Telomeric DNA induces p53-dependent reactive oxygen species and protects against oxidative damage

Abstract

Background: Reactive oxygen species (ROS) are generated by cellular metabolism as well as by exogenous agents. While ROS can promote cellular senescence, they can also act as signaling molecules for processes that do not lead to senescence. Telomere homolog oligonucleotides (T-oligos) induce adaptive DNA damage responses including increased DNA repair capacity and these effects are mediated, at least in part, through p53.

Objective: Studies were undertaken to determine whether such p53-mediated protective responses include enhanced antioxidant defenses.

Methods: Normal human fibroblasts as well as R2F fibroblasts expressing wild type or dominant negative p53 were treated with an 11-base T-oligo, a complementary control oligo or diluents alone and then examined by western blot analysis, immunofluorescence microscopy and various biochemical assays.

Results: We now report that T-oligo increases the level of the antioxidant enzymes superoxide dismutase 1 and 2 and protects cells from oxidative damage; and that telomere-based gammaH2AX (DNA damage) foci that form in response to T-oligos contain phosphorylated ATM and Chk2, proteins known to activate p53 and to mediate cell cycle arrest in response to oxidative stress. Further, T-oligo increases cellular ROS levels via a p53-dependent pathway, and these increases are abrogated by the NAD(P)H oxidase inhibitor diphenyliodonium chloride.

Conclusion: These results suggest the existence of innate telomere-based protective responses that act to reduce oxidative damage to cells. T-oligo treatment induces the same responses and offers a new model for studying intracellular ROS signaling and the relationships between DNA damage, ROS, oxidative stress, and cellular defense mechanisms.

Figure 1. T-oligos stimulate intracellular ROS levels

(A) Normal newborn human fibroblasts were stimulated with T-oligo or diluent and ROS levels were determined using the DCF assay[70, 74, 114, 115]. Within 36 hours intracellular ROS levels were induced only in T-oligo-treated cultures and the levels increased up to 72 hours when the experiment was terminated. One of five representative experiments is shown. (B) Fibroblasts were stimulated as above with increasing T-oligo concentrations and ROS levels were determined 72 hours after stimulation. Maximal induction of ROS was observed at T-oligo concentrations of ≥ 40 μM. One of two reproducible experiments is shown. (C) Fibroblasts were stimulated with T-oligo as above or with 1 mM H₂O₂ for 15 minutes to examine the possibility of DCF probe saturation. H₂O₂ increased fluorescent intensity beyond that of T-oligo-induced fluorescence, confirming that DCF fluorescence was not saturated. One of three reproducible experiments is shown. (D) Fibroblasts were stimulated with T-oligo (40 μM), Cont-oligo (40 μM) or diluent as above and ROS level was determined 72 hours after treatment. Only T-oligo induced intracellular ROS levels. One of five experiments with comparable results shown.

Figure 1. T-oligos stimulate intracellular ROS levels

(A) Normal newborn human fibroblasts were stimulated with T-oligo or diluent and ROS levels were determined using the DCF assay[70, 74, 114, 115]. Within 36 hours intracellular ROS levels were induced only in T-oligo-treated cultures and the levels increased up to 72 hours when the experiment was terminated. One of five representative experiments is shown. (B) Fibroblasts were stimulated as above with increasing T-oligo concentrations and ROS levels were determined 72 hours after stimulation. Maximal induction of ROS was observed at T-oligo concentrations of ≥ 40 μM. One of two reproducible experiments is shown. (C) Fibroblasts were stimulated with T-oligo as above or with 1 mM H₂O₂ for 15 minutes to examine the possibility of DCF probe saturation. H₂O₂ increased fluorescent intensity beyond that of T-oligo-induced fluorescence, confirming that DCF fluorescence was not saturated. One of three reproducible experiments is shown. (D) Fibroblasts were stimulated with T-oligo (40 μM), Cont-oligo (40 μM) or diluent as above and ROS level was determined 72 hours after treatment. Only T-oligo induced intracellular ROS levels. One of five experiments with comparable results shown.

Figure 1. T-oligos stimulate intracellular ROS levels

(A) Normal newborn human fibroblasts were stimulated with T-oligo or diluent and ROS levels were determined using the DCF assay[70, 74, 114, 115]. Within 36 hours intracellular ROS levels were induced only in T-oligo-treated cultures and the levels increased up to 72 hours when the experiment was terminated. One of five representative experiments is shown. (B) Fibroblasts were stimulated as above with increasing T-oligo concentrations and ROS levels were determined 72 hours after stimulation. Maximal induction of ROS was observed at T-oligo concentrations of ≥ 40 μM. One of two reproducible experiments is shown. (C) Fibroblasts were stimulated with T-oligo as above or with 1 mM H₂O₂ for 15 minutes to examine the possibility of DCF probe saturation. H₂O₂ increased fluorescent intensity beyond that of T-oligo-induced fluorescence, confirming that DCF fluorescence was not saturated. One of three reproducible experiments is shown. (D) Fibroblasts were stimulated with T-oligo (40 μM), Cont-oligo (40 μM) or diluent as above and ROS level was determined 72 hours after treatment. Only T-oligo induced intracellular ROS levels. One of five experiments with comparable results shown.

Figure 1. T-oligos stimulate intracellular ROS levels

(A) Normal newborn human fibroblasts were stimulated with T-oligo or diluent and ROS levels were determined using the DCF assay[70, 74, 114, 115]. Within 36 hours intracellular ROS levels were induced only in T-oligo-treated cultures and the levels increased up to 72 hours when the experiment was terminated. One of five representative experiments is shown. (B) Fibroblasts were stimulated as above with increasing T-oligo concentrations and ROS levels were determined 72 hours after stimulation. Maximal induction of ROS was observed at T-oligo concentrations of ≥ 40 μM. One of two reproducible experiments is shown. (C) Fibroblasts were stimulated with T-oligo as above or with 1 mM H₂O₂ for 15 minutes to examine the possibility of DCF probe saturation. H₂O₂ increased fluorescent intensity beyond that of T-oligo-induced fluorescence, confirming that DCF fluorescence was not saturated. One of three reproducible experiments is shown. (D) Fibroblasts were stimulated with T-oligo (40 μM), Cont-oligo (40 μM) or diluent as above and ROS level was determined 72 hours after treatment. Only T-oligo induced intracellular ROS levels. One of five experiments with comparable results shown.

Figure 2. T-oligo induced ROS levels are p53-mediated

p53^DN mouse fibroblasts and isogenic p53 wild type cells were stimulated with T-oligo or diluent for 3 days and ROS levels were examined. Only p53^WT cells displayed increased ROS levels, confirming that functional p53 is required for T-oligo-mediated ROS induction. (One of two representative experiments is shown.)

Figure 3. T-oligo-induced ROS production is NAD(P)H-dependent

Fibroblasts were treated with T-oligo in the presence or absence of the specific NAD(P)H inhibitor DPI for 3 days as per Material and Methods and DCF fluorescence was examined. DPI, added to DCF assay medium, completely abrogated T-oligo mediated ROS induction, verifying that T-oligo induced ROS is NAD(P)H-dependent. (One of six experiments with comparable results is shown.)

Figure 4. T-oligo-induced γH2AX foci contain phospho-ATM and phospho-Chk2

Normal neonatal fibroblasts were grown on glass coverslips and treated for 2 days with 20 μM T-oligo (GTTAGGGTTAGGGTTA). Cells were then fixed in paraformaldehyde and prepared for immunostaining as described in Experimental Procedures. Representative cells are shown. Top row: γH2AX. Middle row: phospho-ATM (left) or phospho Chk2 (right). Bottom row: merged images with DAPI nuclear stain.

Figure 5. T-oligo induces the levels of SOD1 and SOD2 in fibroblasts

Newborn fibroblasts were plated and processed as per Materials and Methods. Total cellular proteins were harvested 16, 24, 48, 72, 96 and 168 hr after oligonucleotide stimulation at time 0. Cultures were provided fresh medium lacking oligonucleotides immediately after the 72 hour timepoint and after 144 hours. Proteins were processed for western blot analysis and the blot was reacted with the following antibodies: SOD1, SOD2, Catalase, GPX and actin, followed by appropriate secondary antibodies. Compared to diluent (D) and control oligo (C), within 16 hours T-oligo (T) induced the levels of SOD1 and SOD2 and the induction persisted through 96 hours. GPX and catalase were not modulated. Differences in SOD1 and SOD2 band intensity between control oligo and diluent appear largely to reflect loading differences as demonstrated by the actin bands.

Figure 6. T-oligo protects fibroblasts from oxidative damage

Newborn fibroblasts were processed as per Materials and Methods, and cells were then treated with 25 μM fresh H₂O₂ or diluent for one hour and then provided fresh medium without T-oligos. Cell yields were determined 8, 16, 24 and 48 hours later. Fibroblasts pretreated with T-oligo and then diluent proliferated more slowly than diluent pretreated cells (p< 0.0001), as expected, given the initial T-oligo induced S-phase arrest[13], but both showed exponential growth after the first 24 hours. Interestingly, fibroblasts pretreated with T-oligo and then challenged with H₂O₂ proliferated significantly better than diluent pretreated H₂O₂-challenged fibroblasts (p< 0.006). Data points are the mean ± SEM for 3 separate experiments with different donor cells.