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How Do Cells Repair Single Stranded Dna

Written By Tuesday, April 5, 2022 Add Comment Edit

doi: 10.1038/ncomms10399.

Defective DNA single-strand intermission repair is responsible for senescence and neoplastic escape of epithelial cells

Sébastien Martien ane , Nathalie Martin one , Emeric Deruy ane , Elisa Tomellini 1 , Nicolas Malaquin 1 , Fatima Bouali 1 , Laure Sabatier 2 , Nicolas Wernert 3 , Sébastien Pinte 1 iv , Eric Gilson 4 , Albin Pourtier i , Olivier Pluquet 1 , Corinne Abbadie 1

Affiliations

  • PMID: 26822533
  • PMCID: PMC4740115
  • DOI: 10.1038/ncomms10399

Free PMC article

Lacking DNA unmarried-strand intermission repair is responsible for senescence and neoplastic escape of epithelial cells

Joe Nassour  et al. Nat Commun. .

Free PMC article

Abstract

The master characteristic of senescence is its stability which relies on the persistence of DNA damage. We show that different fibroblasts, senescent epithelial cells do not activate an ATM-or ATR-dependent DNA impairment response (DDR), but accrue oxidative-stress-induced Dna unmarried-strand breaks (SSBs). These breaks remain unrepaired because of a subtract in PARP1 expression and activity. This leads to the formation of abnormally large and persistent XRCC1 foci that appoint a signalling cascade involving the p38MAPK and leading to p16 upregulation and cell cycle arrest. Chiefly, the default in SSB repair besides leads to the emergence of postal service-senescent transformed and mutated precancerous cells. In man-aged skin, XRCC1 foci accumulate in the epidermal cells in correlation with a turn down of PARP1, whereas DDR foci accrue mainly in dermal fibroblasts. These findings point SSBs equally a Deoxyribonucleic acid impairment encountered by epithelial cells with aging which could fuel the very first steps of carcinogenesis.

Figures

Figure 1
Effigy 1. Growth curve and characteristics of NHEKs and NHDFs.

(a) Growth curve of NHEKs (left console) and NHDFs (right panel; donor 1MC) with representative micrographs of each growth phase; Scale bar, l μm. (b) Cell cycle distribution of exponentially growing and senescent NHEKs and NHDFs. The bar nautical chart represents the ways±south.d. of the means of three experiments performed with 3 unlike NHEKs-NHDFs couples: K1MC (ExpG: iii PDs—Sen: 12.iii PDs), F1MC (ExpG: xi PDs—Sen: 58 PDs), K1320 (ExpG: 3.five PDs—Sen: 10 PDs), F1320 (ExpG: 11 PDs—Sen: 39 PDs), K67FA1 (ExpG: 2 PDs—Sen: 13 PDs) and F67FA1 (ExpG: 10.5 PDs—Sen: 42 PDs). (c) Western absorb analysis of p53, p21, p16, Rb phosphorylated on serine 807 and 811 (p-Rb (S807–811)), Rb and GAPDH (loading command) levels in total cell extracts of NHEKs and NHDFs (donor 1MC). (d) Reverse-transcription quantitative real-fourth dimension PCR (RT–qPCR) analysis of twist and slug transcripts in exponentially growing and PSNE NHEKs (donor 67FA1). Results are mean±s.d. of triplicates. Similar results were obtained with donor 1MC. (east) Western blot assay of F2R, MET, E-cadherin, ADAM10 (pro-ADAM10 and mature-ADAM10) and GAPDH (loading control) levels in full NHEKs extracts (donor 1MC). (f) HPRT assays performed in NHEKs (donor 1MC). The quantification of the results is given in Supplementary Fig. 14. ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The verbal PDs at which cells were taken is indicated.

Figure 2
Figure 2. Senescent NHEKs do not experience massive telomere shortening nor activate the DDR pathway.

(a) Telo-FISH on metaphase chromosome spreads of NHEKs and NHDFs (donor 2F19). Upper console: representative Telo-FISH images. Lower panel: quantification of telomeres loss. The given results are the mean of counts performed on 45–58 metaphases for each case. (b) Telo-FISH on interphasic cells. Upper panel: representative confocal microscopy images for the 1MC donor. Calibration bar, 20 μm. Lower panel: quantification of the fluorescence intensity obtained with three dissimilar NHEKs-NHDFs couples (1MC, 1320 and 67FA1). Scatter dot plots indicate the means±due south.d. of the means of the three experiments. (c) Assay by immunofluorescence of the activation of the DDR pathway in NHEKs and NHDFs (donor 1MC). Left: representative ApoTome microscopy images of DDR foci. Calibration bar, 20 μm. Right: quantification of the number of cells displaying at least iii foci of γH2AX, p-ATM, p-Chk2 or 53BP1, or at to the lowest degree 1 foci of p-ATR or p-Chk1. DDR foci-positive cells were automatically counted with ImageJ in 10 independent microscopic fields for a full of at to the lowest degree 200 cells for each example. The bar nautical chart represents the mean±south.d. of each 10 counts. The results are representative of iii contained experiments. (d) Western absorb analysis of the activation of the DDR pathway in total cell extracts of NHEKs and NHDFs (donor 1MC). PCNA was used as proliferative alphabetize and GAPDH as loading control. (e) p53 and p–p53 (Ser15) immunofluorescences performed on NHEKs and NHDFs (donor 1MC). Upper panel: representative ApoTome microscopy images. Scale bar, 20 μm. Lower panel: quantification of cells displaying p53 and p–p53 (Ser15) nuclear staining. Cells were counted in five independent microscopic fields for a full of at least 100 cells for each case. The bar chart represents the mean±s.d. of each 10 counts. The results are representative of two independent experiments. ExpG. exponentially growing cells; Sen, cells at the senescence plateau. The exact PDs at which cells were taken is indicated.

Figure 3
Figure 3. Senescent NHEKs display a decrease in PARP1 expression and activity and accumulate unrepaired SSBs.

(a) Neutral (pH eight; left) and element of group i (pH 12.three; right) comet assays performed in tandem on exponentially growing and senescent NHEKs and NHDFs (donor 1MC). Microphotographs of the comets are shown in Supplementary Fig. 6A. Tail moments of 30–50 comet-positive cells were quantified. Scatter dot plots represent the hateful±s.d. The results are representative of five independent experiments. (b) XRCC1 immunofluorescence performed on exponentially growing and senescent NHEKs and NHDFs (donor 1MC) using 3 different antibodies raised against different XRCC1 immunogens. The specificity of the antibodies is analysed in Supplementary Fig. 6B and C. Left: representative ApoTome microscopy images. Scale bar, 20 μm. Right: XRCC1 foci-positive cells were automatically counted with ImageJ in 5 independent microscopic fields for a total of at least 100 cells for each instance. The mean±s.d. of the v counts is indicated as inserts. The bar chart represents the means±s.d. of the means obtained with the three antibodies. (c) Contrary-transcription quantitative existent-time PCR (RT–qPCR) analysis of PARP1 transcripts (donor 1MC). Results are means±southward.d. of triplicates. Similar results were obtained with the 67FA1 donor. (d) Western blot analysis of PARP1, PAR, PCNA (proliferative alphabetize) and GAPDH (loading command) levels in total cell extracts of exponentially growing and senescent NHEKs and NHDFs (donor 1 MC) treated or not with 100 μM HiiOtwo at four °C for 10 min and and so placed at 37 °C for 5 min. The specificity of PARP1 and PAR antibodies is analysed in Supplementary Fig. 7B. (e) Double immunofluorescence detection of XRCC1 with BrdU, Ligase1, Ligase3 or PCNA. Upper panel: representative ApoTome microscopy images obtained with the 1MC donor. Calibration bar, x μm. Like results were obtained with the 1320 and 67FA1 donors. Lower console: cells displaying double-positive foci were automatically counted with ImageJ in ten fields for a full of >100 nuclei and the means were calculated. Besprinkle dot plots represents the mean±south.d. of the ways of the three experiments performed with the three different donors. ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The exact PDs at which cells were taken is indicated.

Figure 4
Figure four. Distinctive features of XRCC1 foci at senescence in NHEKs.

(a) Upper panel: follow-up of XRCC1 foci in exponentially growing and senescent NHEKs (donor 1MC) treated by 100 μM HiiO2 at 4 °C for 10 min so placed at 37 °C for 5 to 120 min. The number of foci per cell was counted in >50 cells. Each point represents the mean±s.d. Lower panel: exponentially growing and senescent NHEKs (donor 67FA1) were treated past 100 μM H2O2 at 4 °C for 10 min, placed at 37 °C for 20 min and analysed by western blot for PARP1, XRCC1, phosphorylated XRCC1 (S518/T519/T523), CK2α, PCNA (proliferative index) and GAPDH (loading control). (b) Exponentially growing NHEKs (donor 67FA1) were transfected or not with a puddle of four siRNAs against PARP1 or a puddle of 4 command siRNAs. Forty-eight hours after transfection, the same analyses as in a were performed. (c) Senescent NHEKs (donor 67FA1) were infected with adenoviral vector encoding PARP1 (AdPARP1), adenovirus encoding green fluorescence protein (AdGFP) or kept not-infected (NI). vi h afterwards infection, the same analyses as in a were performed. (d) Exponentially growing and senescent NHEKs (donor 1MC) were treated by 100 μM HtwoOtwo at 4 °C for 10 min and so placed at 37 °C for 5 min. Left panels: representative confocal photomicrographs of PAR and XRCC1 foci. Scale bar, 10 μm. Right panels: measures of fluorescence intensity performed forth the dotted lines. (e) Mensurate of XRCC1 foci area in H2Otwo-treated exponentially growing and non-treated senescent NHEKs. Left: representative confocal photomicrographs of XRCC1 foci. Scale bar, ten μm. Correct: area of at least 100 foci measured past ImageJ. Scatter dot plots represent the mean±s.d. (f) Senescent NHEKs (donor 67FA1) were infected with AdPARP1 or kept non-infected (NI) and stock-still at half dozen, 12, 24 and 48 h postal service-infection. Left console: representative photomicrographs of PARP1, CK2α, phosphorylated XRCC1, XRCC1 and PNKP immunostainings. Scale bar, five μm. Right panel: quantification of cells displaying PARP1 foci, CK2α nuclear staining, phosphorylated XRCC1 foci, total XRCC1 foci and PNKP foci. At to the lowest degree 100 cells were counted for each condition. Each betoken represents the hateful±south.d. ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The exact PDs at which cells were taken is indicated.

Figure 5
Figure 5. Restoring PARP1 expression resumes SSBR and delays senescence.

Exponentially growing NHEKs (donor 1MC; eight.five PDs) were infected with adenoviral vector encoding PARP1 (AdPARP1)/V5 or adenovirus encoding green fluorescence protein (AdGFP) or kept non-infected (NI). (a) Growth curve. (b) Western absorb analysis of the efficacy of infection. PARP1, V5, GFP, PCNA (proliferative alphabetize) and GAPDH (loading control) levels were analysed in full prison cell extracts. (c) Representative images of cell morphology at days 7 and xvi mail-infection. Scale bar=50 μm. (d) Percentage of SA-β-Gal-positive cells at days 3, 7 and 16 post-infection. The bar nautical chart represents the mean±s.d. of five counts. (e) Immunofluorescence detection of XRCC1 and 53BP1. Upper panel: representative ApoTome microscopy images, scale bar, ten μm. Lower panel: XRCC1 foci and 53BP1 foci-positive cells were automatically counted with ImageJ in x independent microscopic fields for a total of at least 200 cells. The bar chart represents the mean±south.d. of each 10 counts. The results are representative of two independent experiments. (f) Senescent NHEKs (donor 1MC) were infected as in a–d. Western absorb analysis of PARP1, V5, p16, p-Rb, p21 and GAPDH (loading command) levels in total cell extracts. ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The exact PDs at which cells were taken is indicated.

Figure 6
Effigy six. XRCC1 foci activate the p16/Rb pathway through p38MAPK.

(a) Pre-senescent NHEKs (donor 1MC; ten PDs) were transfected with a pool of command or anti-XRCC1 siRNAs. Western blot assay of XRCC1, p16, Rb, phosphorylated Rb (p-Rb S807–811), p53 and GAPDH (loading command) levels in full cell extracts on days 2, 4, vi and 8 post-transfection. (b) Western blot analysis of phosphorylated p38MAPK (p-p38MAPK), p38MAPK, p16 and GAPDH (loading control) in exponentially growing and senescent NHEKs (donor 1MC). (c) Senescent NHEKs (donor 1MC) were transfected with a puddle of control or anti-XRCC1 siRNAs and analysed by western blot for XRCC1, phosphorylated ERK1/2 (p-ERK1/2), ERK1/two, phosphorylated p38MAPK (p-p38), p38MAPK, p16 and GAPDH (loading control). ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The exact PDs at which cells were taken are indicated.

Figure 7
Figure 7. Aggregating of unrepaired SSBs is sufficient to cause PSNE.

Exponentially growing NHEKs (donor 1MC; viii PDs) were transfected with iii unlike siRNAs directed against PARP1 or a pool of four command siRNAs. The efficiency of the siRNAs is given in Supplementary Fig. 9A. (a) Growth curves. (b) XRCC1 and 53BP1 immunofluorescences performed at day 6 mail service-transfection. Positive cells were automatically counted with ImageJ in v contained microscopic fields for a full of at least 100 cells for each case. The bar chart represents the mean±southward.d. of each five counts. (c) Neutral (pH viii) and alkaline (pH 12.three) comet assays performed in tandem at solar day 6 post-transfection. Tail moments of fifty comet-positive cells were quantified. Besprinkle dot plots represent the mean±southward.d. of all measures. (d) Upper panel: measure of PSNE frequency performed as described in Methods section at the indicated time. The given results are the mean±due south.d. of counts of PSNE clones performed in five contained culture dishes. Lower panels: representative photomicrographs of PSNE clones stained with Crystal violet. Scale bar, 50 μm. (due east) Upper console: siCTR- and siPARP1-transfected NHEKs were analysed by FACS co-ordinate to their size and granularity. About 30% of the largest and most granular cells were sorted, plated at low density, stained with the filiation tracer CFDA SE and monitored for emergence which occurred nigh one week later. Lower panel: representative images of PSNE clones which accept inherited the fluorescent tracer from their senescent mother cell. Scale bar, fifty μm. (f) Western blot assay of F2R, MET and GAPDH (loading control) levels in total cell extracts at the given time mail-transfection. (g) HPRT assays performed at the given time post-transfection. The quantification of the results is given in Supplementary Fig. 14.

Figure 8
Figure 8. Oxidative stress resumes SSBR default-induced senescence and PSNE.

Exponentially growing NHEKs (donor 1MC; half-dozen.5PDs) were treated daily or non either with ane,000 U ml−i of catalase, 1,000 U ml−ane of catalase-PEG, 1 mM of Due north-acetylcysteine (NAC) or 20 μM H2O2. The efficacy of the antioxidants is given in Supplementary Fig. 12B. (a) Growth curves. (b) Representative images of cell morphologies on days 3, 9 and 18 of the treatment. Calibration bar, 50 μm. (c,east) Assay of XRCC1 and 53BP1 foci-positive cells performed on antioxidant-treated and HiiO2-treated cells at respectively days ix and 3 after the get-go of the treatment. Positive cells were automatically counted with ImageJ in 10 independent microscopic fields for a total of at least 200 cells. The bar chart represents the mean±due south.d. of each 10 counts. The results are representative of two independent experiments. (d,f) Neutral (pH 8) and element of group i (pH 12.three) comet assays performed in tandem on antioxidant-treated and HtwoO2-treated cells at respectively days ix and 3 after the first of the handling. Scatter dot plots correspond the mean±s.d. of l measures. (g) Measure of PSNE frequency in command and H2Otwo-treated NHEKs as described in Methods department. Left: bar nautical chart represents the mean±s.d. of counts performed in five independent culture dishes. Correct: representative photomicrographs of PSNE clones stained with Crystal violet. Scale bar, 50 μm. (h) Western blot analysis of F2R, MET and GAPDH (loading control) levels in total cell extracts of command and H2O2-treated NHEKs at the given days postal service-treatment. (i) HPRT assays performed in HtwoO2-treated NHEKs at the given PDs. The quantification of the results is given in Supplementary Fig. 14.

Figure 9
Figure 9. Detection of Dna damage markers in skin samples of different ages.

PARP1, XRCC1/Vimentin, 53BP1/Vimentin and MnSOD immunohistofluorescences performed in sections of peel samples from healthy human young (northward=3) and old donors (n=4; encounter Supplementary Tabular array ii). Upper panels: representative ApoTome microscopy images for epidermis and dermis of a young (nu 32645/09, 34 years sometime) and an anile donor (nu 9238/09, 75 years old). Scale bar, 50 μm. The foursquare delimits the below image at higher magnification. Lower panels: scatter dot plots indicating the percentage of positive epidermal and dermal cells in immature and aged peel. Positive cells were counted in x contained microscopic fields for a total of at to the lowest degree 150 cells. The given results are the hateful±s.d. of the means in the 3–4 donors.

Figure 10
Effigy 10. Senescence and post-senescence emergence of HMECs.

(a) Growth curve of HMECs. (b) Western blot assay of p16, phosphorylated Rb (p-Rb (S807–811)), p53, PCNA (proliferative alphabetize) and GAPDH (loading control) levels in total cell extracts. (c) Upper panel: morphology of HMECs observed past phase-contrast microscopy. Lower panels: images of SA-β-Gal-stained cells. Scale bar, 50 μm. (d) Western blot analysis of F2R, MET, E-cadherin, vimentin and GAPDH (loading control) levels in full jail cell extracts. (e) HPRT assays. The quantification of the results is given in Supplementary Fig. 14. (f) Western blot analysis of PARP1, PAR, PCNA (proliferative index) and GAPDH (loading control) levels in full extracts of exponentially growing and senescent HMECs treated or not with 100 μM H2Otwo at 4 °C for ten min and then placed at 37 °C for 5 min. (k) Quantification of SA-β-Gal, XRCC1 and 53BP1 foci-positive cells aggregating along the culture of HMECs. SA-β-Gal-positive cells were counted in five independent microscopic fields for a full of at to the lowest degree 100 cells. XRCC1 or 53BP1 foci-positive cells were automatically counted with ImageJ in five–10 independent microscopic fields for a total of at to the lowest degree 100 cells at each point. Each point represents the mean±due south.d. of all counts. ExpG, exponentially growing cells; Sen, cells at the senescence plateau. The exact PD at which cells were taken is indicated.

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