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

<p>Abstract</p> <p>Background</p> <p>In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on <it>Ser</it>139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as <it>ataxia telangiectasia </it>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.</p> <p>Results</p> <p>We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on <it>Ser</it>1981 (ATM-S1981^P) 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-S1981^Pcorrelated well with the increase in expression of phosphorylated H2AX (γH2AX) (R = 0.89). In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.</p> <p>Conclusion</p> <p>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.</p

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells

<p>Abstract</p> <p>Background</p> <p>Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer.</p> <p>Methods</p> <p>Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.</p> <p>Results</p> <p>We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2α) or phosphorylation (i.e., phospho-eIF2α) in a majority of human lung cancers.</p> <p>Conclusion</p> <p>These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2α and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.</p

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Ir. Cells were then placed in fresh media with or without 1 uM thapsigargin or 10 μg/ml tunicamycin for the time periods specified, followed by cell lysis and RNA purification. Spliced and unspliced XBP1 mRNA was detected using PCR methodology. Spliced XBP1 is 398 base pairs and the unspliced variant is 434 base pairs. For each lane the extent of splicing was quantified as described in the Methods section and is presented in additional file – .<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells-9

Ith or without concurrent treatment with NAC or GSH (at 5 and 25 mM concentrations), after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α and GAPDH.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells-6

4F cigarette smoke (CS) for 10 or 20 minutes (35 cc puffs were diluted in 250 cc air), after which the cells were immediately returned to the incubator without a media change for the time periods specified. C = untreated control.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

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Reatment) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Nuclear extracts were prepared as described in the Methods section. Western blots of nuclear fractions were probed with antibodies to ATF4 and Lamin A/C, a nuclear antigen, as a loading control. NHBE cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (CS = smoke treatment) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to GADD34 and α-tubulin as a loading control. A549 cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF3 and GAPDH.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

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Reatment) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. C = untreated control. NHBE cells were treated with either 1 uM thapsigargin in DMSO (T), or DMSO (VC = vehicle control) for the times specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH. C = untreated control.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells-1

Tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

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M 2R4F cigarettes, after which the cells were placed in fresh medium and returned to the incubator for the time periods specified. Vapor phase was created by passing whole smoke through a Cambridge pad filter to trap the particulate matter. Particulate 'tar' phase was achieved by passing the whole smoke through 450 mg activated carbon to remove vapor phase components. Duration of whole smoke and vapor phase exposures was 20 minutes. Duration of particulate 'tar' phase exposures was 25 minutes in order to deliver an amount of particulate matter equivalent to that of the whole smoke exposure as some particulate matter is lost upon transit through the activated carbon. In all exposures the 35 cc puffs were diluted in 250 cc air. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH.<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p

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, then treated with 2 mM DTT for 1 h (lanes 3 and 4) or left untreated (lanes 1 and 2). Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. All lanes show lysates from A549 cells transfected with a plasmid expressing ATF6. Cells were exposed to air (M = mock treatment) or 2R4F cigarette smoke (CS = smoke treatment) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to ATF6 and α-tubulin. Gel bands were quantified as described in the Methods section. CS treatment resulted in a 23% and 26% decrease in ATF6 90 kDa protein at 2 and 4 h respectively (lane 5 compared to lane 6, lane 7 compared to lane 8).<p><b>Copyright information:</b></p><p>Taken from "Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells"</p><p>http://www.biomedcentral.com/1471-2407/8/229</p><p>BMC Cancer 2008;8():229-229.</p><p>Published online 11 Aug 2008</p><p>PMCID:PMC2527015.</p><p></p