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. 2014 Feb 15;33(1):19.
doi: 10.1186/1756-9966-33-19.

Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity

Tamara Fernández-Marcelo, Cristina Frías, Irene Pascua, Carmen de Juan, Jacqueline Head, Ana Gómez, Florentino Hernando, Jose-Ramon Jarabo, Eduardo Díaz-Rubio, Antonio-Jose Torres, Michèle Rouleau, Manuel Benito, Pilar Iniesta  1
Affiliations

Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity

Tamara Fernández-Marcelo et al. J Exp Clin Cancer Res. .

Abstract

Background: Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity.

Methods: We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay.

Results: In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control.

Conclusion: Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability.

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Figures

Figure 1
Figure 1
Time course of PARP3 expression levels by qRT-PCR after transient transfection, in A549 cells. Bars are the average of three experiments, media ± standard error.
Figure 2
Figure 2
Telomerase activity in A549 cells after transient transfection. Time course of telomerase activity ratios [Absorbance (450 nm) of the protein extracts from A549 cells transfected with pcDNA/GW-53/PARP3 vector]/[Absorbance (450 nm) of the protein extracts from A549 cells transfected with pcDNA-DEST53], after transient transfection. (Data are the average of four experiments, media ± standard error).
Figure 3
Figure 3
Representative examples of telomerase activity on Polyacrylamide Gel Electrophoresis (PAGE) in A549 transfectants are shown. (A) 24 and 48 hours after trasfection. (B) 96 hours after transfection.
Figure 4
Figure 4
Western-blot assay for testing PARP3 protein levels in A549 cells after transient transfection. Bars are the average of three experiments, media ± standard error.
Figure 5
Figure 5
PARP3 mRNA expression and protein levels in Saos-2 cells after transfection. (A) Analysis of PARP3 expression levels by qRT-PCR, after shRNA transfection (data are the average of triplicate experiments, media ± standard error). (B) Western-blot assay for testing PARP3 protein levels in Saos-2 cell line (bars are the average of three experiments, media ± standard error).
Figure 6
Figure 6
Telomerase activity in Saos-2 cells after transfection. (A) Telomerase activity ratios [Absorbance (450 nm) of the protein extracts from Saos-2 cells with PARP3 down-regulated]/[Absorbance (450 nm) of the protein extracts from Saos-2 cells control] (data are the average of three experiments, media ± standard error). (B) Telomerase activity on Polyacrylamide gel Electrophoresis (PAGE).
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