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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 the expression of TCAB1 and hTERT. (A) WB and RT-PCR analyses demonstrate the effect of TCAB1 siRNA transfection on TCAB1 and hTERT expression in A549 cells. (B) Densitometric quantification of the relative TCAB1 and hTERT levels of the cells. Levels of TCAB1 and hTERT were normalized to actin levels. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). (C) A TRAP assay was performed to evaluate the effect of TCAB1 siRNA treatment on the activity of telomerase in A549 cells. The 56-bp bands represent the IC. (D) The bar graph presents the densitometry-quantified data of the TRAP products in the single lane/PC (line 1) of the TRAP reaction ratios from three independent experiments. Telomerase activities were quantified by comparing the mean band intensity of each lane with the PC. The PC mean band intensity was defined as 100% telomerase-positive. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). TCAB1, telomerase Cajal body protein 1; con siRNA, control small interfering RNA; WT, wild-type; WB, western blot; hTERT, telomerase reverse transcriptase; RT-PCR, reverse transcription polymerase chain reaction; PC, positive control; IC, internal control; SD, standard deviation; TRAP, telomeric repeat amplification protocol.
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f1-ol-08-03-1009: Effect of TCAB1 siRNA on the expression of TCAB1 and hTERT. (A) WB and RT-PCR analyses demonstrate the effect of TCAB1 siRNA transfection on TCAB1 and hTERT expression in A549 cells. (B) Densitometric quantification of the relative TCAB1 and hTERT levels of the cells. Levels of TCAB1 and hTERT were normalized to actin levels. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). (C) A TRAP assay was performed to evaluate the effect of TCAB1 siRNA treatment on the activity of telomerase in A549 cells. The 56-bp bands represent the IC. (D) The bar graph presents the densitometry-quantified data of the TRAP products in the single lane/PC (line 1) of the TRAP reaction ratios from three independent experiments. Telomerase activities were quantified by comparing the mean band intensity of each lane with the PC. The PC mean band intensity was defined as 100% telomerase-positive. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). TCAB1, telomerase Cajal body protein 1; con siRNA, control small interfering RNA; WT, wild-type; WB, western blot; hTERT, telomerase reverse transcriptase; RT-PCR, reverse transcription polymerase chain reaction; PC, positive control; IC, internal control; SD, standard deviation; TRAP, telomeric repeat amplification protocol.

Mentions: TCAB1 expression was investigated in A549 cells. The protein and total RNA were isolated from A549 cells treated with TCAB1 siRNA and control siRNA and the levels of TCAB1 and hTERT were analyzed by WB analysis and RT-PCR. TCAB1 expression was downregulated by ~45% without altering hTERT expression (Fig. 1A and B). Telomeric repeat amplification protocol (TRAP) was also conducted to analyze telomerase activity and the results showed that the telomerase activity remained unchanged compared with the control groups (Fig. 1C).


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 the expression of TCAB1 and hTERT. (A) WB and RT-PCR analyses demonstrate the effect of TCAB1 siRNA transfection on TCAB1 and hTERT expression in A549 cells. (B) Densitometric quantification of the relative TCAB1 and hTERT levels of the cells. Levels of TCAB1 and hTERT were normalized to actin levels. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). (C) A TRAP assay was performed to evaluate the effect of TCAB1 siRNA treatment on the activity of telomerase in A549 cells. The 56-bp bands represent the IC. (D) The bar graph presents the densitometry-quantified data of the TRAP products in the single lane/PC (line 1) of the TRAP reaction ratios from three independent experiments. Telomerase activities were quantified by comparing the mean band intensity of each lane with the PC. The PC mean band intensity was defined as 100% telomerase-positive. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). TCAB1, telomerase Cajal body protein 1; con siRNA, control small interfering RNA; WT, wild-type; WB, western blot; hTERT, telomerase reverse transcriptase; RT-PCR, reverse transcription polymerase chain reaction; PC, positive control; IC, internal control; SD, standard deviation; TRAP, telomeric repeat amplification protocol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ol-08-03-1009: Effect of TCAB1 siRNA on the expression of TCAB1 and hTERT. (A) WB and RT-PCR analyses demonstrate the effect of TCAB1 siRNA transfection on TCAB1 and hTERT expression in A549 cells. (B) Densitometric quantification of the relative TCAB1 and hTERT levels of the cells. Levels of TCAB1 and hTERT were normalized to actin levels. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). (C) A TRAP assay was performed to evaluate the effect of TCAB1 siRNA treatment on the activity of telomerase in A549 cells. The 56-bp bands represent the IC. (D) The bar graph presents the densitometry-quantified data of the TRAP products in the single lane/PC (line 1) of the TRAP reaction ratios from three independent experiments. Telomerase activities were quantified by comparing the mean band intensity of each lane with the PC. The PC mean band intensity was defined as 100% telomerase-positive. Each bar represents triplicate analyses of the mean ± SD. *P<0.05 vs. con siRNA and WT (n=3). TCAB1, telomerase Cajal body protein 1; con siRNA, control small interfering RNA; WT, wild-type; WB, western blot; hTERT, telomerase reverse transcriptase; RT-PCR, reverse transcription polymerase chain reaction; PC, positive control; IC, internal control; SD, standard deviation; TRAP, telomeric repeat amplification protocol.
Mentions: TCAB1 expression was investigated in A549 cells. The protein and total RNA were isolated from A549 cells treated with TCAB1 siRNA and control siRNA and the levels of TCAB1 and hTERT were analyzed by WB analysis and RT-PCR. TCAB1 expression was downregulated by ~45% without altering hTERT expression (Fig. 1A and B). Telomeric repeat amplification protocol (TRAP) was also conducted to analyze telomerase activity and the results showed that the telomerase activity remained unchanged compared with the control groups (Fig. 1C).

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