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Targeted Knockdown of the Kinetochore Protein D40/Knl-1 Inhibits Human Cancer in a p53 Status-Independent Manner.

Urata YN, Takeshita F, Tanaka H, Ochiya T, Takimoto M - Sci Rep (2015)

Bottom Line: The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis.These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status.Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Gene Regulation, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53- cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

No MeSH data available.


Related in: MedlinePlus

D40 siRNA induces Growth Inhibition and Apoptotic Cell death in the p53- cancer cell line.Confirmation of the p53- state of the PC-3M-luc cell line and depletion of the D40 protein by D40 siRNA. Left: The human p53- cancer cell line PC-3M-luc and human wild-type p53 cancer cell line MCF-7 were treated with adriamycin at a final concentration of 1.0 μg/ml for 17 hrs. Cell lysate preparation and WB analyses were performed as described above. The gels for p53 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-p53 antibody, and the other was probed with the anti-β-actin antibody. p53 protein was absent in PC-3M-luc cells, but p53 protein expression was highly induced in MCF-7 cells. Right: PC-3M-luc cells were transfected with D40 or control siRNA and harvested 60 hrs after transfection for WB analyses. The gels for D40 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-D40 antibody, and the other was probed with the anti-β-actin antibody. (b) Growth Inhibition of the PC-3M-luc cell line by D40 siRNA. Left: Microphotographs of the PC-3M-luc cell line transfected with control RNA (upper) or D40 siRNA (lower) 60 hrs after transfection. Right: Growth curves of the transfected PC-3M-luc cell lines. Cell counting kit-8 was used to determine viable cell numbers. The number of viable cells 96 hrs and 144 hrs after transfection was significantly lower in the D40 siRNA-transfected PC-3M-luc cell line than in the respective control cell lines. (c) D40 siRNA induced the activation of caspase 3/7 and increased cytoplasmic cytochrome c levels in the PC-3M-luc cell line. The PC-3M-luc cell line was transfected with siRNAs as described. Left: Caspase 3/7 activities in the transfected PC-3M-luc cells were determined 96 hrs after transfection. Right: The amount of cytoplasmic cytochrome c was quantitatively determined 70 hrs after transfection.
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f3: D40 siRNA induces Growth Inhibition and Apoptotic Cell death in the p53- cancer cell line.Confirmation of the p53- state of the PC-3M-luc cell line and depletion of the D40 protein by D40 siRNA. Left: The human p53- cancer cell line PC-3M-luc and human wild-type p53 cancer cell line MCF-7 were treated with adriamycin at a final concentration of 1.0 μg/ml for 17 hrs. Cell lysate preparation and WB analyses were performed as described above. The gels for p53 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-p53 antibody, and the other was probed with the anti-β-actin antibody. p53 protein was absent in PC-3M-luc cells, but p53 protein expression was highly induced in MCF-7 cells. Right: PC-3M-luc cells were transfected with D40 or control siRNA and harvested 60 hrs after transfection for WB analyses. The gels for D40 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-D40 antibody, and the other was probed with the anti-β-actin antibody. (b) Growth Inhibition of the PC-3M-luc cell line by D40 siRNA. Left: Microphotographs of the PC-3M-luc cell line transfected with control RNA (upper) or D40 siRNA (lower) 60 hrs after transfection. Right: Growth curves of the transfected PC-3M-luc cell lines. Cell counting kit-8 was used to determine viable cell numbers. The number of viable cells 96 hrs and 144 hrs after transfection was significantly lower in the D40 siRNA-transfected PC-3M-luc cell line than in the respective control cell lines. (c) D40 siRNA induced the activation of caspase 3/7 and increased cytoplasmic cytochrome c levels in the PC-3M-luc cell line. The PC-3M-luc cell line was transfected with siRNAs as described. Left: Caspase 3/7 activities in the transfected PC-3M-luc cells were determined 96 hrs after transfection. Right: The amount of cytoplasmic cytochrome c was quantitatively determined 70 hrs after transfection.

Mentions: We first assessed the growth inhibitory effects of D40 siRNA on the p53- cell line, PC-3M-luc, which represents a loss-of-function (LOF) p53 mutants cell line24. To confirm that this cell line did not express the p53 protein, Western blot analysis was performed on the lysates of cells that were treated with adriamycin, a DNA-damaging reagent. While the p53 protein was highly induced the adriamycin treatment in the wild-type p53 cell line MCF-7, it was undetectable in the PC-3M-luc cell line, even under the same treatment conditions (Fig. 3a Left). We subsequently referred to the PC-3M-luc cell line used in this study as p53-. We next determined whether D40 siRNA could reduce D40 protein expression in this cell line. The results demonstrated that D40 protein expression in the PC-3M-luc cell line was sensitive to D40 siRNA (Fig. 3a Right). Therefore, the effects of D40 siRNA on the growth of this cell line were examined. The results showed that growth was significantly inhibited in the cells transfected with D40 siRNA, as shown in the microphotographs (Fig. 3b Left) and growth curves (Fig. 3b Right).


Targeted Knockdown of the Kinetochore Protein D40/Knl-1 Inhibits Human Cancer in a p53 Status-Independent Manner.

Urata YN, Takeshita F, Tanaka H, Ochiya T, Takimoto M - Sci Rep (2015)

D40 siRNA induces Growth Inhibition and Apoptotic Cell death in the p53- cancer cell line.Confirmation of the p53- state of the PC-3M-luc cell line and depletion of the D40 protein by D40 siRNA. Left: The human p53- cancer cell line PC-3M-luc and human wild-type p53 cancer cell line MCF-7 were treated with adriamycin at a final concentration of 1.0 μg/ml for 17 hrs. Cell lysate preparation and WB analyses were performed as described above. The gels for p53 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-p53 antibody, and the other was probed with the anti-β-actin antibody. p53 protein was absent in PC-3M-luc cells, but p53 protein expression was highly induced in MCF-7 cells. Right: PC-3M-luc cells were transfected with D40 or control siRNA and harvested 60 hrs after transfection for WB analyses. The gels for D40 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-D40 antibody, and the other was probed with the anti-β-actin antibody. (b) Growth Inhibition of the PC-3M-luc cell line by D40 siRNA. Left: Microphotographs of the PC-3M-luc cell line transfected with control RNA (upper) or D40 siRNA (lower) 60 hrs after transfection. Right: Growth curves of the transfected PC-3M-luc cell lines. Cell counting kit-8 was used to determine viable cell numbers. The number of viable cells 96 hrs and 144 hrs after transfection was significantly lower in the D40 siRNA-transfected PC-3M-luc cell line than in the respective control cell lines. (c) D40 siRNA induced the activation of caspase 3/7 and increased cytoplasmic cytochrome c levels in the PC-3M-luc cell line. The PC-3M-luc cell line was transfected with siRNAs as described. Left: Caspase 3/7 activities in the transfected PC-3M-luc cells were determined 96 hrs after transfection. Right: The amount of cytoplasmic cytochrome c was quantitatively determined 70 hrs after transfection.
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f3: D40 siRNA induces Growth Inhibition and Apoptotic Cell death in the p53- cancer cell line.Confirmation of the p53- state of the PC-3M-luc cell line and depletion of the D40 protein by D40 siRNA. Left: The human p53- cancer cell line PC-3M-luc and human wild-type p53 cancer cell line MCF-7 were treated with adriamycin at a final concentration of 1.0 μg/ml for 17 hrs. Cell lysate preparation and WB analyses were performed as described above. The gels for p53 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-p53 antibody, and the other was probed with the anti-β-actin antibody. p53 protein was absent in PC-3M-luc cells, but p53 protein expression was highly induced in MCF-7 cells. Right: PC-3M-luc cells were transfected with D40 or control siRNA and harvested 60 hrs after transfection for WB analyses. The gels for D40 and β-actin were run under the same experimental conditions, and then the proteins were transferred to a filter membrane. The membrane was cut and divided into two parts. One was probed with the anti-D40 antibody, and the other was probed with the anti-β-actin antibody. (b) Growth Inhibition of the PC-3M-luc cell line by D40 siRNA. Left: Microphotographs of the PC-3M-luc cell line transfected with control RNA (upper) or D40 siRNA (lower) 60 hrs after transfection. Right: Growth curves of the transfected PC-3M-luc cell lines. Cell counting kit-8 was used to determine viable cell numbers. The number of viable cells 96 hrs and 144 hrs after transfection was significantly lower in the D40 siRNA-transfected PC-3M-luc cell line than in the respective control cell lines. (c) D40 siRNA induced the activation of caspase 3/7 and increased cytoplasmic cytochrome c levels in the PC-3M-luc cell line. The PC-3M-luc cell line was transfected with siRNAs as described. Left: Caspase 3/7 activities in the transfected PC-3M-luc cells were determined 96 hrs after transfection. Right: The amount of cytoplasmic cytochrome c was quantitatively determined 70 hrs after transfection.
Mentions: We first assessed the growth inhibitory effects of D40 siRNA on the p53- cell line, PC-3M-luc, which represents a loss-of-function (LOF) p53 mutants cell line24. To confirm that this cell line did not express the p53 protein, Western blot analysis was performed on the lysates of cells that were treated with adriamycin, a DNA-damaging reagent. While the p53 protein was highly induced the adriamycin treatment in the wild-type p53 cell line MCF-7, it was undetectable in the PC-3M-luc cell line, even under the same treatment conditions (Fig. 3a Left). We subsequently referred to the PC-3M-luc cell line used in this study as p53-. We next determined whether D40 siRNA could reduce D40 protein expression in this cell line. The results demonstrated that D40 protein expression in the PC-3M-luc cell line was sensitive to D40 siRNA (Fig. 3a Right). Therefore, the effects of D40 siRNA on the growth of this cell line were examined. The results showed that growth was significantly inhibited in the cells transfected with D40 siRNA, as shown in the microphotographs (Fig. 3b Left) and growth curves (Fig. 3b Right).

Bottom Line: The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis.These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status.Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

View Article: PubMed Central - PubMed

Affiliation: Division of Cancer Gene Regulation, Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

ABSTRACT
The D40 gene encodes a kinetochore protein that plays an essential role in kinetochore formation during mitosis. Short inhibitory RNA against D40, D40 siRNA, has been shown to deplete the D40 protein in the human cancer cell line HeLa, which harbors wild-type p53, and this activity was followed by the significant inhibition of cell growth and induction of apoptotic cell death. The p53- cancer cell line, PC-3M-luc, is also sensitive to the significant growth inhibition and cell death induced by D40 siRNA. The growth of PC-3M-luc tumors transplanted into nude mice was inhibited by the systemic administration of D40 siRNA and the atelocollagen complex. Furthermore, D40 siRNA significantly inhibited growth and induced apoptotic cell death in a cell line with a gain-of-function (GOF) mutation in p53, MDA-MB231-luc, and also inhibited the growth of tumors transplanted into mice when administered as a D40 siRNA/atelocollagen complex. These results indicated that D40 siRNA induced apoptotic cell death in human cancer cell lines, and inhibited their growth in vitro and in vivo regardless of p53 status. Therefore, D40 siRNA is a potential candidate anti-cancer reagent.

No MeSH data available.


Related in: MedlinePlus