GSE4, a Small Dyskerin- and GSE24.2-Related Peptide, Induces Telomerase Activity, Cell Proliferation and Reduces DNA Damage, Oxidative Stress and Cell Senescence in Dyskerin Mutant Cells

<div><p>Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.</p></div

DNA-damage protective effect, telomerase activation and cell proliferation induction of one small peptide, GSE4, derived from GSE24.2.

<p>Panel A. One small peptide derived from GSE24.2, GSE4, and GSE24.2 were expressed in F9_A353V cells that were transfected with the pRRL-CMV-IRES-EGFP vector, either empty (Vector) or expressing GSE24.2 or GSE4. Twenty four hours later cells were lysed and the presence of γH2AX and α-tubulin (loading control) analyzed by western blot. Un-transfected F9 and F9-A353V cells were used as controls. The values at the bottom of the panel indicate the estimated ratio between γH2AX and α-tubulin expression levels referred to those found in cells transfected with the empty vector (F9-A353V vector). The amino acid sequences of GSE24.2 and GSE4 are indicated at the lower part of the panel. Panel B. The telomerase activity of F26IIB cells transfected with the pRRL-CMV-IRES-GFP vector empty (vector), expressing GSE24.2 (GSE24.2) or GSE4 (GSE4) was determined using the Telomeric Repeat Amplification Protocol (TRAP) assay. The amplification products obtained using three decreasing amounts of cell extracts for each cell line are shown in the right panel. Quantification of the amplification products, normalized to the internal control provided in the assay (indicated by an arrow at the right panel) is shown in the left panel. Panel C. Expression of Ki67 was determined by immunocytochemistry in F26IIB cells transfected as described in panel B. The percentage of cells expressing Ki67 is represented for each type of transfected cells. The experiments were repeated three times with similar results. Asterisks indicated the statistical significance (* p<0.05, **p<0.01, ***p<0.001).</p

Localization of 53BP1 foci to telomeres in X-DC patient cells.

<p>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.</p

F9A353V cells show enhanced, basal and bleomycin induced, DNA damage response.

<p>(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.</p

Localization of 53BP1 foci to telomeres in F9A353V cells.

<p>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.</p

Determination of Histone-macroH2A.1-associated heterochromatin and senescence in X-DC cells.

<p>(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.</p

Oxidative stress analysis in X-DC fibroblasts after GSE24.2 transfection.

<p>(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.</p

Activity of the GSE24.2 peptide expressed in bacteria or chemically-synthesized.

<p>(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.</p

Proliferative cells can be isolated from adult human white matter.

<p><b>A</b>. Diagram depicting the different procedures followed in the present work for the isolation of proliferative cells from human white matter. <b>B</b>. The cell isolation protocol without (w/o) the sucrose centrifugation step gives rise to a monolayer culture, which could be expanded more than seven passages. However, only 28.57% of samples generated viable cultures with this procedure (<b>B</b>). Therefore, we added a final sucrose centrifugation, which allowed the obtaining of sphere-like colonies lightly adhered to the culture plate from all samples processed (<b>D</b>). These cells generated an adherent culture that could be expanded independently for more than five passages (<b>E</b>). When the initial spheres were passaged and reseeded at a cell density of 3000 cells/cm² or higher, new spheres were obtained (<b>C</b>), which, in turn, generated a new monolayer culture. These spheres could be passaged up to tertiary spheres, similarly to other proliferative cells isolated from adult human white matter. The scale bars represent 50 µm.</p

Brain white matter tissue obtained from epileptic patients does not show histopathological abnormalities.

<p><b>A.</b> The DTI imaging analysis of the temporal areas used in our experiments showed diffusion tensors with elongated ellipsoidal form and grouped colouring, indicating a conserved white matter ultrastructure similar to the contralateral areas. <b>B–C.</b> The anisotropy of water molecules, quantified by the fractional anisotropy mean (FA) and the apparent diffusion coefficient mean (ADC), showed no significant differences between both epileptic (black) and contralateral (grey) white matter zones (p-value: 0.155 and 0.439, respectively) <b>D.</b> No neuronal heterotopia was observed within the white matter, with only a few neurons NeuN⁺ rarely spread throughout white matter parenchyma (white arrow). The scale bar represents 20 µm. <b>E.</b> Microglial Iba-1⁺ cells showed mainly quiescent morphology, with long branching processes and small cellular bodies. The scale bar represents 100 µm. <b>F.</b> Cortical disorganisation was also discarded by NeuN immunohistochemistry, as the neuronal layer could be perfectly differentiated (indicated by the roman numerals). The scale bar represents 20 µm. <b>G–J.</b> Haematoxylin-eosin staining revealed a normal cellularity (<b>G</b>) and the Ki67 recounts showed a normal number of cells in active phases of the cell cycle in all samples (<b>H</b>). Glioma tissue was used as positive control (<b>I–J</b>)<b>.</b> The scale bars represent 50 µm.</p