Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
Ataxia telangiectasia (AT) is a rare autosomal syndrome due to inactivation of ATM (AT-mutated) a Ser/Thr protein kinase from the PIKK family [1, 2]. AT is characterized by progressive cerebellar ataxia, immunodeficiency, radiosensitivity, and increased cancer incidence. Functions assigned to ATM include, cell cycle regulation, DNA repair, and cellular redox status . The best understood function of ATM is the initial phosphorylation of the DNA double-strand breaks (DSBs) damage response pathway. Accordingly, AT patients and deficient cells display increased radiosensitivity, strongly suggesting a link between DSBs sensitivity and this pathology [2, 3]. ATM is involved in efficient and accurate DSB repair, preventing cell death and genomic instability exclusively when DNA ends are irreversibly blocked. Therefore, blocked DNA ends may be the pathogenic trigger of AT . After DSB, ATM is rapidly recruited to DNA damage sites , and dissociates from homodimers into active monomers  by autophosphorylation. ATM phosphorylates multiple substrates  involved in recognition of DSBs in DNA and in cell cycle checkpoint activation among other functions . Key events in this initial ATM-mediated DNA damage response (DDR) are phosphorylation of histone variant H2A.X in Ser139 (γ-H2A.X) in the chromatin surrounding the DSB, and recruitment of 53BP1. Both events can be cytologically visualized by their accumulation in nuclear foci, and are widely used for the quantification of DSBs.
Another key ATM function is to sense and regulate cellular redox. In ATM deficiency, cellular redox homeostasis is severely disrupted resulting in high levels of reactive oxygen species (ROS), which may contribute to the progressive neurodegeneration displayed by AT patients . In response to oxidative stress (OS), p38 mitogen-active protein kinase (p38) is activated in ATM-depleted cells  and treatment with the specific p38 inhibitor SB203580 restores normal proliferation. Persistent DNA damage leads AT cells to permanent mitotic arrest or senescence . Several inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, are secreted by these senescent cells and enforce senescence  allowing damaged cells to communicate their compromised state to the surrounding tissue. Therefore, damaged cells can contribute to tissue dysfunction promoting inflammation, angiogenesis, or other inducers of cancer progression .
GSE24.2 is a peptide corresponding to an internal domain of dyskerin . Dyskerin mutations cause the rare disease X-linked dyskeratosis congenita (X-DC) . Individuals with X-DC show premature ageing, bone marrow failure, pulmonary fibrosis, and increased susceptibility to cancer [15, 16]. GSE24.2 expression increases telomerase reverse transcriptase (TERT) mRNA and TERC levels and the recovery of telomerase activity [13,14,15,16,17,18]. Expression of GSE24.2 and GSE4 (composed by the 11 N-terminal amino acids of GSE24.2) reduce DNA damage in X-DC cell lines, decreasing OS and increasing expression of detoxifying genes. This results in decreased cell senescence and increased proliferation . Due to GSE24.2 and GSE4 properties, we aimed to test their function in AT cells. Expression of both peptides reduced basal levels of DNA damage in AT cells probably due to reduced oxidative DNA damage, and not to increased DSB repair capacity. Further, AT cells expressing both peptides were able to divide for longer periods of time and showed reduced acid-β-galactosidase activity as a senescence marker, expression of IL6, increased expression of TERT and telomerase activity, elongation of telomeres, and decreased oxidation of telomere and mitochondrial DNA. Altogether our results support a potential therapeutic role of GSE24.2/GSE4 in AT cells mainly by decreasing OS and increasing telomerase activity with positive effects on cell growth.
Expression of GSE24.2 and GSE4 in AT cells decreases spontaneous DNA damage signaling
The effect of GS24.4/GSE4 on DDR, was studied in human fibroblast cell lines from AT patients GMO3487 (AT-3487) and controls GMO1787 (C-1787, C-3490), measuring γ-H2A.X accumulation. AT fibroblasts rapidly senesce in vitro, therefore we also used AT (AT-3189, AT-719, AT-137IIB) and control lymphoblast cell lines (C-736, C-JY). We generated stable cell lines by transduction of GSE24.2 (LV-CMV-GSE24.2-eGFP), GSE4 (LV-CMV-GSE4-eGFP), control vectors (LV-CMV-eGFP), and GSE-scramble (LV-CMV-scramble-eGFP). Expression levels of GSE4 and GSE24.2 in transduced cells are presented in Fig S1. We first studied fibroblast and found that the number of γ-H2A.X foci/cell was higher in AT-3487 than in control C-1787 and C-3490 cells (Fig. 1a and Fig S2). Damage was partially reverted in AT-3487 cells expressing GSE24.2 and GSE4. AT-3487-GSE24.2 and AT-3487-GSE4 cells showed increased number of cells with low signal (0–4) γ-H2A.X-associated foci. We also investigated the presence of 53BP1 foci, more specific than γ-H2A.X for DSBs. The average number of foci/cell was similar to that observed for γ-H2AX, higher in AT-3487 than in control C-1787 and C-3490 cells, and also decreased in AT-3487-GSE24.2 and AT-3487-GSE4 cells (Fig. 1a and Fig S3). Finally, we treated AT-3487 cells with empty, GSE24.2 or GSE4-PLGA-PEI nanoparticles  and evaluated the number of γ-H2A.X-associated foci/cell (Fig. 1b). GSE24.2 and GSE4 loaded nanoparticles reduced the γ-H2A.X signal obtained, strongly suggesting that both peptides have similar effects in AT cells. Similar results were obtained determining γ-H2AX levels by western blot in C-736 and C-JY control cells in comparison with AT-719, AT-3189, and AT-L137IIB cells or those expressing GSE24.2 or GSE4 (Fig. 1c).
Expression of GSE24.2 or GSE4 reduces oxidative damage caused by ATM loss but does not affect DSB signaling and repair deficiencies
Accumulation of γ-H2A.X can be influenced by three factors: (i) the level of endogenous DNA damage, (ii) the signaling of this damage, and (iii) the rate of repair. DDR activation was analyzed determining γ-H2A.X foci formation induced by the chemotherapeutic agent etoposide, which generates DSBs by impairing the activity of DNA topoisomerase II (TOP2) . These breaks present covalent binding of TOP2-derived peptides to 5′-DNA ends, which, in the absence of the enzyme TDP2 results in irreversible blockage, requiring nucleolytic activity for repair through a pathway that specifically requires ATM function . As previously described, etoposide treatment induced γ-H2A.X foci formation in Tdp2−/− mouse embryonic fibroblasts (MEFs), which were smaller and less bright upon treatment with ATM inhibitor (ATMi) (Fig. 2a). This effect was independent of GSE24.2 (Fig. 2a) or GSE4 (data not shown) expression, indicating that these peptides do not affect DDR signaling in response to DSBs. Finally, to analyze DSB repair, we monitored the disappearance of etoposide-induced γ-H2A.X foci in Tdp2−/− MEFs (Fig. 2b), highly depends on ATM function. DSB repair was not improved (if something slightly impaired) by expression of either GSE24.2 or GSE4, with >10 γ-H2A.X foci/cell remaining after 6 h of repair. Overall, we conclude that the effect of GSE24.2/GSE4 on the spontaneous accumulation of γ-H2A.X in AT cells is unlikely related to modulation of DDR signaling or DSB repair. We also used human control cell lines C-1787 and C-3490 expressing either GFP or GSE4 in a similar experiment, treating the cells with bleomycin and ATMi (Fig. 2c). We observed that damage increased at 6 h and remained to the same level at 24 h in the absence of ATMi, with a small decrease in those expressing GSE4. But in the presence of ATMi, there was an increase in γ-H2A.X foci/cell at 24 h. This difference should be due to DDR signaling in response to DSBs, not repaired by GSE4.
We next studied if the decrease in DNA damage may be due to decreased spontaneous oxidative DNA damage investigating if GSE4 was able to reduce this damage in AT cells . We treated AT-3487 cells with PLGA-PEI-GSE4-nanoparticles and evaluated the levels of 8-oxoguanine by immunofluorescence (Fig. 2d) finding that GSE4 treatment was able to reduce the signal. This finding strongly suggests that GSE4-mediated rescue of DDR activation in AT cells corresponds, at least in part, to an inhibition of DNA oxidative damage.
Increased ROS is a hallmark of AT cells responsible for the reduced repopulation capacity of ATM−/− hematopoietic stem cells (HSCs) [22, 23]. Therefore, we compared ROS levels in AT cells (AT-3189) expressing GSE24.2 or GSE4 with C-736 control cells. ROS levels were elevated in AT-3189 cells (Fig. 3a) and decreased upon GSE24.2 or GSE4 expression. We previously described that GSE24.2 or GSE4 increased the expression of three antioxidant enzyme genes, SOD1, SOD2 and catalase in DC cells [18, 19]. Expression of SOD1 and catalase, but not SOD2 were reduced in three AT patient cell lines (Fig. 3b) in comparison with C-736 and C-JY cells. Expression of GSE24.2 and GSE4 in AT-719, AT-L137IIB, or AT-3189 cells increased the mRNA levels of these genes, specially SOD1 (Fig. 3b), suggesting that SOD1 may be involved in the reduction in ROS levels. However, expression of GSE4 in control C-736 or C-JY cells did not induce significant changes in the expression of those genes. SOD1 protein levels also increased in AT-719 cells expressing GSE24.2 or GSE4 (Fig S4a). GSE4 induced the expression of SOD1 in AT cells by stimulating its promoter revealing the mechanism of SOD1 activation (Fig. 4a). To further understand the role of SOD1, we transduced C-736 and AT-3189 cells with an shRNA targeting SOD1 (Fig S4b) and measured ROS levels. The results clearly indicated an increase in ROS levels in these cell lines after SOD1 depletion but this increase was higher in AT-3189 cells expressing GSE4 (Fig. 4b and. Fig S4c). We also tested the effect of GSE4 on cell viability after treating control and AT cells with H2O2 during 72 h. The results indicated increased viability of GSE4-expressing cells, although in C-JY cells the effect was lower (Fig. 4c). Altogether the results indicated that expression of GSE peptides by inducing expression of SOD1 should increase survival to the high ROS levels present in AT cells.
Inhibition of IL-6 expression, p38 phosphorylation, and increased viability by GSE24.2 and GSE4 in AT cells
Human cells harboring increased persistent chromatin lesions as AT cells secrete inflammatory cytokines such as IL-6 . Given the previous results, we studied the expression of IL-6 and found that was increased in the AT cell lines and diminished upon GSE24.2 or GSE4 expression (Fig. 4d).
Increased levels of ROS in AT cells induce p38 mitogen activated protein (MAP) kinase activation and cell death [24, 25]. Basal levels of phosphorylated p38 MAP kinase were higher in AT cells AT-719, AT-L137IIB, AT-3189, and AT-3487 than in control cells C-736, C-JY, and C-3490, and decreased after expression of GSE24.2 or GSE4 in AT cells (Fig. 5a). These results support that AT cells expressing GSE24.2 or GSE4 show an important reduction of the pro-apoptotic pathway mediated by activated p38.
ATM deficiency contributes to in vivo senescence in the hematopoietic system  probably as a result of persistent DNA damage. AT-3487 fibroblasts expressing GSE24.2 or GSE4, showed a marked reduction in the expression of the senescence-associated β-galactosidase (SA-β-gal), more evident after 8 days of peptide expression (Fig. 5b). Since AT fibroblasts grow very poorly in vitro, control and AT lymphoblasts expressing enhanced green fluorescent protein (eGFP), GSE24.2, or GSE4 were allowed to grow along 2 months and the number of accumulated cells expressing GSE24.2 or GSE4 estimated in relation with eGFP-expressing cells (Fig. 5c and Fig S5a). The results indicated that expression of GSE24.2 or GSE4 was able to prolong growth of AT lymphoblasts probably due to the lower levels of ROS and activated p38 in AT cells expressing these peptides.
AT cells are very sensitive to agents inducing DSBs such as ionizing radiation or bleomycin. We investigated if there was any protective effect to bleomycin with GSE24.2 or GSE4. Control and AT cells expressing either eGFP, GSE24.2, or GSE4 were treated with increasing doses of bleomycin and cell viability determined (Fig. 5d and Fig S5b). Even though there was almost no effect in the viability of control cells expressing GSE24.2 or GSE4, the IC50 of AT cells expressing either peptides was always higher than that of AT-GFP cells, which was lower than in control cells.
Recovery in TERT expression and telomerase activity in AT lymphoblast cells after GSE24.2/GSE4 expression
AT patients have shortened telomeres and increased frequency of chromosome end-to-end fusions (CEFs) [26, 27]. We have evaluated the expression of TERT, TERC, and DKC1 and found that expression of TERC (Fig. 6a) was higher in some AT cell lines than in control cells, expression of DKC1 did not show major differences (Fig S6a), but the expression of TERT was very low in most AT cell lines studied (Fig. 6a). Expression of GSE24.2 or GSE4 in AT cells resulted in non-homogeneous changes in TERC (Fig. 6b) or DKC1 (Fig S6a), but TERT expression was increased in all AT cells (Fig. 6b). Dyskerin protein levels showed no significant changes in control and AT cells expressing either GSE24.2 or GSE4 (Fig S6b). As dyskerin is involved in pseudouridylation and processing of ribosomal RNAs (rRNAs), we determined the expression levels of rRNAs (45S, 28S, 18S, and 5.8S) in control and AT cells expressing GFP, GSE24.2, or GSE4 (Fig S7). There was an increase in the expression of 28S rRNA in most AT cells expressing GSE24.2 or GSE4 but there was no correlation with decreased expression level of 45S rRNA or increased levels of 18S or 5.8S rRNAs as could be expected if GSE peptides would affect rRNA processing.
In agreement with the results on TERT expression, telomerase activity in AT-3189 cells was 40% lower than in control C-736 cells (Fig. 6c). Expression of GSE4 in AT-3189 cells increased telomerase activity by 60%, correlating with increased TERT expression (Figs. 6b, c).
We next studied telomere length (TL) in two AT cell lines (AT-3189 and AT-719) by Southern blot. The results clearly showed that, the mean TL was around 5 and 4.5 kbp in AT patient-derived cells and 10–12 kbp in a control cell line derived from an older proband (Fig. 6d). GSE4 expression induced changes in TL after 2 months of in vitro culture (Fig. 6d). We used quantitative polymerase chain reaction (qPCR) to measure those changes, as it is more sensitive than Southern blot for small TL changes . We observed an increase in the T/S ratio (the ratio of telomere (T) repeat to single-copy (S) gene (36B4) copy numbers) in AT-3189 GSE4 cells after 60 days of culture. Using a conversion from T/S ratio to kbp obtained with a reference DNA from MCF-7 cells, we determined that, in AT-3189-GSE4 there was an increase of almost 0.5 kbp of TL (Fig. 6d table). Similar results were obtained using another single-copy gene human beta globin (HBG) (data not shown). Therefore, the increase in TERT expression and in telomerase activity by GSE4 expression resulted in telomere elongation.
Changes in telomere and mitochondrial DNA oxidation in AT cells induced by expression of GSE4
OS accelerates telomere shortening [28, 29] during DNA replication and ultimately promote cellular senescence . In vitro studies have shown that the 5′-GGG-3′ sequence in the 5′-TTAGGG-3′ telomeric-DNA repeats is more prone to oxidation than other DNA sequences . We next studied if GSE4 expression may modify telomere oxidation. To that end, we adapted the procedure described by O’Callaghan et al.  by measuring base excision repair (BER)-related lesions at telomeres and at mitochondrial DNA. This qPCR method is based on differences in PCR kinetics between DNA-template digested by formamidopyrimidine-DNA-glycosylase (FPG) and undigested DNA. We have modified the original protocol by incorporating the human glycosidase from the BER pathway OGG1 that eliminates 8-oxoguanine in double-stranded DNA . The results showed that the ratio of PCR amplification with vs. without OGG1 digestion (an indicator of 8-oxoguanine levels) in telomere regions were higher in cells after 60 days of culture. Basal levels of oxidation in the control cell line C-736 (T0) was higher than in AT-3189 (T0) probably due to the fact that TL in C-736 cells was twice longer than that of AT-3189 and therefore the abundance of G triplets. After 60 days, GSE4-expressing cells either control or AT had lower oxidation levels than at T0, but the effect was stronger in AT-T60 cells (Fig. 7a). Similar results were obtained when a probe to amplify the mitochondrial gene COX1 (Fig. 7b) was used. Expression of GSE4 resulted in a marked decrease in oxidation in COX1 gene, which was more evident in C-736-T60 and AT-3189-T0 and even more at T60. These results indicate that expression of GSE4 in AT cells decreases the level of 8-oxoguanine at telomeres and mitochondrial DNA suggesting an important decrease in oxidative DNA damage at different compartments.
Several lines of evidence suggest that high levels of OS contribute to neurodegeneration in AT . First, AT cells and tissues display abnormally high levels of OS and ROS in the brain (particularly the cerebellum) [35, 36], oxidative DNA damage, and lipid oxidation products . Second, AT cells and tissues are especially sensitive to ROS-inducing agents (such as hydrogen peroxide and ionizing radiation) and have impaired DNA damage checkpoint responses, persistent DNA damage, and a lowered apoptosis threshold [38,39,40]. Finally, treatment of AT cells and tissues with antioxidants can improve cell and tissue survival.
Here we describe that expression of GSE24.2 or GSE4 peptides in AT cells mitigate many of the pathologic consequences of high ROS levels including telomere shortening. Indicators of DNA damage such as the histone γ-H2AX and 53BP1 foci decreased in AT cells expressing GSE24.2 or GSE4. Levels of 8-oxoguanine residues also decreased, indicating reduced oxidative damage. Our previous results indicate that expression of both peptides decreased ROS and 8-oxoguanine levels in X-DC cells with high oxidative damage generated by telomere shortening [18, 19]. Interestingly, expression of both peptides was unable to rescue the DDR and repair defect caused by ATM inhibition in Tdp2−/− MEFs or primary human cells. Therefore, these peptides cannot rescue the effects of ATM inactivation on DSB signaling and repair, but importantly inhibit the accumulation of DNA oxidative damage.
ROS are endogenous agents causing DNA damage, and perform critical signaling functions in apoptosis, stress responses, and proliferation . Cellular mechanisms protecting from ROS, include detoxifying enzymes such as superoxide dismutases (SODs), and small-molecule scavengers such as glutathione . We have found lower SOD1 and catalase mRNA levels in AT than in control cells. Interestingly, expression levels increased in AT cells expressing GSE24.2 or GSE4, even in cells not showing deficient SOD2 expression. GSE4 was able to activate the SOD1 promoter revealing a possible mechanism for regulation of ROS levels by GSE peptides. Knock-down of SOD1 expression in AT-GSE4-expressing cells resulted in increased ROS levels, strongly suggesting that increased expression of SOD1 was at least partially responsible of the decrease in ROS levels. This cellular defense induced by GSE peptides results in protection to ROS scavengers such as H202. In agreement with our results, treatment of AT cells with antioxidant therapy corrects clinically important aspects of the disease .
We found increased levels of IL-6 in human AT cell lines in agreement with previous results . Expression of GSE24.2 or GSE4 in AT cells decreased IL-6 expression. IL-6 mediates inflammation and contributes to the maintenance of the senescent phenotype, which might induce malignancy in neighboring cells specially in AT patients . Increased p38-MAPK activation is also related with increased proinflammatory cytokine induction under continuous DNA damage conditions . Expression of either GSE24.2 or GSE4 decreases the ratio of p38-activated/unphoshorylated-p38 in AT cells. AT fibroblast cells expressing of GSE24.2 or GSE4 also showed lower expression of SA-β-gal, strongly indicating that the expression of both peptides can trigger inhibition of senescence-associated secretory phenotype (SASP)-related pathways activated in AT cells.
Immunodeficiency and bone marrow failure are severe effects of ATM loss in AT patients. We found that AT lymphoblasts expressing both peptides, were able to continuously divide during 2 months in contrast to unmodified AT cells suggesting beneficial effects counteracting the loss of B or T cells found in AT patients.
AT patients exhibit radiosensitivity and higher risk of cancers  between 10% and 38% of them developing at least one malignancy during lifetime . AT patients also have high risk of developing secondary malignancies and serious chemotherapy-associated side effects such as severe mucosal inflammation or life-threatening infections. Therefore, radiotherapy is strongly discouraged in AT patients. Expression of GSE24.2 or GSE4 decreased sensitivity of AT cells to bleomycin, which may indicate a lower toxicity of radiation. Since increased ROS are also induced in response to radiomimetic drugs, we suggest that the antioxidant capacity of both peptides may contribute to this protective effect.
AT is classified as a secondary telomere disease even if mutations are not detected in telomere regulatory genes . We have found that decreased TL in AT cells is probably partially due to decreased expression of the catalytic telomere subunit TERT. In addition, 8-oxoguanine is present in high levels, which might contribute to telomere attrition. Expression of GSE24.2 and GSE4 induced a small but reproducible increase in TERT expression, resulting in a 50% increase in telomerase activity and a rate of telomere elongation of 0.5 kbp in 2 months. This protective effect is also associated with lower oxidation in telomere and mitochondrial DNA. This last finding may explain how GSE4 expression could contribute to less ROS production at mitochondria thus enforcing the protective effect of GSE peptides.
In summary, our results indicate that GSE4 could be used in the treatment of AT patients, inducing protection to OS, decreasing oxidative damage at DNA, inflammation-associated parameters, senescence and increasing the viability to ROS scavengers, and radiomimetic drugs. Decreased oxidative DNA damage may result in attenuation of malignancies associated with AT and also to decreased bone marrow failure, a pathology that increased morbidity in AT patients. A second therapeutic advantage would be the effects of telomerase activation, reduced telomere oxidative damage, and telomere elongation that would result in maintenance of cell proliferation and reduction of genomic instability.
Materials and methods
Cell lines and cell culture
Cell lines, both control and those derived from AT patients, were obtained from Coriell Cell Repository (Camden, NJ). We used control human fibroblasts GMO1787 (C-1787) and C-3490 and AT fibroblasts GMO3487 (AT-3487, 8 years). This cell line carries an A > T substitution at nucleotide 8266 (8266A > T) in exon 58 resulting in a truncation at codon 2756 [Lys2756Ter (K2756X)], whereas the paternal allele carries a 4-bp insertion at nucleotide 1141 in exon 11 resulting in a frameshift at codon 381 leading to a truncation (S381fsX). Lymphoblast cells were also obtained from Coriell: GMO736 (C-736, 30 years) from a healthy donor and C-JY cells (ATCC, Manassas, VA USA), and two from AT patients GMO3189 (A > T substitution at nucleotide 8266 (8266A > T, 7 years) and GMO719 (AT-719, 13 years) . AT-L137IIB lymphoblast cells were obtained in our lab from an AT patient (ATM:c.103C > T; p.R35X; rs55861249, 11 years). Primary MEFs were isolated from Tdp2−/− embryos  at day 13 post coitum (p.c.) and cultured at 37 °C, 5% CO2, 3% O2. All primary fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS), gentamicin and l-glutamine and lymphoblasts cells in RPMI (Roswell Park Memorial Institute medium) supplemented with 20% FBS, gentamicin and l-glutamine.
Lentiviral vectors for GSE24.2, GSE4, and shSOD1 expression
All lentiviral vectors (LVs) used in these experiments were third-generation sin-LVs. The fragments scramble peptide, GSE24.2, and GSE4 were cloned into the pRRLsin18.ppt.CMV.IRES.eGFP transfer vector using the PstI and BamHI restriction sites. GSE4 was cloned as double-stranded oligonucleotides as described . The DNA coding for a scrambled peptide containing the same amino acids as GSE4 arranged randomly was cloned between the PstI and BamHI restriction sites of the same vector using the annealed oligonucleotides 5′-GATGCTTTCTAAGGGTAATAACATTCCCGACTTCCCCTAAG-3′ and 5′-GATCCTTAGGGGAAGTCGGGAATGTTATTACCCTTAGAAAGCATCTGCA-3′. The original pRRLsin18.ppt.CMVeGFPWpre transfer vector, pMDLg-pRRE and pRSV-REV packaging vectors, and pMD2-VSVG envelope vector were kindly provided by Dr. Luigi Naldini (San Raffaele Telethon Institute for Gene Therapy, University Medical School, Milan, Italy). The LVs supernatants (LV-CMV-eGFP, LV-CMV-scramble-eGFP, LV-CMV-GSE24.2-eGFP, LV-CMV-GSE4-eGFP) and pGKshSOD1 (Sigma-Aldrich-Merck, St. Louis, MO, USA) were produced using a viral packaging system that includes the packaging and envelope vectors in 293T cells and used for transduction of the indicated cell lines. Efficiency of transduction was monitored by flow cytometry detecting expression of eGFP. Expression of GSE24.2, GSE4, and SOD1 was quantified by RT-qPCR using oligonucleotides previously reported .
Nanoparticle synthesis and loading of GSE24.2 and GSE4 peptides
Chemically synthesized GSE4 and GSE24.2 peptides labeled with fluorescein-5-isothiocyanate (FITC) were obtained from China Peptides (Hangzhou, China) and purified by high-performance liquid chromatography (HPLC). poly(lactic-co-glycolic acid/Poly(ethylenimine) (PLGA/PEI) nanoparticle preparation and peptide loading was performed as previously described . Internalization efficiency was estimated by determination of intracellular fluorescence using a fluorescence enzyme-linked immunoSorbent assay (ELISA) reader.
Determination of ROS content with dihydroethidium
Cells were washed two times with pre-warmed phosphate-buffered saline (PBS) medium and 2 µL/mL of diluted dihydroethidium (D7008-Sigma-Aldrich-Merck, St. Louis, MO, USA) was added to the plate. Cells were incubated at 37 °C for 20 min and then washed with PBS. Medium was replaced and cells cultured for an additional 1 h at 37 °C. The fluorescence was measured using spectraMAX GEMINIS (Molecular Device, Sunnyvale, USA), with 530 nm of excitation wavelength and 610 nm of emission wavelength. Mean fluorescence intensity (MFI) for each cell line was normalized by the cell number.
Real-time quantitative PCR
RNA isolation and cDNA synthesis
Total cellular RNA was extracted using Trizol (Invitrogen-Thermo, Carlsbad, USA) according to the manufacturer’s instructions. Reverse transcription (RT) reactions were made using 1 µg of the purified RNA using random hexamers and the High-Capacity cDNA Archive kit (Applied Biosystems-Thermo Fisher, P/N: 4322171; Foster City, CA) according to the manufacturer’s instructions.
Measurement of mRNA levels
The mRNA levels were determined by quantitative real-time PCR analysis using an ABI Prism Step One Plus PCR System (Applied Biosystems-Thermo Fisher, Foster City, CA). Gene-specific primer pairs and probe for IL-6 (Hs00985639_m1), β-actin: Hs99999903_m1, catalase Hs00156308_m1, SOD1: Hs00533490_m1, SOD2: Hs00167309_m1 were used together with TaqMan® Universal PCR Master Mix (Applied Biosystems-Thermo Fisher, P/N 4304437, Foster City, CA) and 2 µl of reverse transcribed RNA in 20 µl reaction volume. PCR conditions were 10 min at 95 °C for enzyme activation, followed by 40 two-step cycles (15 s at 95 °C; 1 min at 60 °C). The levels of beta-actin (β-act) expression were measured in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality, and reverse transcription efficiency. Expression of TERT, TERC, DKC1, GAPDH and 45S, 28S, 18S and 2.5S rRNAS were performed with the following oligonucleotides:
TERT F 5′-CGGAAGAGTGTCTGGAGCAA-3′; TERT R 5′-GGATGAAGCGGAGTCTGG-3′;
TERC F 5′-TCTAACCCTAACTGAGAAGGGCGTAG-3′; TERC R 5′-GTTTGCTCTAGAATGAACGGTGGAAG-3′;
DKC1 F 5′-GCTCCTCAGTTGATCAAGAAGG-3′; DKC1 R 5′-CTCAGAAACCAATTCTACCTC-3′;
45S F 5′-GGACGTGCCGCGCCAGGAAG-3′; 45S R 5′-CGACGCTCAGACAGGCGTAG-3′;
28S F 5′-GCCAAGCGTTCATAGCGACG-3′; 28S R 5′-CTGAGCAGGATTACCATGGC-3′;
18S F 5′-GGACACGGACAGGATTGACAG-3′; 18S R 5′-CAATCTCGGGTGGCTGAACG-3′;
5.8S F 5′-GACTCTTAGCGGTGGATCAC-3′; 5.8S R 5′-CGACGCTCAGACAGGCGTAG-3′.
GAPDH F 5′-GAGAGACCCTCACTGCTG-3′; GAPDH R 5′-GATGGTACATGACAAGGTGC-3′. Specific oligonucleotides were used together with Power Sybrgreen PCR mastermix (Applied Biosystems-Thermo Fisher, P/N 4304437, Foster City, CA) and 2 µl of reverse transcribed RNA in 20 µl reaction volume. PCR conditions were 10 min at 95 °C for enzyme activation, followed by 40 two-step cycles (15 s at 95 °C; 1 min at 60 °C). The levels of GAPDH expression were determined in all samples to normalize gene expression for sample-to-sample differences in RNA input, RNA quality and reverse transcription efficiency. Each sample was analyzed in triplicate, and the expression was calculated according to the 2−ΔΔCt method.
In all, 3 × 105 Tdp2−/− primary MEFs were infected with LV-CMV-eGFP, LV-CMV-GSE24.2-eGFP, or LV-CMV-GSE4-eGFP (MOI 10) in T25 flasks, incubated for 3 days, and then grown on coverslips for 7 days until confluence. Cells were then treated 30 min with etoposide (10 μM) (Sigma) in the presence or absence of 10 μM ATMi (KU55933) (Tocris Bioscience). ATMi was maintained at 3 and 6 h after washing the etoposide. Repair analysis was carried out by monitoring the time course of γ-H2AX foci disappearance following drug (etoposide) removal. C-1787 and C-3490 cells were treated 30 min with bleomycin (1 μM) (Sigma-Aldrich-Merck) in the presence or absence of 10 μM ATMi (KU55933) (Tocris Bioscience). ATMi was maintained at 6 and 24 h after washing the bleomycin. Repair analysis was carried out by monitoring the time course of γ-H2AX foci persistence following drug (bleomycin) removal.
Protein localization was carried out by fluorescence microscopy. For this purpose, cells were grown on coverslips, transfected and fixed in 3.7% formaldehyde solution (47608; Fluka, Sigma-Aldrich-Merck, St. Louis, MO) at room temperature for 15 min. After washing with 1× PBS, cells were permeabilized with 0.2% Triton X-100 in PBS and blocked with 10% horse serum before overnight incubation with γ-H2A.X (05-636; Merck Millipore), 53BP1 (4937; Cell Signaling), and 8-oxoguanine antibodies (MAB2560; Merck Millipore). Finally, cells were washed and incubated with secondary antibodies coupled to fluorescent dyes (Alexa fluor 488 or/and Alexa fluor 647). Images were acquired with a Confocal Spectral Leica TCS SP5 using a HCX PL APO Lambda blue 6361.40 OIL UV, zoom 2.3 lens. Images were acquired using LAS-AF 1.8.1 Leica software and processed using LAS-AF 1.8.1. Leica software and Adobe Photoshop CS. Each experiment was performed in triplicates and at least 200 cells were quantified for each experimental condition.
Western blot and antibodies
Whole-cell extracts were prepared essentially as described previously . Western blotting was performed using standard methods . Protein concentration was measured by using the Bio-Rad protein assay.
The source of antibodies was as follows: phospho-Histone H2A.X Ser139 (2577) Cell Signaling, monoclonal anti-β-actin (A2228) from Sigma-Aldrich-Merck, SOD1 (HPA001401) Sigma-Aldrich-Merck, p38 (C-20) and dyskerin (H-300) from Santa Cruz Biotechnology and p38 (Th180/tyr182 (9211) from Cell Signaling.
Control and AT fibroblasts (1 × 104 cells) were plated onto six-well plates and fixed after 4 days to assay the SA-β-gal activity (Senescence Detection Kit, BioVision, Milpitas, California). The percentage of senescent cells was calculated in six images per sample taken by bright field microscopy at ×100 magnification (Nikon Eclipse TS100 Microscope, Melville, NY, USA).
Telomeric repeat amplification protocol (TRAP) assay
Telomerase activity was determined under recommended standard conditions of the TRAPEZE® Telomerase Detection S7700 Kit (Merck Millipore) for TRAP using radioisotopic detection. Telomerase activity was determined in each sample using 0.5, 0.25, and 0.125 µg of protein extract and normalized with the internal control included in the assay .
TL was determined from DNA of cells, either control or AT, by Southern blot analysis of telomere restriction fragment (TeloTAGGG Telomere Length Assay, Sigma-Aldrich-Merck), which is considered the gold standard to determine TL as previously described . The relative TL was assessed by qPCR as previously described . The qPCR determines the ratio of telomere (T) repeat copy number to single-copy (S) gene (36B4) copy number (T/S ratio) in experimental samples, as compared with a reference DNA sample (MCF-7 cells).
Oxidative DNA damage within telomeres and mitochondrial DNA
We adapted the procedure described by O’Callaghan et al.  to measure oxidative DNA damage at telomeres, 36B4 genomic region and at mitochondrial DNA. This is a qPCR method based on differences in PCR kinetics between DNA template digested by FPG and undigested DNA. We have modified the original protocol to incorporate another purified human DNA glycosylase from the BER OGG1 (kindly provided by Professor Thomas Helleday’s Lab, Karolinska Institutet, Sweden) to quantify the accumulation of base lesions specific for OGG1, 8-oxoguanine. This enzyme recognizes and cuts 8-oxoguanine producing apurinic sites (AP) that are converted in single-strand breaks (SSBs) by its AP lyase activity. These SSBs inhibit the PCR, thus, the ΔCT after digesting DNA by 8-oxoguanine (Ct digested–Ct undigested) is proportional to the oxidative damage in the amplified region. Conditions used for incubation were 2.4 μM OGG1 during 4 h. The quantitative real-time amplification of genomic DNA was performed as described by O’Callaghan et al. . Each 20 μL qPCR were composed as follows: 40 ng of digested or undigested genomic DNA, 1 × GoTaq® qPCR Master Mix (Promega), specific primers for each genomic region at a final concentration of 100 nM (Mitochondrial COX1 F: 5′-CACAGCCCATGCATTTGTAA-3′; COX1 R: 5′-GATGCGAGCAGGAGTAGGAG-3′ and Telo1/2 and 36B4 F/R)  Amplification cycling conditions were 10 min at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. ΔCT method was run in an ABI quant studio 7 and all samples were loaded and analyzed in triplicate.
Cells were seeded in 24-well plates (75,000 cells per well), cultured for 16 h, and transfected with a DNA mixture consisting of 100 ng of the appropriate luciferase construct (pGL3basic or pGL3basic-sod1-1499 containing the complete SOD1 promoter)  and 400 ng of the GSE or scramble plasmid vectors, using Lipofectamine reagent (Invitrogen-Thermo Fisher, Carlsbad, CA) according to the manufacturer’s recommendations. After 24 h from transfection, the cells were serum starved for 16 h, lysed, and assayed for luciferase activity with the Luciferase Assay System (Promega) according to the manufacturer’s instructions; relative light units were measured in a BG1 Optocomp I, GEM Biomedical luminometer (Sparks, NV).
Viability and cell growth assays
Briefly, cells were treated in 96-multiwell dishes with increasing concentrations of bleomycin or H2O2. Cell viability was estimated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) hydrolysis using a kit from Promega (Promega Corporation, Madison, WI, USA). Assays were made in triplicate. For the growth assays, cells were grown in culture media and counted every week. Growth was represented as cumulative growth of cells infected with Lenti-CMV-GSE24.2-eGFP or LV-CMV-GSE4-eGFP in relation of growth of Lenti-CMV-eGFP-infected cells. Growth was monitored until 60 days after infection.
For the statistical analysis of the results, the mean was taken as the measurement of the main tendency, whereas standard deviation was taken as the dispersion measurement. Student's t-test was performed. The significance has been considered at *p < 0.05. **p < 0.01 and ***p < 0.001. GraphPad Software v5.0 was used for statistical analysis and graphic representations.
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RP laboratory is funded by grant P14-01495 and P17-01401 (Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain supported by FEDER funds) and CIBER 576/805_ER16PE06P2016 supported by FEDER funds. GG grant “Ministerio de Economía, Comercio y Competitividad y Fondo Europeo de Desarrollo Regional (FEDER)” (SAF2015-68073-R). CM-G is granted by the CIBERER. Work in FC-L laboratory is funded with grants from the Spanish Government (SAF2014-55532-R, Ministerio de Economía, Industria y Competitividad), the regional Andalusian Government (CVI-7948), the Fundación Ramón Areces (XVII Convocatoria Ciencias de la Vida y Materia), the European Research Council (ERC-CoG-2014-647359), and with a Predoctoral Fellowship from AEFAT (Asociación Española Familia Ataxia Telangiectasia) to AS-B. BER OGG1 was kindly provided by Professor Thomas Helleday’s Lab, Karolinska Institutet, Sweden. We gratefully acknowledge to Dr. Antonio Cuadrado for the SOD1 promoter, to Monica Martinez-Belinchon, and Dolores Morales-Garcia for valuable help in the confocal studies. We also acknowledge Ana Sastre-Perona for useful comments.
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Pintado-Berninches, L., Fernandez-Varas, B., Benitez-Buelga, C. et al. GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells. Cell Death Differ 26, 1998–2014 (2019). https://doi.org/10.1038/s41418-018-0272-7
Immunity & Ageing (2022)