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Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion.

Rodier F, Coppé JP, Patil CK, Hoeijmakers WA, Muñoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J - Nat. Cell Biol. (2009)

Bottom Line: Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs).ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence.Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

View Article: PubMed Central - PubMed

Affiliation: Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.

ABSTRACT
Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

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ATM modulates a SASP subset(a) Factors secreted by presenescent (PRE) or senescent HCA2 (10 d following 10 Gy) were analyzed using antibody arrays6. Signals above the PRE baseline are in yellow, signals below baseline in blue. Factors that differed significantly between PRE and XRA cells are displayed (p<0.05, student unpaired T-Test).(b) SASP factors in (A) were analyzed in X-irradiated ATM-depleted cells (XRA(shATM)) and compared to irradiated-senescent HCA2-shGFP cells (XRA). Changes are reported as XRA(shATM) divided by XRA levels. Signals below the XRA baseline are in blue, factors not substantially modulated by ATM-deficiency in gray.(c) T47D invasion stimulated by presenescent (PRE) HCA2 CM was given a value of 1. Compared to PRE and serum-free (SF) media, invasion was significantly (p<0.05) stimulated by 10% serum (FBS), and CM from irradiated HCA2 (XRA), HCA2-shGFP (XRA-shGFP), and HCA2-shATM plus IL-6 (XRA-shATM+IL-6), but not by CM from HCA2-shATM (XRA-shATM) (n=number of samples analyzed). Recombinant IL-6 was added to levels detected by ELISA in XRA-shGFP (3ng/ml).(d) Human breast tissues were analyzed for ATM/ATR serine threonine kinase substrates (p-STK Sub) or IL-6 by immunofluorescence. Data are reported as arbitrary units, with the average normal signal=1 (Signal Intensity). Significance was determined by two-tailed student T-test for unpaired samples using representative fields from 21 normal and 37 cancer tissues.(e) HCA2 cells were untreated or infected with lentiviruses expressing RAS and shRNAs against GFP (shGFP2) or ATM (shATM2), and pulsed with BrdU for 24 h 9−10 d after infection.(f) HCA2 or A-T cells were untreated or infected as in (e) and photographed 6 d later.(g) HCA2 cells infected as in (e) were fixed 10 d later and analyzed for 53BP1 (red), phospho-ATM (green) and DNA (DAPI, blue). Top panel=merge; bottom panels=grayscale for 53BP1 or p-ATM.(h) A-T fibroblasts were untreated or infected as in (e). CM was collected for 24 h 9−10 d after infection and analyzed for IL-6 (fold increase relative to untreated cells; magnitude is given over the bars).(i) HCA2 cells were untreated or infected as in (e) and IL-6 secretion analyzed as above.
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Figure 5: ATM modulates a SASP subset(a) Factors secreted by presenescent (PRE) or senescent HCA2 (10 d following 10 Gy) were analyzed using antibody arrays6. Signals above the PRE baseline are in yellow, signals below baseline in blue. Factors that differed significantly between PRE and XRA cells are displayed (p<0.05, student unpaired T-Test).(b) SASP factors in (A) were analyzed in X-irradiated ATM-depleted cells (XRA(shATM)) and compared to irradiated-senescent HCA2-shGFP cells (XRA). Changes are reported as XRA(shATM) divided by XRA levels. Signals below the XRA baseline are in blue, factors not substantially modulated by ATM-deficiency in gray.(c) T47D invasion stimulated by presenescent (PRE) HCA2 CM was given a value of 1. Compared to PRE and serum-free (SF) media, invasion was significantly (p<0.05) stimulated by 10% serum (FBS), and CM from irradiated HCA2 (XRA), HCA2-shGFP (XRA-shGFP), and HCA2-shATM plus IL-6 (XRA-shATM+IL-6), but not by CM from HCA2-shATM (XRA-shATM) (n=number of samples analyzed). Recombinant IL-6 was added to levels detected by ELISA in XRA-shGFP (3ng/ml).(d) Human breast tissues were analyzed for ATM/ATR serine threonine kinase substrates (p-STK Sub) or IL-6 by immunofluorescence. Data are reported as arbitrary units, with the average normal signal=1 (Signal Intensity). Significance was determined by two-tailed student T-test for unpaired samples using representative fields from 21 normal and 37 cancer tissues.(e) HCA2 cells were untreated or infected with lentiviruses expressing RAS and shRNAs against GFP (shGFP2) or ATM (shATM2), and pulsed with BrdU for 24 h 9−10 d after infection.(f) HCA2 or A-T cells were untreated or infected as in (e) and photographed 6 d later.(g) HCA2 cells infected as in (e) were fixed 10 d later and analyzed for 53BP1 (red), phospho-ATM (green) and DNA (DAPI, blue). Top panel=merge; bottom panels=grayscale for 53BP1 or p-ATM.(h) A-T fibroblasts were untreated or infected as in (e). CM was collected for 24 h 9−10 d after infection and analyzed for IL-6 (fold increase relative to untreated cells; magnitude is given over the bars).(i) HCA2 cells were untreated or infected as in (e) and IL-6 secretion analyzed as above.

Mentions: To identify which SASP components respond to DDR signaling, we used antibody arrays to interrogate 120 cytokines and other factors secreted by senescent HCA2 cells. We focused on 16 factors that were significantly modulated by X-irradiation, the majority being upregulated (Fig. 5a). We compared the secretion levels of these 16 factors in control and ATM-depleted cells induced to senesce by X-irradiation (10 Gy). ATM depletion reduced the secretion of 7 of these 16 SASP factors, reducing IL-6 secretion 50-fold and IL-8 secretion 10-fold. Nine factors were unchanged by ATM depletion (≤1.4-fold the secretion level of non-depleted cells) (Fig.5b). Thus, ATM signaling does not regulate the entire SASP, but is required for a subset of SASP components, including the major inflammatory cytokines.


Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion.

Rodier F, Coppé JP, Patil CK, Hoeijmakers WA, Muñoz DP, Raza SR, Freund A, Campeau E, Davalos AR, Campisi J - Nat. Cell Biol. (2009)

ATM modulates a SASP subset(a) Factors secreted by presenescent (PRE) or senescent HCA2 (10 d following 10 Gy) were analyzed using antibody arrays6. Signals above the PRE baseline are in yellow, signals below baseline in blue. Factors that differed significantly between PRE and XRA cells are displayed (p<0.05, student unpaired T-Test).(b) SASP factors in (A) were analyzed in X-irradiated ATM-depleted cells (XRA(shATM)) and compared to irradiated-senescent HCA2-shGFP cells (XRA). Changes are reported as XRA(shATM) divided by XRA levels. Signals below the XRA baseline are in blue, factors not substantially modulated by ATM-deficiency in gray.(c) T47D invasion stimulated by presenescent (PRE) HCA2 CM was given a value of 1. Compared to PRE and serum-free (SF) media, invasion was significantly (p<0.05) stimulated by 10% serum (FBS), and CM from irradiated HCA2 (XRA), HCA2-shGFP (XRA-shGFP), and HCA2-shATM plus IL-6 (XRA-shATM+IL-6), but not by CM from HCA2-shATM (XRA-shATM) (n=number of samples analyzed). Recombinant IL-6 was added to levels detected by ELISA in XRA-shGFP (3ng/ml).(d) Human breast tissues were analyzed for ATM/ATR serine threonine kinase substrates (p-STK Sub) or IL-6 by immunofluorescence. Data are reported as arbitrary units, with the average normal signal=1 (Signal Intensity). Significance was determined by two-tailed student T-test for unpaired samples using representative fields from 21 normal and 37 cancer tissues.(e) HCA2 cells were untreated or infected with lentiviruses expressing RAS and shRNAs against GFP (shGFP2) or ATM (shATM2), and pulsed with BrdU for 24 h 9−10 d after infection.(f) HCA2 or A-T cells were untreated or infected as in (e) and photographed 6 d later.(g) HCA2 cells infected as in (e) were fixed 10 d later and analyzed for 53BP1 (red), phospho-ATM (green) and DNA (DAPI, blue). Top panel=merge; bottom panels=grayscale for 53BP1 or p-ATM.(h) A-T fibroblasts were untreated or infected as in (e). CM was collected for 24 h 9−10 d after infection and analyzed for IL-6 (fold increase relative to untreated cells; magnitude is given over the bars).(i) HCA2 cells were untreated or infected as in (e) and IL-6 secretion analyzed as above.
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Figure 5: ATM modulates a SASP subset(a) Factors secreted by presenescent (PRE) or senescent HCA2 (10 d following 10 Gy) were analyzed using antibody arrays6. Signals above the PRE baseline are in yellow, signals below baseline in blue. Factors that differed significantly between PRE and XRA cells are displayed (p<0.05, student unpaired T-Test).(b) SASP factors in (A) were analyzed in X-irradiated ATM-depleted cells (XRA(shATM)) and compared to irradiated-senescent HCA2-shGFP cells (XRA). Changes are reported as XRA(shATM) divided by XRA levels. Signals below the XRA baseline are in blue, factors not substantially modulated by ATM-deficiency in gray.(c) T47D invasion stimulated by presenescent (PRE) HCA2 CM was given a value of 1. Compared to PRE and serum-free (SF) media, invasion was significantly (p<0.05) stimulated by 10% serum (FBS), and CM from irradiated HCA2 (XRA), HCA2-shGFP (XRA-shGFP), and HCA2-shATM plus IL-6 (XRA-shATM+IL-6), but not by CM from HCA2-shATM (XRA-shATM) (n=number of samples analyzed). Recombinant IL-6 was added to levels detected by ELISA in XRA-shGFP (3ng/ml).(d) Human breast tissues were analyzed for ATM/ATR serine threonine kinase substrates (p-STK Sub) or IL-6 by immunofluorescence. Data are reported as arbitrary units, with the average normal signal=1 (Signal Intensity). Significance was determined by two-tailed student T-test for unpaired samples using representative fields from 21 normal and 37 cancer tissues.(e) HCA2 cells were untreated or infected with lentiviruses expressing RAS and shRNAs against GFP (shGFP2) or ATM (shATM2), and pulsed with BrdU for 24 h 9−10 d after infection.(f) HCA2 or A-T cells were untreated or infected as in (e) and photographed 6 d later.(g) HCA2 cells infected as in (e) were fixed 10 d later and analyzed for 53BP1 (red), phospho-ATM (green) and DNA (DAPI, blue). Top panel=merge; bottom panels=grayscale for 53BP1 or p-ATM.(h) A-T fibroblasts were untreated or infected as in (e). CM was collected for 24 h 9−10 d after infection and analyzed for IL-6 (fold increase relative to untreated cells; magnitude is given over the bars).(i) HCA2 cells were untreated or infected as in (e) and IL-6 secretion analyzed as above.
Mentions: To identify which SASP components respond to DDR signaling, we used antibody arrays to interrogate 120 cytokines and other factors secreted by senescent HCA2 cells. We focused on 16 factors that were significantly modulated by X-irradiation, the majority being upregulated (Fig. 5a). We compared the secretion levels of these 16 factors in control and ATM-depleted cells induced to senesce by X-irradiation (10 Gy). ATM depletion reduced the secretion of 7 of these 16 SASP factors, reducing IL-6 secretion 50-fold and IL-8 secretion 10-fold. Nine factors were unchanged by ATM depletion (≤1.4-fold the secretion level of non-depleted cells) (Fig.5b). Thus, ATM signaling does not regulate the entire SASP, but is required for a subset of SASP components, including the major inflammatory cytokines.

Bottom Line: Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs).ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence.Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

View Article: PubMed Central - PubMed

Affiliation: Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA.

ABSTRACT
Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.

Show MeSH
Related in: MedlinePlus