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Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

Yuan P, Wang Z, Lv W, Pan H, Yang Y, Yuan X, Hu J - Oncol Lett (2014)

Bottom Line: This recruitment is dependent on TCAB1 binding to a telomerase RNA component.In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes.In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.

ABSTRACT
Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

No MeSH data available.


Related in: MedlinePlus

Effect of TCAB1 siRNA on cell cycle and apoptosis regulation. (A) Each bar of the bar graph represents the mean percentage of cells in the G1 and S phases. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (B) The cell cycle distribution was determined by the detection of DNA degradation. A549 cells were treated with TCAB1 siRNA and stained with PI and analyzed by flow cytometry. The cell count versus PI staining is shown (n=10,000 per treatment). (C) Expression of CDK6 was determined by RT-PCR analysis of the total isolated RNA. Actin served as a gel-loading control. The densitometric quantifications of the CDK6 levels were normalized against the actin levels. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (D) Annexin V staining of TCAB1 siRNA-treated cells. Dot-plot data of FITC and PI staining measured by fluorescence-activated cell sorting analysis and graphical representation of apoptosis in cells treated with TCAB1 siRNA. *P<0.05 vs. control siRNA and WT (n=3). WT, wild-type; con siRNA, control small interfering RNA; TCAB1, telomerase Cajal body protein 1; RT-PCR, reverse transcription-polymerase chain reaction; CDK6, cyclin-dependent kinase 6; FITC, fluorescein isothiocyanate; PI, propidium iodide; SD, standard deviation.
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f4-ol-08-03-1009: Effect of TCAB1 siRNA on cell cycle and apoptosis regulation. (A) Each bar of the bar graph represents the mean percentage of cells in the G1 and S phases. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (B) The cell cycle distribution was determined by the detection of DNA degradation. A549 cells were treated with TCAB1 siRNA and stained with PI and analyzed by flow cytometry. The cell count versus PI staining is shown (n=10,000 per treatment). (C) Expression of CDK6 was determined by RT-PCR analysis of the total isolated RNA. Actin served as a gel-loading control. The densitometric quantifications of the CDK6 levels were normalized against the actin levels. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (D) Annexin V staining of TCAB1 siRNA-treated cells. Dot-plot data of FITC and PI staining measured by fluorescence-activated cell sorting analysis and graphical representation of apoptosis in cells treated with TCAB1 siRNA. *P<0.05 vs. control siRNA and WT (n=3). WT, wild-type; con siRNA, control small interfering RNA; TCAB1, telomerase Cajal body protein 1; RT-PCR, reverse transcription-polymerase chain reaction; CDK6, cyclin-dependent kinase 6; FITC, fluorescein isothiocyanate; PI, propidium iodide; SD, standard deviation.

Mentions: Further study was conducted to elucidate the mechanism of action for the antiproliferative activity of TCAB1 depletion on A549 cells. Flow cytometry was used to analyze the A549 cell cycle distribution and apoptosis. Cancer cells are generally immortal and divide uncontrollably, and the replication of telomeres occurs in the S phase (24). Thus, it was predicted that the number of A549 cells would increase in the G1 phase and decrease in the S phase as a result of TCAB1 depletion. Consistent with this hypothesis, TCAB1 siRNA was found to induce G1 cell cycle arrest, and the percentage of cells with G1 DNA content increased from 70 to 88% and the percentage of cells in the S phase decreased from 25 to 9%, when compared with that of the control groups (Fig. 4A). However, cells were not found to accumulate in the sub G1 peak (Fig. 4B). In addition, the G1 cell cycle checkpoint protein, cyclin-dependent kinase 6 (CDK6), was suppressed (Fig. 4C). Accordingly, the Annexin V staining apoptosis assay of A549 cells treated with TCAB1 siRNA demonstrated no evidence of apoptotic cell death (Fig. 4D). These observations indicated that the antiproliferative effects of TCAB1 siRNA in A549 cells is caused by cell cycle arrest without the induction of apoptosis.


Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

Yuan P, Wang Z, Lv W, Pan H, Yang Y, Yuan X, Hu J - Oncol Lett (2014)

Effect of TCAB1 siRNA on cell cycle and apoptosis regulation. (A) Each bar of the bar graph represents the mean percentage of cells in the G1 and S phases. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (B) The cell cycle distribution was determined by the detection of DNA degradation. A549 cells were treated with TCAB1 siRNA and stained with PI and analyzed by flow cytometry. The cell count versus PI staining is shown (n=10,000 per treatment). (C) Expression of CDK6 was determined by RT-PCR analysis of the total isolated RNA. Actin served as a gel-loading control. The densitometric quantifications of the CDK6 levels were normalized against the actin levels. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (D) Annexin V staining of TCAB1 siRNA-treated cells. Dot-plot data of FITC and PI staining measured by fluorescence-activated cell sorting analysis and graphical representation of apoptosis in cells treated with TCAB1 siRNA. *P<0.05 vs. control siRNA and WT (n=3). WT, wild-type; con siRNA, control small interfering RNA; TCAB1, telomerase Cajal body protein 1; RT-PCR, reverse transcription-polymerase chain reaction; CDK6, cyclin-dependent kinase 6; FITC, fluorescein isothiocyanate; PI, propidium iodide; SD, standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114580&req=5

f4-ol-08-03-1009: Effect of TCAB1 siRNA on cell cycle and apoptosis regulation. (A) Each bar of the bar graph represents the mean percentage of cells in the G1 and S phases. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (B) The cell cycle distribution was determined by the detection of DNA degradation. A549 cells were treated with TCAB1 siRNA and stained with PI and analyzed by flow cytometry. The cell count versus PI staining is shown (n=10,000 per treatment). (C) Expression of CDK6 was determined by RT-PCR analysis of the total isolated RNA. Actin served as a gel-loading control. The densitometric quantifications of the CDK6 levels were normalized against the actin levels. Data are presented as the mean ± SD of triplicate analyses. *P<0.05 vs. con siRNA and WT (n=3). (D) Annexin V staining of TCAB1 siRNA-treated cells. Dot-plot data of FITC and PI staining measured by fluorescence-activated cell sorting analysis and graphical representation of apoptosis in cells treated with TCAB1 siRNA. *P<0.05 vs. control siRNA and WT (n=3). WT, wild-type; con siRNA, control small interfering RNA; TCAB1, telomerase Cajal body protein 1; RT-PCR, reverse transcription-polymerase chain reaction; CDK6, cyclin-dependent kinase 6; FITC, fluorescein isothiocyanate; PI, propidium iodide; SD, standard deviation.
Mentions: Further study was conducted to elucidate the mechanism of action for the antiproliferative activity of TCAB1 depletion on A549 cells. Flow cytometry was used to analyze the A549 cell cycle distribution and apoptosis. Cancer cells are generally immortal and divide uncontrollably, and the replication of telomeres occurs in the S phase (24). Thus, it was predicted that the number of A549 cells would increase in the G1 phase and decrease in the S phase as a result of TCAB1 depletion. Consistent with this hypothesis, TCAB1 siRNA was found to induce G1 cell cycle arrest, and the percentage of cells with G1 DNA content increased from 70 to 88% and the percentage of cells in the S phase decreased from 25 to 9%, when compared with that of the control groups (Fig. 4A). However, cells were not found to accumulate in the sub G1 peak (Fig. 4B). In addition, the G1 cell cycle checkpoint protein, cyclin-dependent kinase 6 (CDK6), was suppressed (Fig. 4C). Accordingly, the Annexin V staining apoptosis assay of A549 cells treated with TCAB1 siRNA demonstrated no evidence of apoptotic cell death (Fig. 4D). These observations indicated that the antiproliferative effects of TCAB1 siRNA in A549 cells is caused by cell cycle arrest without the induction of apoptosis.

Bottom Line: This recruitment is dependent on TCAB1 binding to a telomerase RNA component.In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes.In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, First Affiliated Hospital of Zhejiang University, Hangzhou, Zhejiang 310003, P.R. China.

ABSTRACT
Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

No MeSH data available.


Related in: MedlinePlus