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ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke.

Tanaka T, Huang X, Jorgensen E, Gietl D, Traganos F, Darzynkiewicz Z, Albino AP - BMC Cell Biol. (2007)

Bottom Line: Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations.As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis.Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY 10595, USA. toshiki_tanaka@nymc.edu <toshiki_tanaka@nymc.edu>

ABSTRACT

Background: In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.

Results: We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (gammaH2AX) (R = 0.89). In addition, we note that while CS-induced gammaH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.

Conclusion: These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

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Effect of treatment with caffeine and NU7026 on ATM and H2AX phosphorylation. A549 cells were exposed to CS for 20 min either in the presence of PBS, 4 mM caffeine or 10 uM NU7026 or were collected in the absence of smoke treatment (mock exposure). Cells exposed to caffeine and NU7026 were also treated for one hour prior to exposure and one hour subsequent to cell harvest. Only treatment with caffeine lowered the level of expression of ATM-A1981P and γH2AX IF.
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Figure 6: Effect of treatment with caffeine and NU7026 on ATM and H2AX phosphorylation. A549 cells were exposed to CS for 20 min either in the presence of PBS, 4 mM caffeine or 10 uM NU7026 or were collected in the absence of smoke treatment (mock exposure). Cells exposed to caffeine and NU7026 were also treated for one hour prior to exposure and one hour subsequent to cell harvest. Only treatment with caffeine lowered the level of expression of ATM-A1981P and γH2AX IF.

Mentions: As can be seen in Figure 6, CS from IM16 induced a considerable level of γH2AX IF in A549 cells. The values in the figure represent the difference between the CS-treated sample and the mock-treated control (Δ). Inclusion of the vehicle DMSO had no appreciable effect on the level of γH2AX IF generated by CS from IM16. Caffeine, at a concentration of 4 mM, reduced the average level of γH2AX IF by approximately 37%. In parallel cultures on the same slide, ATM-S1981P IF was reduced by 45% in the presence of caffeine. Nu7026 had little or no inhibitory effect on γH2AX IF (reduced by 3%) while ATM-S1981P IF was actually increased slightly (Fig. 6). No cell cycle phase specificity associated with the caffeine-induced decrease in either ATM-S1981P or γH2AX IF was apparent.


ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke.

Tanaka T, Huang X, Jorgensen E, Gietl D, Traganos F, Darzynkiewicz Z, Albino AP - BMC Cell Biol. (2007)

Effect of treatment with caffeine and NU7026 on ATM and H2AX phosphorylation. A549 cells were exposed to CS for 20 min either in the presence of PBS, 4 mM caffeine or 10 uM NU7026 or were collected in the absence of smoke treatment (mock exposure). Cells exposed to caffeine and NU7026 were also treated for one hour prior to exposure and one hour subsequent to cell harvest. Only treatment with caffeine lowered the level of expression of ATM-A1981P and γH2AX IF.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919366&req=5

Figure 6: Effect of treatment with caffeine and NU7026 on ATM and H2AX phosphorylation. A549 cells were exposed to CS for 20 min either in the presence of PBS, 4 mM caffeine or 10 uM NU7026 or were collected in the absence of smoke treatment (mock exposure). Cells exposed to caffeine and NU7026 were also treated for one hour prior to exposure and one hour subsequent to cell harvest. Only treatment with caffeine lowered the level of expression of ATM-A1981P and γH2AX IF.
Mentions: As can be seen in Figure 6, CS from IM16 induced a considerable level of γH2AX IF in A549 cells. The values in the figure represent the difference between the CS-treated sample and the mock-treated control (Δ). Inclusion of the vehicle DMSO had no appreciable effect on the level of γH2AX IF generated by CS from IM16. Caffeine, at a concentration of 4 mM, reduced the average level of γH2AX IF by approximately 37%. In parallel cultures on the same slide, ATM-S1981P IF was reduced by 45% in the presence of caffeine. Nu7026 had little or no inhibitory effect on γH2AX IF (reduced by 3%) while ATM-S1981P IF was actually increased slightly (Fig. 6). No cell cycle phase specificity associated with the caffeine-induced decrease in either ATM-S1981P or γH2AX IF was apparent.

Bottom Line: Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations.As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis.Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, NY 10595, USA. toshiki_tanaka@nymc.edu <toshiki_tanaka@nymc.edu>

ABSTRACT

Background: In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.

Results: We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (gammaH2AX) (R = 0.89). In addition, we note that while CS-induced gammaH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.

Conclusion: These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

Show MeSH
Related in: MedlinePlus