Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.

Maturation-promoting factor (MPF) is well-known as cell cycle regulator during oocyte maturation and fertilization. MPF activity maintains high levels and arrest the cell cycle progression until fertilization. After fertilization, Anaphase-promoting complex/cyclosome (APC/C) mediated degradation of cyclin B causes decrease of MPF activity. One of the cytostatic factor (CSF), Emi2 inhibits APC/C activity by binding to APC/C-cdc20, therefore blocks the proteolysis of cyclin B. Degradation of Emi2 requires phosphorylation by Polo-like kinase 1 (Plk1). Thus recognition and phosphorylation of Emi2 by Plk1 are essential step for meiotic cell cycle resumption. In our previous research, we found that two phosphorylated threonine regions at amino acid position 152 and 176 in Emi2 are respectively contributed for recognition by polo-box domain of Plk1. Peptidomimetics 103-8 can block the interaction between Plk1-PBD and Emi2, and therefore meiotic maturation and meiosis resumption via parthenogenetic activation were impaired. However, major drawback of 103-8 was the limitation of penetration through the cell membrane. We synthesized the new peptidomimetics and checked bioavailability in mammalian oocyte by injection and media treatment. Medium treatment with peptidomimetics C-4, meiotic maturation has significantly decreased and meiotic resumption via parthenogenetic activation has perfectly impaired. For the next experiment, we are preparing X-ray crystallography to identify the binding modes between Plk1-PBD and peptidomimetics C-4.

Chromosomal abnormality s uch as aneuploidy is one of the main factors to cause cancers This abnormality is caused by defects in centrosomal duplication‘ and most cancer cells have extra copies of centrosomes, r esulting in t he formation of multipolar spindles. Several kinases playing in mitotic phase have been implicated in regulating the centrosomal cycle‘ spindle checkpoint‘ and chromosome co ndensation and segregation. They also have Lhe ability to act as oncogenes. FOl studying the relationship between rnitotic kinase and oral cancers, the kinase activity of polo-like kinase-1, which is one of mitosis-specific kinases, is analyzed in oral carcinoma cells originated differently. Kinase activity was increased in these cancer cells compared to normal human gingival fibroblast primary cultured cells Moreover. the mitotic cell populations were a lso increased in these cell lines. Whereas the inhibition of Polo-like kinase-1 by C-terrninal domain in human gingival fibroblast cells induced multiploidy‘ the apoptotic cell population was increased in oral cancer cells overexpressed C-terminal domain 0 1' Polo- Ii ke kinase-1. These data suggested that polo-like kinase-1 might be involved in the on cogenic effect in oral cancer like other solid human carcinomas, and be target molecules for anti-cancer drug.

Polo-Like Kinase(PLK) is a cell cycle-regulated, cyclin-independent serine/threonine protein kinase. Recent reports have shown a critical role for PLK during tumorigenesis. To explore whether PLK plays a general role as a tumor marker of oral squamous cell carcinomas, we examined the expression of PLK mRNA and protein in oral squamous cell carcinoma cells and immortalized normal oral keratinocytes(INOK). We also investigated that PLK mRNA was expressed in specimens from 4NQO-induced SD rat tongue carcinomas using in situ RT-PCR methods. Immunocytochemically, most of the PLK was highly expressed in the nucleus of carcinoma cells, but not INOK. RT-PCR revealed PLK1 mRNA was detected in the FaDu and Hep2 cancer cells, but no detected in the INOK. In situ RT-PCR revealed PLK1 mRNA expression increased sequentially from hyperplasia to dysplasia, and squamous cell carcinoma during the malignant progression. PLK1 expression could reflect the degree of malignancy and proliferation in oral squamous cell carcinomas. Thus, in addition to being of diagnostic value, modulation of PLK1 activity in the tumors by chemotherapeutic agents or gene therapy may prove to be of therapeutic value.