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 PF-01367338 datasheet in Saos-2 cell line (bars are the average of three experiments, media ± standard error). The clone of

Saos-2 cells with the highest decrease of PARP3 expression showed a significant (P-value: 0.003, Paired Samples T Test) increase in telomerase activity (2.3-fold increase), compared to the control, which was transfected with a non-functional shRNA (Figure 6A). As before, telomerase activity results on PAGE are shown (Figure 6B). 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).

Discussion The considerable progress in the science of PARPs in the last years has introduced these proteins function as a key mechanism regulating in a wide variety of cellular processes including, among others, telomere homeostasis. Recently, De Vos et al. have suggested that one of the major missions for MK-1775 solubility dmso the coming years in the PARP field is to further dissect the biological activities of the emerging DNA-dependent PARPs (i.e. PARP3, Tankyrase), and to exploit their known structural features for the rational

design of selective and potent PARP inhibitors [12]. Recent results identified PARP3, the third member of the PARP family, as a newcomer in DBS repair [13, 14]. PARP3 has been found to regulate mitotic progression by stimulating the Tankyrase 1 catalyzed auto (ADP-ribosyl) ation and hetero (ADP-ribosyl) ation of the mitotic factor NuMA N-acetylglucosamine-1-phosphate transferase (nuclear mitotic apparatus protein 1) [14]. Tankyrase 1 is denoted as a telomere associated PARP involved in the release of the telomeric protein TRF1, via its PARsylation to control access and elongation of telomeres by telomerase [15]. In this work, we observed that PARP3 depletion in lung cancer cells resulted in increased telomerase activity. Moreover, in cancer cells with low telomerase activity, PARP3 showed high expression levels. These results seem to indicate an inverse correlation between telomerase activity and PARP3 expression in cancer cells. According to our data, in A549 cells the highest mRNA PARP3 levels were detected 24 h after transfection.