Dataset Information

Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells.

ABSTRACT: Melanoma, one of the most lethal forms of skin cancer, remains resistant to currently available treatments. Therefore, additional target-based approaches are needed for the management of this neoplasm. Polo-like kinase 1 (Plk1) has been shown to be a crucial regulator of mitotic entry, progression, and exit. Elevated Plk1 level has been associated with aggressiveness of several cancer types and with poor disease prognosis. However, the role of Plk1 in melanoma is not well established. Here, we show that Plk1 is overexpressed in both clinical tissue specimens and cultured human melanoma cells (WM115, A375, and HS294T) when compared with normal skin tissues and cultured normal melanocytes, respectively. Furthermore, Plk1 gene knockdown through Plk1-specific shRNA or its activity inhibition by a small-molecule inhibitor resulted in a significant decrease in the viability and growth of melanoma cells without affecting normal human melanocytes. In addition, Plk1 inhibition resulted in a significant (i) decrease in clonogenic survival, (ii) multiple mitotic errors, (iii) G(2)/M cell-cycle arrest, and (iv) apoptosis of melanoma cells. This study suggests that Plk1 may have a functional relevance toward melanoma development and/or progression. We suggest that the targeting of Plk1 may be a viable approach for the treatment of melanoma.

SUBMITTER: Schmit TL

PROVIDER: S-EPMC2799787 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells.

Schmit Travis L TL Zhong Weixiong W Setaluri Vijayasaradhi V Spiegelman Vladimir S VS Ahmad Nihal N

The Journal of investigative dermatology 20090625 12

Melanoma, one of the most lethal forms of skin cancer, remains resistant to currently available treatments. Therefore, additional target-based approaches are needed for the management of this neoplasm. Polo-like kinase 1 (Plk1) has been shown to be a crucial regulator of mitotic entry, progression, and exit. Elevated Plk1 level has been associated with aggressiveness of several cancer types and with poor disease prognosis. However, the role of Plk1 in melanoma is not well established. Here, we s ...[more]

PMID: 19554017

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Project description:Mitotic catastrophe, which refers to cell death or its prologue triggered by aberrant mitosis, can be induced by a heterogeneous group of stimuli, including chromosome damage or perturbation of the mitotic apparatus. We investigated the mechanism of mitotic catastrophe and cell death induced by depletion of centrosomal proteins that perturbs microtubule organization. We transfected cells harboring wild-type or mutated p53 with siRNAs targeting Aurora A, ninein, TOG, TACC3, ?-tubulin, or pericentriolar material-1, and monitored the effects on cell death. Knockdown of Aurora A, ninein, TOG, and TACC3 led to cell death, regardless of p53 status. Knockdown of Aurora A, ninein, and TOG, led to aberrant spindle formation and subsequent cell death, which was accompanied by several features of apoptosis, including nuclear condensation and Annexin V binding in HeLa cells. During this process, cleavage of poly(ADP-ribose) polymerase-1, caspase-3, and caspase-9 was detected, but cleavage of caspase-8 was not. Cell death, monitored by time-lapse imaging, occurred during both interphase and M phase. In cells depleted of a centrosomal protein (Aurora A, ninein, or TOG), the rate of cell death was higher if the cells were cotransfected with siRNA against BubR1 or Mad2 than if they were transfected with siRNA against Bub1 or a control siRNA. These results suggest that metaphase arrest is necessary for the mitotic catastrophe and cell death caused by depletion of centrosomal proteins. Knockdown of centrosomal proteins led to increased phosphorylation of Chk2. Enhanced p-Chk2 localization was also observed at the centrosome in cells arrested in M phase, as well as in the nuclei of dying cells. Cotransfection of siRNAs against Chk2, in combination with depletion of a centrosomal protein, decreased the amount of cell death. Thus, Chk2 activity is indispensable for apoptosis after mitotic catastrophe induced by depletion of centrosomal proteins that perturbs microtubule organization.

Project description:BACKGROUND: We have previously reported that p53 mutated radioresistant lymphoma cell lines undergo mitotic catastrophe after irradiation, resulting in metaphase arrest and the generation of endopolyploid cells. A proportion of these endopolyploid cells then undergo a process of de-polyploidisation, stages of which are partially reminiscent of meiotic prophase. Furthermore, expression of meiosis-specific proteins of the cancer/testis antigens group of genes has previously been reported in tumours. We therefore investigated whether expression of meiosis-specific genes was associated with the polyploidy response in our tumour model. METHODS: Three lymphoma cell lines, Namalwa, WI-L2-NS and TK6, of varying p53 status were exposed to a single 10 Gy dose of gamma radiation and their responses assessed over an extended time course. DNA flow cytometry and mitotic counts were used to assess the kinetics and extent of polyploidisation and mitotic progression. Expression of meiotic genes was analysed using RT-PCR and western blotting. In addition, localisation of the meiotic cohesin REC8 and its relation to centromeres was analysed by immunofluorescence. RESULTS: The principal meiotic regulator MOS was found to be significantly post-transcriptionally up-regulated after irradiation in p53 mutated but not p53 wild-type lymphoma cells. The maximum expression of MOS coincided with the maximal fraction of metaphase arrested cells and was directly proportional to both the extent of the arrest and the number of endopolyploid cells that subsequently emerged. The meiotic cohesin REC8 was also found to be up-regulated after irradiation, linking sister chromatid centromeres in the metaphase-arrested and subsequent giant cells. Finally, RT-PCR revealed expression of the meiosis-prophase genes, DMC1, STAG3, SYCP3 and SYCP1. CONCLUSION: We conclude that multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation. Furthermore, we suggest that the coordinated expression of MOS and REC8 regulate the extent of arrested mitoses and polyploidy.

Dataset Information

Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells.

Publications

Targeted depletion of Polo-like kinase (Plk) 1 through lentiviral shRNA or a small-molecule inhibitor causes mitotic catastrophe and induction of apoptosis in human melanoma cells.

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