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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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Induction of ATM activation and H2AX phosphorylation in A549 cells following their exposure to IM16 CS. Bivariate distributions (scattergrams; DNA content, DI vs. ATM-S1981P IF, or vs. γH2AX IF, respectively) of the A549 cells, mock treated (Ctrl) or exposed 20 min to CS (CS), as described in the Materials and Methods. After exposure to CS or mock treatment the cells were incubated for an additional hour, then fixed and subjected to immunocytochemical detection of ATM-S1981P or γH2AX, their DNA counterstained with DAPI and the intensity of IF- and DAPI-fluorescence measured by iCys. The inset shows the cellular DNA content histogram representing cells from these cultures. Based on differences in DI the cells can be subdivided into G1, S and G2M compartments, as shown in the left panel. Mitotic (M) A549 cells constitutively express higher level of ATM-S1981P and γH2AX [14,38,70,71] and their position on the scattergrams is marked within dashed oval boundaries. The dashed line represents the upper threshold of ATM-S1981Por γH2AX IF level for 97% of interphase cells.
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Figure 1: Induction of ATM activation and H2AX phosphorylation in A549 cells following their exposure to IM16 CS. Bivariate distributions (scattergrams; DNA content, DI vs. ATM-S1981P IF, or vs. γH2AX IF, respectively) of the A549 cells, mock treated (Ctrl) or exposed 20 min to CS (CS), as described in the Materials and Methods. After exposure to CS or mock treatment the cells were incubated for an additional hour, then fixed and subjected to immunocytochemical detection of ATM-S1981P or γH2AX, their DNA counterstained with DAPI and the intensity of IF- and DAPI-fluorescence measured by iCys. The inset shows the cellular DNA content histogram representing cells from these cultures. Based on differences in DI the cells can be subdivided into G1, S and G2M compartments, as shown in the left panel. Mitotic (M) A549 cells constitutively express higher level of ATM-S1981P and γH2AX [14,38,70,71] and their position on the scattergrams is marked within dashed oval boundaries. The dashed line represents the upper threshold of ATM-S1981Por γH2AX IF level for 97% of interphase cells.

Mentions: Exposure of A549 cells to CS led to a marked rise in expression of ATM-S1981P and γH2AX (Fig. 1). The bivariate analysis of cellular DNA content and intensity of immunofluorescence made it possible to correlate expression of ATM-S1981P or γH2AX with cell position in the cell cycle. Expression of ATM-S1981P and γH2AX was markedly increased in cells exposed to CS compared to the mock treated cells. In fact, nearly all cells from the CS-treated cultures expressed ATM-S1981P or γH2AX IF above the level representing the upper threshold of expression of these phospho-antibodies for 97% of the cells from the mock-treated cultures. It is also apparent from the raw data shown in the scattergrams (Fig. 1), that exposure to CS led to induction of ATM-S1981P or γH2AX expression at a comparable level in all phases of the cell cycle.


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)

Induction of ATM activation and H2AX phosphorylation in A549 cells following their exposure to IM16 CS. Bivariate distributions (scattergrams; DNA content, DI vs. ATM-S1981P IF, or vs. γH2AX IF, respectively) of the A549 cells, mock treated (Ctrl) or exposed 20 min to CS (CS), as described in the Materials and Methods. After exposure to CS or mock treatment the cells were incubated for an additional hour, then fixed and subjected to immunocytochemical detection of ATM-S1981P or γH2AX, their DNA counterstained with DAPI and the intensity of IF- and DAPI-fluorescence measured by iCys. The inset shows the cellular DNA content histogram representing cells from these cultures. Based on differences in DI the cells can be subdivided into G1, S and G2M compartments, as shown in the left panel. Mitotic (M) A549 cells constitutively express higher level of ATM-S1981P and γH2AX [14,38,70,71] and their position on the scattergrams is marked within dashed oval boundaries. The dashed line represents the upper threshold of ATM-S1981Por γH2AX IF level for 97% of interphase cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Induction of ATM activation and H2AX phosphorylation in A549 cells following their exposure to IM16 CS. Bivariate distributions (scattergrams; DNA content, DI vs. ATM-S1981P IF, or vs. γH2AX IF, respectively) of the A549 cells, mock treated (Ctrl) or exposed 20 min to CS (CS), as described in the Materials and Methods. After exposure to CS or mock treatment the cells were incubated for an additional hour, then fixed and subjected to immunocytochemical detection of ATM-S1981P or γH2AX, their DNA counterstained with DAPI and the intensity of IF- and DAPI-fluorescence measured by iCys. The inset shows the cellular DNA content histogram representing cells from these cultures. Based on differences in DI the cells can be subdivided into G1, S and G2M compartments, as shown in the left panel. Mitotic (M) A549 cells constitutively express higher level of ATM-S1981P and γH2AX [14,38,70,71] and their position on the scattergrams is marked within dashed oval boundaries. The dashed line represents the upper threshold of ATM-S1981Por γH2AX IF level for 97% of interphase cells.
Mentions: Exposure of A549 cells to CS led to a marked rise in expression of ATM-S1981P and γH2AX (Fig. 1). The bivariate analysis of cellular DNA content and intensity of immunofluorescence made it possible to correlate expression of ATM-S1981P or γH2AX with cell position in the cell cycle. Expression of ATM-S1981P and γH2AX was markedly increased in cells exposed to CS compared to the mock treated cells. In fact, nearly all cells from the CS-treated cultures expressed ATM-S1981P or γH2AX IF above the level representing the upper threshold of expression of these phospho-antibodies for 97% of the cells from the mock-treated cultures. It is also apparent from the raw data shown in the scattergrams (Fig. 1), that exposure to CS led to induction of ATM-S1981P or γH2AX expression at a comparable level in all phases of the cell cycle.

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