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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

Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Nuclear chromatin condensation and fragmentation induced by D40 siRNA in HeLa cells. HeLa cells were transfected with D40 siRNA (left) or control siRNA (right), and were stained with Hoechst 33342 48 hrs after transfection. Cells with fragmented chromatin were regarded as apoptotic. Arrows in the left figure indicate the apoptotic D40 siRNA-transfected cells. (b) Increase in the sub-G1 fraction of HeLa cells transfected with D40 siRNA. DNA content analyses were performed as described in the Materials and Methods section. The upper figures show histograms of the DNA contents of cells 48 hrs after transfection, while the lower table shows the percentage of the cell population in each fraction. A significant increase in the sub-G1 fraction was detected in the D40 siRNA-transfected HeLa cells but not in control siRNA-transfected cells.
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f2: Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Nuclear chromatin condensation and fragmentation induced by D40 siRNA in HeLa cells. HeLa cells were transfected with D40 siRNA (left) or control siRNA (right), and were stained with Hoechst 33342 48 hrs after transfection. Cells with fragmented chromatin were regarded as apoptotic. Arrows in the left figure indicate the apoptotic D40 siRNA-transfected cells. (b) Increase in the sub-G1 fraction of HeLa cells transfected with D40 siRNA. DNA content analyses were performed as described in the Materials and Methods section. The upper figures show histograms of the DNA contents of cells 48 hrs after transfection, while the lower table shows the percentage of the cell population in each fraction. A significant increase in the sub-G1 fraction was detected in the D40 siRNA-transfected HeLa cells but not in control siRNA-transfected cells.

Mentions: The nuclear staining of siRNA-transfected HeLa cells was performed using Hoechst 33342, and the results obtained revealed that a greater population of the cells with chromatin condensation and fragmentation, which are features of apoptotic cells, was observed in the D40 siRNA-transfected cells compared with control cells (Fig. 2a). To confirm cell death in D40 siRNA-transfected cells, DNA content analyses of the transfected cells were performed using a flow cytometer. The population of cells with a DNA content less than 2n, the sub-G1 population in the M1 fraction, was significantly higher in D40 siRNA-transfected cells than in cells transfected with control siRNA, as shown in Fig. 2b. As the sub-G1 population is characteristic of apoptosis, we concluded that D40 siRNA induced apoptotic cell death in HeLa cells by apoptosis.


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)

Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Nuclear chromatin condensation and fragmentation induced by D40 siRNA in HeLa cells. HeLa cells were transfected with D40 siRNA (left) or control siRNA (right), and were stained with Hoechst 33342 48 hrs after transfection. Cells with fragmented chromatin were regarded as apoptotic. Arrows in the left figure indicate the apoptotic D40 siRNA-transfected cells. (b) Increase in the sub-G1 fraction of HeLa cells transfected with D40 siRNA. DNA content analyses were performed as described in the Materials and Methods section. The upper figures show histograms of the DNA contents of cells 48 hrs after transfection, while the lower table shows the percentage of the cell population in each fraction. A significant increase in the sub-G1 fraction was detected in the D40 siRNA-transfected HeLa cells but not in control siRNA-transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562263&req=5

f2: Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Nuclear chromatin condensation and fragmentation induced by D40 siRNA in HeLa cells. HeLa cells were transfected with D40 siRNA (left) or control siRNA (right), and were stained with Hoechst 33342 48 hrs after transfection. Cells with fragmented chromatin were regarded as apoptotic. Arrows in the left figure indicate the apoptotic D40 siRNA-transfected cells. (b) Increase in the sub-G1 fraction of HeLa cells transfected with D40 siRNA. DNA content analyses were performed as described in the Materials and Methods section. The upper figures show histograms of the DNA contents of cells 48 hrs after transfection, while the lower table shows the percentage of the cell population in each fraction. A significant increase in the sub-G1 fraction was detected in the D40 siRNA-transfected HeLa cells but not in control siRNA-transfected cells.
Mentions: The nuclear staining of siRNA-transfected HeLa cells was performed using Hoechst 33342, and the results obtained revealed that a greater population of the cells with chromatin condensation and fragmentation, which are features of apoptotic cells, was observed in the D40 siRNA-transfected cells compared with control cells (Fig. 2a). To confirm cell death in D40 siRNA-transfected cells, DNA content analyses of the transfected cells were performed using a flow cytometer. The population of cells with a DNA content less than 2n, the sub-G1 population in the M1 fraction, was significantly higher in D40 siRNA-transfected cells than in cells transfected with control siRNA, as shown in Fig. 2b. As the sub-G1 population is characteristic of apoptosis, we concluded that D40 siRNA induced apoptotic cell death in HeLa cells by apoptosis.

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