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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

Growth Inhibition and Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Depletion of D40 protein by D40 siRNA. The human cancer cell line with wild-type p53, HeLa, was transfected with D40 or control siRNA. The transfected HeLa cells were harvested at the indicated time points after transfection, and cell lysates were prepared with TNE buffer. The gels for D40 and β-actin were run under the same experimental conditions, and 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. Significant depletion of D40 protein was observed in D40 siRNA-transfected HeLa cells. (b) Growth and Death Rate Curves of HeLa cells transfected with D40 siRNA. HeLa cells were transfected with D40 or control siRNA and harvested at the indicated times after transfection. The number of viable cells and dead cells was significantly lower and higher, respectively, in D40 siRNA-transfected HeLa cells than in control siRNA-transfected cells 72 hrs after transfection.
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f1: Growth Inhibition and Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Depletion of D40 protein by D40 siRNA. The human cancer cell line with wild-type p53, HeLa, was transfected with D40 or control siRNA. The transfected HeLa cells were harvested at the indicated time points after transfection, and cell lysates were prepared with TNE buffer. The gels for D40 and β-actin were run under the same experimental conditions, and 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. Significant depletion of D40 protein was observed in D40 siRNA-transfected HeLa cells. (b) Growth and Death Rate Curves of HeLa cells transfected with D40 siRNA. HeLa cells were transfected with D40 or control siRNA and harvested at the indicated times after transfection. The number of viable cells and dead cells was significantly lower and higher, respectively, in D40 siRNA-transfected HeLa cells than in control siRNA-transfected cells 72 hrs after transfection.

Mentions: RNA interference targeting D40 was used to determine whether inhibiting D40 protein expression affected the growth of human cancer cell lines. Three different D40 mRNA sequences that were 21 bases in length were selected to synthesize short inhibitory RNA (siRNA). A mixture of the siRNAs (D40 siRNA) was transfected into the wild-type p53 cell line HeLa. D40 siRNA, but not control siRNA, significantly reduced D40 protein expression at the indicated hours after transfection (Fig. 1a). As the specific depletion of the D40 protein was induced by D40 siRNA, the effects of D40 siRNA on the growth of the HeLa cell line were assessed. The growth of HeLa cells was significantly inhibited by D40 siRNA, but not by control siRNA (Fig. 1b Left). The proportion of dead cells was higher in D40 siRNA-transfected HeLa cells than in control HeLa cells (Fig. 1b Right). Each of the three D40 siRNAs also significantly depleted D40 protein expression and inhibited the growth of HeLa cells (Supplementary Figures 1 and 2). The mixture of three different D40 siRNAs was labelled as D40 siRNA Trio in Supplementary Figures 1 and 2. However, this trio was described as D40 siRNA in all of the other parts of the manuscript and figures.


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)

Growth Inhibition and Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Depletion of D40 protein by D40 siRNA. The human cancer cell line with wild-type p53, HeLa, was transfected with D40 or control siRNA. The transfected HeLa cells were harvested at the indicated time points after transfection, and cell lysates were prepared with TNE buffer. The gels for D40 and β-actin were run under the same experimental conditions, and 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. Significant depletion of D40 protein was observed in D40 siRNA-transfected HeLa cells. (b) Growth and Death Rate Curves of HeLa cells transfected with D40 siRNA. HeLa cells were transfected with D40 or control siRNA and harvested at the indicated times after transfection. The number of viable cells and dead cells was significantly lower and higher, respectively, in D40 siRNA-transfected HeLa cells than in control siRNA-transfected cells 72 hrs after transfection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Growth Inhibition and Induction of Apoptotic Cell death by D40 siRNA in the wild-type p53 cancer cell line.(a) Depletion of D40 protein by D40 siRNA. The human cancer cell line with wild-type p53, HeLa, was transfected with D40 or control siRNA. The transfected HeLa cells were harvested at the indicated time points after transfection, and cell lysates were prepared with TNE buffer. The gels for D40 and β-actin were run under the same experimental conditions, and 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. Significant depletion of D40 protein was observed in D40 siRNA-transfected HeLa cells. (b) Growth and Death Rate Curves of HeLa cells transfected with D40 siRNA. HeLa cells were transfected with D40 or control siRNA and harvested at the indicated times after transfection. The number of viable cells and dead cells was significantly lower and higher, respectively, in D40 siRNA-transfected HeLa cells than in control siRNA-transfected cells 72 hrs after transfection.
Mentions: RNA interference targeting D40 was used to determine whether inhibiting D40 protein expression affected the growth of human cancer cell lines. Three different D40 mRNA sequences that were 21 bases in length were selected to synthesize short inhibitory RNA (siRNA). A mixture of the siRNAs (D40 siRNA) was transfected into the wild-type p53 cell line HeLa. D40 siRNA, but not control siRNA, significantly reduced D40 protein expression at the indicated hours after transfection (Fig. 1a). As the specific depletion of the D40 protein was induced by D40 siRNA, the effects of D40 siRNA on the growth of the HeLa cell line were assessed. The growth of HeLa cells was significantly inhibited by D40 siRNA, but not by control siRNA (Fig. 1b Left). The proportion of dead cells was higher in D40 siRNA-transfected HeLa cells than in control HeLa cells (Fig. 1b Right). Each of the three D40 siRNAs also significantly depleted D40 protein expression and inhibited the growth of HeLa cells (Supplementary Figures 1 and 2). The mixture of three different D40 siRNAs was labelled as D40 siRNA Trio in Supplementary Figures 1 and 2. However, this trio was described as D40 siRNA in all of the other parts of the manuscript and figures.

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