Figure 1.
DNA damage signaling in X-DC patient cells.
(A) Immunofluorescence staining of DNA damage proteins. Control X-DC-1787-C and patient X-DC-1774-P cells were, either not treated (-Bleo) or treated (+Bleo) with bleomycin (10 µg/ml) for 24 hours, fixed and incubated with antibodies against γ-H2AX, 53BP1, p-ATM or p-CHK1 and secondary fluorescent antibodies. Nuclear DNA was counterstained with DAPI (blue). (B). Quantification of γ-H2A.X foci, pATM, 53BP1 and pCHK2 associated foci in X-DC-1787-C and X-DC-1774-P cells. More than 200 cells were analyzed in each cell line and indicated as the average number of foci/cell. Asterisks indicate significant differences in relation to control cells lines or to untreated cells. Average values and standard deviations of two independent experiments are shown. Experiments were repeated 3 times with similar results.
Figure 2.
Determination of Histone-macroH2A.1-associated heterochromatin and senescence in X-DC cells.
(A) Histone-macroH2A.1-associated heterochromatin detection in X-DC cells. X-DC-1787-C and X-DC-1774-P cells were either not treated (-Bleo) or treated (+Bleo) with bleomycin (10 mg/ml) for 24 hours, fixed and incubated with an antibody against Histone-macroH2A.1 followed by a secondary fluorescence labeled antibody. (B) Quantification of Histone-macroH2A.1-associated heterochromatin. More than 200 cells were analyzed in each cell line and grouped to the area presenting Macro H2A.1 foci per cell. Asterisks indicate significant differences between cells lines. Average values and standard deviations of two independent experiments are shown. (C) SA-β-gal activity in X-DC-1787-C and X-DC-1774-P cells either untreated (-Bleo) or treated (+Bleo) with bleomycin (10 µg/ml). Senescent cells were quantified in 6 images of random regions. Experiments were repeated 3 times with similar results. Asterisks indicate significant differences in response to bleomycin.
Figure 3.
Localization of 53BP1 foci to telomeres in X-DC patient cells.
X-DC-1787-C and X-DC-1774-P cells untreated (-Bleo) or treated (+Bleo) with bleomycin (10 µg/ml) and incubated with γ-H2A.X and PNA-FISH probe. (A) Colocalization of 53BP1 foci (green) and telomeres as identified by hybridizing with a PNA-FISH probe (red). DNA was counterstained with DAPI (blue). Magnified views of merged images showing details of the colocalization are shown in the two lower series of panels (B) Colocalized 53BP1 foci and PNA-FISH probe at telomeres was quantified. More than 200 cells were analyzed in each cell line in an experiment performed three times with similar results. Asterisks indicate significant differences in relation to different cell lines.
Figure 4.
F9A353V cells show enhanced, basal and bleomycin induced, DNA damage response.
(A) F9, F9A353V and F9A353V cells transfected with GSE24.2 (10 µg DNA per million cells). F9A353V 24.2 cells were treated with bleomycin (10 µg/ml). After 0, 15 or 30 minutes of treatment cells were lysed and the experiment analyzed by western blot with antibodies against γ-H2A.X or α-tubulin as a loading control. (B) Immunofluorescence staining of γ-H2A.X (green) in F9, F9A353V and F9A353V 24.2 cells (10 µg DNA per million cells). Nuclear DNA was counterstained with DAPI (blue). (C) Quantification of γ-H2AX foci in F9, F9A353V or F9A353V 24.2 cells. More than 200 cells were analyzed in each cell line and grouped to the number of γ-H2A.X foci observed per cell. Experiments were repeated 3 times with similar results. Asterisks indicate significant differences in relation to different cell lines.
Figure 5.
Localization of 53BP1 foci to telomeres in F9A353V cells.
F9, F9A353V and F9A353V cells transfected with GSE24.2 (F9A353V 24.2) (F9 cells were treated with bleomycin,10 µg/ml for 24 hours) and incubated with 53BP1 antibodies and with a PNA-FISH probe. (A) Colocalization of 53BP1 foci (green) and PNA-FISH probe that identified telomeres (PNA-Tel, red). DNA was counterstained with DAPI (blue). Magnified views of merged images showing details of the colocalization are shown in the lower panels. (B) Quantification of the colocalization of 53BP1 foci and telomere signals shown in panel A. More than 200 cells were analyzed in each cell line and grouped to the number of 53BP1 foci associated to telomeres (PNA-Tel) per cell. Experiments were repeated 3 times with similar results. Asterisks indicate significant differences in relation to different cell lines.
Figure 6.
Activity of the GSE24.2 peptide expressed in bacteria or chemically-synthesized.
(A) F9A353V cells were transfected with 15 µg of β-galactosidase as a control (galactosidase), or GSE24.2 purified from E. Coli (GSE24.2E.coli) or obtained by chemical synthesis (GSE24.2 synthetic). After 24 hours cells were lysed and the levels of γ-H2AX and α-tubulin determined by western blot. The values at the bottom were obtained after quantification of the blot and show the ration between expression levels of γ-H2AX and α-tubulin in each line and referred to those found in β-galactosidase transfected cells. (B) Same experiment described in A, performed in X-DC3 cells transfected with β-galactosidase or chemically synthesized GSE24.2. (C) Reactivation of telomerase activity by chemically synthesized GSE24.2. X-DC3cells were transfected with β-galactosidase or chemically synthesized GSE24.2 and telomerase activity determined by TRAP assay (right). Different amounts extract were used for each TRAP assay as indicated. The activity was quantified by evaluating the intensity of the bands in relation with the internal control (TEL/IC) (left panel). The values for GSE24.2 transfected cells were referred to the β-galactosidase transfected cells. The experiments were repeated at least three times with similar results. Asterisks indicate significant differences between the two different transfected peptides.
Figure 7.
Oxidative stress analysis in X-DC fibroblasts after GSE24.2 transfection.
(A) ROS levels were determined in fibroblasts from the carrier DC1787, and fibroblasts from the patient X-DC1774-P. Levels were determined using the fluorescent probe dihydroethidium in confluent cells (left panel). RNA expression was determined for CuZnSOD, MnSOD, and GPX1 by qRT-PCR (A right panels). B) Enzymatic activities of CuZnSOD,MnSOD, and Glutathione peroxidase 1 were also determined. C) ROS levels were studied in X-DC1774-P fibroblasts (expressing pLNCX vector) and X-DC-1774-P cells expressing GSE24.2 (X-DC1774-PGSE24.2, left panel). Cu/ZnSOD, MnSOD, and catalase expression levels were determined by qRT-PCR. D) Cu/ZnSOD, MnSOD, and catalase activities in confluent pLNCX and 24.2 cells are shown in left panels. E) X-DC1774-P and X-DC1774-PGSE24.2, cells were transfected with GSE24.2 synthetic peptide and levels of 8-oxoguanine studied by immunofluorescence. The 8-oxoguanine foci signal was expressed as the average number of foci/cell in 200 cells. Results are expressed as mean ± standard deviation from three independent experiments. Statistical significance is expressed as (*) p<0.05.
Figure 8.
Proposed biological activity of GSE24.2 on DNA damage and oxidative stress.
Dyskeratosis congenita cells display high basal DNA damage detected by increased γH2AX, p-ATM, p-CHK2 and 53BP1 foci. Additionally there are increased levels of ROS, and decreased expression and activity of antioxidant enzymes resulting in higher oxidative damage and senescence (left panel). GSE24.2 peptide increases expression of antioxidant enzymes and as a consequence decreased ROS levels (right panel). In parallel there is increased telomerase activity that may help to decrease global and telomeric DNA damage. Globally these two activities of GSE24.2 might result in increased viability and growth of DC cells (26).