these Plk1-null embryos might have a cytokinesis defect, which could eventually allow them to exit mitosis but be arrested at the tetraploidy G1
phase. Nonetheless, our Plk1 KO data clearly suggest that different Plks have overlapping yet distinct functions in mammalian cells.
Similar to Plk1 heterozygotes, Plk4 heterozygotes display an augmented frequency of tumors at advanced age (18 to 24 months). In comparison, Plk1 heterozygotes develop tumors at 13 to 14 months of age on average. This could be due to the fact that Plk1 is essential for mitosis; null embryos die at E3.5 without entering the blastocyst stage. Loss of one allele of Plk1 can perhaps cause a delay in mitosis or failed chromosome segregation, eventually leading to aneuploidy and tumorigenesis, which is supported by our observation that premalignant splenocytes from Plk1+/− mice harbor increased levels of aneuploidy. On the other hand, the phenotypes that occur in the absence of Plk4 are less severe. Plk4-null embryos are able to undergo mitosis but die at a later stage due to an elevated number of mitotic errors and the delayed progression of anaphase. This difference could be the reason why Plk4 heterozygous mice develop tumors at an advanced age, since the accumulation of mitotic errors may be less rapid in these mice.
It is intriguing that we observed increased tumor susceptibility in mice lacking one allele of Plk1, since Plk1 is normally considered to be an oncogene, due to its enhanced expression in a variety of human cancers. In fact, Plk1 inhibitors have been developed for potential use as anticancer agents (10
). Our study with Plk1 KO mice leads us to speculate that the levels of Plk1s must be tightly regulated in the cell; too much can tip the balance toward the promotion of tumorigenesis and, even when reduced by half, can also license tumor progression. Therefore, using Plk1 as a therapeutic target may not be as straightforward as previously thought. The potential negative impact of reduced Plk1 activity should be carefully considered and assessed before Plk1 inhibitors are used for the treatment of human cancers.