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Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe.

Mu R, Wang YB, Wu M, Yang Y, Song W, Li T, Zhang WN, Tan B, Li AL, Wang N, Xia Q, Gong WL, Wang CG, Zhou T, Guo N, Sang ZH, Li HY - Cell Death Dis (2014)

Bottom Line: Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage.We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down.Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.

ABSTRACT
Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy.

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Related in: MedlinePlus

Cdc5L deficiency causes mitotic arrest and chromosome misalignment. (a and b) HeLa cells were transfected with control siRNA or two independent siRNAs against Cdc5L. (a) FACS analysis of cell cycle distribution by PI staining at indicated times post transfection. (b)The proportion of mitotic cells in total cells were analyzed by FACS using phosphorylated histone H3 (Ser-10) (p-H3)-PI staining at indicated times post transfection. (c) Selected frames from time-lapse movies of representative HeLa/GFP-H2B cells transfected with control or Cdc5L siRNA. The time on the images is in minutes. A, anaphase; M, metaphase; NEB, nuclear envelope breakdown. Scale bar, 10 μm. (d) The percentage of mitotic incompetent cells (mitotic duration >90 min) in control and Cdc5L-knockdown cells entering mitosis. Data are representative of three independent experiments, error bars indicate S.D. (e) The percentage of mitotic cells with misaligned chromosome in control and Cdc5L-knockdown cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D. (f) Complementation of red fluorescent protein (RFP)-Cdc5L in knockdown HeLa/GFP-H2B cells rescues chromosome misalignment. The Cdc5L-knockdown cells were transfected with Cdc5L siRNA-resistant expression construct or control vector. The data show the percentage of mitotic cells with misaligned chromosome in RFP-positive cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D.
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fig1: Cdc5L deficiency causes mitotic arrest and chromosome misalignment. (a and b) HeLa cells were transfected with control siRNA or two independent siRNAs against Cdc5L. (a) FACS analysis of cell cycle distribution by PI staining at indicated times post transfection. (b)The proportion of mitotic cells in total cells were analyzed by FACS using phosphorylated histone H3 (Ser-10) (p-H3)-PI staining at indicated times post transfection. (c) Selected frames from time-lapse movies of representative HeLa/GFP-H2B cells transfected with control or Cdc5L siRNA. The time on the images is in minutes. A, anaphase; M, metaphase; NEB, nuclear envelope breakdown. Scale bar, 10 μm. (d) The percentage of mitotic incompetent cells (mitotic duration >90 min) in control and Cdc5L-knockdown cells entering mitosis. Data are representative of three independent experiments, error bars indicate S.D. (e) The percentage of mitotic cells with misaligned chromosome in control and Cdc5L-knockdown cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D. (f) Complementation of red fluorescent protein (RFP)-Cdc5L in knockdown HeLa/GFP-H2B cells rescues chromosome misalignment. The Cdc5L-knockdown cells were transfected with Cdc5L siRNA-resistant expression construct or control vector. The data show the percentage of mitotic cells with misaligned chromosome in RFP-positive cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D.

Mentions: Dysregulation of mitosis results in mitotic cell death or the generation of tumorigenic aneuploidy daughter cells, which lead to cancer.25, 26 In a separate mitosis screening study, we found that Cdc5L knockdown markedly impaired the ability of cells to resume mitotic progression after release from treatment with the mitotic inhibitor nocodazole (Supplementary Figures 1a and b). To confirm if Cdc5L was involved in mitotic progression, we transfected asynchronized HeLa cells with two individual siRNAs against Cdc5L. Cell cycle distribution and the proportion of mitotic cells (mitotic index) were analyzed. We found that the percentage of cells in G2/M phase increased from 14.16% in control siRNA-transfected cells to 24.63% and 21.71% in Cdc5L siRNA no.1- and no.2-transfected cells, respectively (Figure 1a). The mitotic index of Cdc5L siRNA-transfected cells was much higher than that of control siRNA-transfected cells (Figure 1b). Consistently, the expression of the mitotic marker Ser 10-phosphorylated histone H3 was dramatically increased in Cdc5L-knockdown cells (Supplementary Figure 1c). To further investigate the function of Cdc5L in mitosis, we monitored mitotic progression by time-lapse imaging of HeLa cells stably expressing green fluorescent protein (GFP)-tagged histone H2B, which revealed that Cdc5L knockdown caused prolonged mitotic arrest compared with control siRNA (Figure 1c). The percentage of mitosis-incompetent cells that were still in M phase 90 min after nuclear envelope breakdown (NEB) increased from 7.60% in control siRNA-transfected cells to 90.79% and 94.76% in Cdc5L siRNA no.1- and Cdc5L siRNA no. 2-transfected cells, respectively (Figure 1d). Interestingly, we observed that Cdc5L knockdown induced severe chromosome misalignment compared with control siRNA (Figure 1c). More than 70% of Cdc5L-knockdown cells entering mitosis were unable to properly align their chromosomes at the spindle equator to form a stable metaphase plate (Figure 1e). To rule out off-target effects of Cdc5L siRNA, HeLa/GFP-H2B cells were co-transfected with red fluorescent protein-tagged siRNA-resistant wild-type Cdc5L or control vector together with Cdc5L siRNA. The misalignment of chromosomes during prometaphase induced by Cdc5L knockdown was reversed by the expression of siRNA-resistant wild-type Cdc5L (Figure 1f and Supplementary Figure 2a). The levels of ectopically expressed Cdc5L protein were comparable to those of endogenous Cdc5L (Supplementary Figure 2b). The no.1 siRNA was used in the Cdc5L RNAi experiments in this study unless otherwise indicated. Taken together, these data suggest that Cdc5L knockdown causes severe chromosome alignment defects and mitotic arrest.


Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic progression and triggers mitotic catastrophe.

Mu R, Wang YB, Wu M, Yang Y, Song W, Li T, Zhang WN, Tan B, Li AL, Wang N, Xia Q, Gong WL, Wang CG, Zhou T, Guo N, Sang ZH, Li HY - Cell Death Dis (2014)

Cdc5L deficiency causes mitotic arrest and chromosome misalignment. (a and b) HeLa cells were transfected with control siRNA or two independent siRNAs against Cdc5L. (a) FACS analysis of cell cycle distribution by PI staining at indicated times post transfection. (b)The proportion of mitotic cells in total cells were analyzed by FACS using phosphorylated histone H3 (Ser-10) (p-H3)-PI staining at indicated times post transfection. (c) Selected frames from time-lapse movies of representative HeLa/GFP-H2B cells transfected with control or Cdc5L siRNA. The time on the images is in minutes. A, anaphase; M, metaphase; NEB, nuclear envelope breakdown. Scale bar, 10 μm. (d) The percentage of mitotic incompetent cells (mitotic duration >90 min) in control and Cdc5L-knockdown cells entering mitosis. Data are representative of three independent experiments, error bars indicate S.D. (e) The percentage of mitotic cells with misaligned chromosome in control and Cdc5L-knockdown cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D. (f) Complementation of red fluorescent protein (RFP)-Cdc5L in knockdown HeLa/GFP-H2B cells rescues chromosome misalignment. The Cdc5L-knockdown cells were transfected with Cdc5L siRNA-resistant expression construct or control vector. The data show the percentage of mitotic cells with misaligned chromosome in RFP-positive cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D.
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Related In: Results  -  Collection

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fig1: Cdc5L deficiency causes mitotic arrest and chromosome misalignment. (a and b) HeLa cells were transfected with control siRNA or two independent siRNAs against Cdc5L. (a) FACS analysis of cell cycle distribution by PI staining at indicated times post transfection. (b)The proportion of mitotic cells in total cells were analyzed by FACS using phosphorylated histone H3 (Ser-10) (p-H3)-PI staining at indicated times post transfection. (c) Selected frames from time-lapse movies of representative HeLa/GFP-H2B cells transfected with control or Cdc5L siRNA. The time on the images is in minutes. A, anaphase; M, metaphase; NEB, nuclear envelope breakdown. Scale bar, 10 μm. (d) The percentage of mitotic incompetent cells (mitotic duration >90 min) in control and Cdc5L-knockdown cells entering mitosis. Data are representative of three independent experiments, error bars indicate S.D. (e) The percentage of mitotic cells with misaligned chromosome in control and Cdc5L-knockdown cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D. (f) Complementation of red fluorescent protein (RFP)-Cdc5L in knockdown HeLa/GFP-H2B cells rescues chromosome misalignment. The Cdc5L-knockdown cells were transfected with Cdc5L siRNA-resistant expression construct or control vector. The data show the percentage of mitotic cells with misaligned chromosome in RFP-positive cells entering mitosis. Data are representative of four independent experiments, error bars indicate S.D.
Mentions: Dysregulation of mitosis results in mitotic cell death or the generation of tumorigenic aneuploidy daughter cells, which lead to cancer.25, 26 In a separate mitosis screening study, we found that Cdc5L knockdown markedly impaired the ability of cells to resume mitotic progression after release from treatment with the mitotic inhibitor nocodazole (Supplementary Figures 1a and b). To confirm if Cdc5L was involved in mitotic progression, we transfected asynchronized HeLa cells with two individual siRNAs against Cdc5L. Cell cycle distribution and the proportion of mitotic cells (mitotic index) were analyzed. We found that the percentage of cells in G2/M phase increased from 14.16% in control siRNA-transfected cells to 24.63% and 21.71% in Cdc5L siRNA no.1- and no.2-transfected cells, respectively (Figure 1a). The mitotic index of Cdc5L siRNA-transfected cells was much higher than that of control siRNA-transfected cells (Figure 1b). Consistently, the expression of the mitotic marker Ser 10-phosphorylated histone H3 was dramatically increased in Cdc5L-knockdown cells (Supplementary Figure 1c). To further investigate the function of Cdc5L in mitosis, we monitored mitotic progression by time-lapse imaging of HeLa cells stably expressing green fluorescent protein (GFP)-tagged histone H2B, which revealed that Cdc5L knockdown caused prolonged mitotic arrest compared with control siRNA (Figure 1c). The percentage of mitosis-incompetent cells that were still in M phase 90 min after nuclear envelope breakdown (NEB) increased from 7.60% in control siRNA-transfected cells to 90.79% and 94.76% in Cdc5L siRNA no.1- and Cdc5L siRNA no. 2-transfected cells, respectively (Figure 1d). Interestingly, we observed that Cdc5L knockdown induced severe chromosome misalignment compared with control siRNA (Figure 1c). More than 70% of Cdc5L-knockdown cells entering mitosis were unable to properly align their chromosomes at the spindle equator to form a stable metaphase plate (Figure 1e). To rule out off-target effects of Cdc5L siRNA, HeLa/GFP-H2B cells were co-transfected with red fluorescent protein-tagged siRNA-resistant wild-type Cdc5L or control vector together with Cdc5L siRNA. The misalignment of chromosomes during prometaphase induced by Cdc5L knockdown was reversed by the expression of siRNA-resistant wild-type Cdc5L (Figure 1f and Supplementary Figure 2a). The levels of ectopically expressed Cdc5L protein were comparable to those of endogenous Cdc5L (Supplementary Figure 2b). The no.1 siRNA was used in the Cdc5L RNAi experiments in this study unless otherwise indicated. Taken together, these data suggest that Cdc5L knockdown causes severe chromosome alignment defects and mitotic arrest.

Bottom Line: Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage.We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down.Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, Beijing, China.

ABSTRACT
Disturbing mitotic progression via targeted anti-mitotic therapy is an attractive strategy for cancer treatment. Therefore, the exploration and elucidation of molecular targets and pathways in mitosis are critical for the development of anti-mitotic drugs. Here, we show that cell division cycle 5-like (Cdc5L), a pre-mRNA splicing factor, is a regulator of mitotic progression. Depletion of Cdc5L causes dramatic mitotic arrest, chromosome misalignments and sustained activation of spindle assembly checkpoint, eventually leading to mitotic catastrophe. Moreover, these defects result from severe impairment of kinetochore-microtubule attachment and serious DNA damage. Genome-wide gene expression analysis reveals that Cdc5L modulates the expression of a set of genes involved in the mitosis and the DNA damage response. We further found that the pre-mRNA splicing efficiency of these genes were impaired when Cdc5L was knocked down. Interestingly, Cdc5L is highly expressed in cervical tumors and osteosarcoma. Finally, we demonstrate that downregulation of Cdc5L decreases the cell viability of related tumor cells. These results suggest that Cdc5L is a key regulator of mitotic progression and highlight the potential of Cdc5L as a target for cancer therapy.

Show MeSH
Related in: MedlinePlus