university of groningen gene-environment interactions in ... · pdf filecigarettes (lexington,...

University of Groningen

Gene-environment interactions in Inflammatory Bowel DiseaseRegeling, Anouk

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2014

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Regeling, A. (2014). Gene-environment interactions in Inflammatory Bowel Disease: Emphasis on smokingand autophagy ([S.l.] ed.) [S.n.]

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 11-05-2018

https://www.rug.nl/research/portal/en/publications/geneenvironment-interactions-in-inflammatory-bowel-disease(9a90ab07-00cb-4466-84fa-7360ef6f5dce).html

Chapter 3

ciGarette smoke enhances resistance to apoptosis; relevance for its opposite

effects in inflammatory Bowel Diseases

Anouk Regeling1*, Elise M.J. van der Logt1*, Tjasso Blokzijl2, Dirk Jan Slebos3, Maikel P. Peppelenbosch4,

Klaas Nico Faber1# and Gerard Dijkstra1#

1University of Groningen, University Medical Center Groningen, Department of Gastroenterology and Hepatology, Groningen, The Netherlands,

2University of Groningen, University Medical Center Groningen, Department of Laboratory Medicine, Groningen, The Netherlands,

3University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases, Groningen, The Netherlands,

4Erasmus MC, Erasmus University of Rotterdam, Department of Gastroenterology and Hepatology, Rotterdam, The Netherlands

*Both authors contributed equally to this work#Both last authors contributed equally to this work

Submitted

proefschrift Anouk Regeling.indb 47 02-09-14 20:21

48

CHAPTER 3

chapter

3chapter

3

ABSTRACT

BACKGROUND & AIMS

Cigarette smoking is associated with infl ammatory bowel diseases (IBD), but with a remarkable opposite eff ect in Crohn’s disease (CD) and ulcerative colitis (UC). Smoking aggravates CD and ameliorates UC. Here, we investigated the smoke-induced signaling pathways in order to explain this dichotomy.

METHODS

Intestinal epithelial cells (DLD-1) and T-cells (Jurkat) were exposed to cigarette smoke extract (CSE) and cellular changes in signaling pathways were identifi ed using kinome profi ling. Cells were exposed to infl ammatory triggers (anti-Fas, cytokines, menadione) in presence or absence of CSE, nicotine, acrolein or carbon monoxide-releasing molecules (CORMs). Apoptosis was quantifi ed by caspase-3 assays and PARP cleavage. Contribution of apoptotic pathways was studied using specifi c inhibitors. Necrosis and cell viability were determined by Sytox Green nuclear staining and WST-1 assays, respectively.

RESULTS

Kinome analyses revealed that the canonical apoptotic pathway was inhibited by CSE, while multiple survival pathways were stimulated simultaneously, including AMPK, PI3K and ERK1/2. CSE dose-dependently (from 0-20% CSE) suppressed caspase-3 activity provoked by anti-Fas, cytokine mixture or menadione in both DLD-1 and Jurkat cells with a concomitant increase in cellular metabolic activity. CSE concentrations above 30% induced necrosis. Inhibition of individual CSE-activated anti-apoptotic signaling partially reversed CSE eff ects on survival. All tested cigarette smoke compounds showed anti-apoptotic eff ects, with CORMS being the most potent.

CONCLUSION

Smoke components exert multiple anti-apoptotic eff ects in epithelial cells and T-lymphocytes, which may aggravate CD that is characterized by apoptosis-resistant T-cells. Conversely, it may prevent excessive damage to the colonic epithelium and thereby ameliorate UC.


49

SMOKING INHIBITS APOPTOSIS IN IBD

chapter

3chapter

3

INTRODUCTION

Crohn’s disease (CD) and ulcerative colitis (UC) are the two major types of infl ammatory bowel disease (IBD). CD is characterized by transmural discontinuous infl ammation with granuloma and may aff ect the whole gastrointestinal tract. UC is characterized by a superfi cial continuous infl ammation of the mucosa of the colon ascending from the rectum1. Both CD and UC develop as a consequence of a complex interplay between genetic and environmental factors. Recent genome-wide association studies have provided a comprehensive map of genes that are linked to the propensity to these diseases2. Though instrumental for defi ning the cellular processes that may promote CD or UC, genetic predisposition only explains about 20% of CD and <10% of UC disease development2,3. This implies that IBD is primarily caused by environmental factors. The most prominent environmental factor that is implicated in the development of IBD is cigarette smoke and was fi rst documented in the early 1980s, with smokers being overrepresented in CD and underrepresented in UC compared to the healthy control population4,5. Since then, many studies have confi rmed that smoking aggravates CD and ameliorates UC. Smoking is associated with a higher prevalence of ileal disease in CD, more complex disease, increased need for steroids and immunosuppressive therapy as well as poorer response to therapy with infl iximab6,7. In contrast, smoking is associated with less hospitalizations in UC and decreased need for steroids and surgery7,8. Recently, we found that also passive smoking is detrimental for CD patients and that active smoking showed dose-dependent benefi cial eff ects in UC8.How smoking modulates disease course and therapeutic outcome in IBD is still unclear. Several potential mechanisms, including smoke-provoked alterations in mucosal immune responses, modifi cations in intestinal mediators and cytokines and modulation of gut permeability have been proposed6,9, but none of these off ers a satisfying explanation for the dichotomal eff ects of smoking on CD and UC. The intestinal tract is composed of several barriers to prevent pathogenic invasion. However, bacteria and dietary antigens constantly pass the epithelium leading to a state of controlled infl ammation. Disruption of the barrier allows luminal pathogens to pass the epithelium and ongoing exposure of pathogens triggers the immune system and causes infl ammation of the colon leading to epithelial damage. To limit infl ammation to a subclinical state, a delicate balance between continuous cell replacement and repair of the epithelium is required. Regulatory mechanisms are essential for eliminating damaged cells during tissue remodeling and infl ammation, processes that appear to be modulated by cigarette smoke with opposite outcomes


50

CHAPTER 3

chapter

3chapter

3

in CD and UC. Therefore, we set out to identify the regulatory mechanisms provoked by cigarette smoke and studied their putative role in the dichotomal eff ects on the two main forms of IBD. Defi ning the cigarette smoke-induced cellular (dys)functions will help to understand disease progression in smoking and non-smoking patients with CD or UC and may unveil novel therapeutic targets for these diseases.

MATERIALS AND METHODS

CELL LINES AND CULTURE CONDITIONS

Human colonic epithelial cell lines (DLD-1, SW480, HCT116, Caco-2) and T-lymphocyte cell lines (Jurkat, MOLT-4) were cultured in T75 fl asks (Greiner bio-one, Alphen aan den Rijn, The Netherlands) as described previously10. Cells were exposed to various apoptotic stimuli in presence or absence of diff erent concentrations cigarette smoke extract (CSE; 0-100% v/v) as specifi ed in the result section. CSE was prepared as described11 by passing smoke from 2 standard 3R4F research cigarettes (Lexington, KY, USA) through 25 ml culture medium, which was defi ned as 100% CSE. Death receptor-mediated apoptosis was induced by anti-Fas (1 µg/ml)12 or cytokine mixture (CM; consisting of TNF-α, IFN-γ and IL-1β, 10 ng/ml each)10. Oxidative stress-mediated apoptosis was induced by the superoxide anion donor menadione (50 mmol/l)13. Anti-Fas-induced caspase-3 activity is maximal at 9 and 3 hours in DLD-1 and Jurkat cells, respectively12. For CM- and menadione-induced apoptosis, caspase-3 activity peaks at 16 hours in DLD-1 cells. Therefore, these time-points were chosen to study the eff ect of CSE. In addition, cells were exposed to a water-soluble fraction of CSE, which was obtained by a “water pipe” setting. Smoke from 2 cigarettes was fi rst passed through 25 ml HBSS-buff er (= 100% water pipe medium) and subsequently led through 25 ml culture medium (= 100% CSE after water pipe medium). Furthermore, cells were exposed to various pure compounds of cigarette smoke, e.g. nicotine, acrolein and carbon monoxide (CO), to study their eff ect on anti-Fas-, CM-, or menadione-induced apoptosis. Nicotine (#N5260; Sigma-Aldrich, MO, USA) was added at concentrations 0.1-10 µM. Acrolein (#A1679; Sigma) was used at concentrations of 25-100 µM. CO was either diff used through 25 ml medium, similar as preparing CSE, or by adding CO-releasing molecule-2 (CORM-2, tricarbonyldichlororuthenium II dimer, #288144; Sigma) at concentrations of 50-200 µM. In parallel, cells were incubated with inactivated (incubated at 37°C for 24 hour and saturated with nitrogen for 1 hour just before use) CORM-2.


51


chapter

3chapter

3

KINOME PROFILING AND DATA ACQUISITION

The peptide array data analysis was done as described earlier14. Spot density and background from three technical replicates of each individual experimental sample were analyzed using Scanalyze (http://rana.lbl.gov/EisenSoftware). For each peptide the average and standard deviation of phosphorylation was determined and plotted in an amplitude-based hierarchical fashion. Background phosphorylation of the array was determined from the exponent describing the amplitude behavior of the 500 least phosphorylated peptides (which are assumed not to contain phosphorylation derived from a relevant biological signal). Peptides of which the average phosphorylation minus 1.96 times the standard deviation was higher than background were considered to represent true phosphorylation events (P<0.05). Peptide sets that showed statistically signifi cant changes compared to the background were subsequently subjected to elective Markov analysis and the resulting binary values were also analyzed in cohorts of peptides representing a read out for the same signal transduction element, collapsing results towards human interpretable format. Signifi cance analysis was performed using a minimal modifi cation for the algorithm originally developed for microarray analysis (www-stat.stanford.edu/~tibs/SAM/) and was essentially done as described earlier15.

PROTEIN EXTRACTION AND CONCENTRATION DETERMINATION

Cells were harvested in lysis buff er (25 mmol/l HEPES, 2 mmol/l EDTA, 150 mmol/l KAc, 0.1% NP40, 10 mmol/l NaF, 1 mmol/l PMSF, 1 mmol/l DTT, aprotinine, leupeptide and prostatine (each 1 µg/ml) pH 7.2) followed by four cycles of freezing (liquid nitrogen) and thawing (37°C). Lysates were centrifuged (10 min, 12.500xg, RT) and supernatant was stored at -80°C until use. Protein concentrations were determined using the BioRad DC protein assay (BioRad, CA, USA). Absorbance was measured at 750 nm on an EL800 universal microplate reader (Bio-Tek instruments, VT, USA).

WESTERN BLOTTING

Total protein extracts (20 µg) from control and treated cells were separated on 10% sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gels and transferred to nitrocellulose membrane (GE Healthcare Europe, Diegem, Belgium). After blocking with 5% skim milk, membranes were probed with primary antibodies (Supplementary table S1). The target proteins were visualized using an enhanced chemiluminescence SuperSignal West Dura detection system (#34076; Pierce, IL, USA). GAPDH (#CB1001; Calbiochem, CA, USA) was used as loading control.


52

CHAPTER 3

chapter

3chapter

3

QUANTIFICATION OF APOPTOTIC AND NECROTIC CELL DEATH Caspase-3 activity in 20 µg total cellular protein was measured as described previously16 using an FL600 microplate fl uorescence reader (Bio-Tek instruments) with excitation and emission set at 380 and 460 nm, respectively. To detect necrotic cell death (leaky plasma membrane), cells were incubated for 15 min with sytox green nucleic acid stain (Molecular Probes, OR, USA), as previously described13.

METABOLIC ACTIVITY

Metabolic activity of cells was determined using WST-1 reagent following the instructions of the manufacturer (Roche, CA, USA). Cells exposed to 5 mmol/l H2O2 were used as a control for complete suppression of metabolic activity.

INHIBITION OF PHOSPHORYLATION

To determine the contribution of the survival/proliferation PI3K/Akt and/or autophagy/AMPK pathways to CSE-mediated protection of DLD-1 and Jurkat cells to apoptosis, we used the PI3K inhibitor LY294002 (50 µM for DLD-1, 25 µM for Jurkat), the ERK1/2 inhibitor U01260 (10 µM) and the AMPK inhibitor 5-iodotubercidin (0.1 µM) in the absence or presence of CSE and/or anti-Fas. Cells were pre-treated with the inhibitors for 30 min.

STATISTICAL ANALYSIS

Data are expressed as the means ± SD of at least 3 independent experiments that were each performed in duplicate. One way analysis of variance (ANOVA) in combination with a Bonferroni correction was used to determine the statistical signifi cance of the diff erences between groups. A P value of <0.05 was considered statistically signifi cant.

RESULTS

DISSECTION OF THE MOLECULAR PATHWAYS MODULATED BY CIGARETTE SMOKE

First, we determined the eff ect of various concentrations cigarette smoke-saturated medium (e.g. cigarette smoke extract; CSE) on cell viability and the metabolic activity of DLD-1 cells (model for human intestinal epithelial cells) and Jurkat cells (model for human T-lymphocytes) (Figure 1). Concentrations up to 20% CSE did not cause signifi cant levels of necrosis in either cell type (Figure 1A). In fact, these conditions signifi cantly enhanced the metabolic activity of both cell types compared to untreated control cells (Figure 1B and C, for DLD-1 and Jurkat, respectively). High


53


chapter

3chapter

3

concentrations of 50 and 100% CSE strongly reduced the metabolic activity of DLD-1 cells and Jurkat cells and causes massive necrosis.

DISSECTION OF THE CELL SIGNALING PATHWAYS MODULATED BY CIGARETTE SMOKE

To identify possible molecular signaling pathways that are modulated by CSE, we resorted to kinome profi ling, an unbiased approach allowing the generation of comprehensive description of cellular signaling without the need of a priori assumptions as to the pathways aff ected by smoke. DLD-1 and Jurkat cells incubated with or without 20% CSE for 30 min were used for this analysis. The data were subjected to Markov analysis and the results reveal the complement of kinase changes upon CSE challenge. For 20% CSE 141 DLD-1 and 184 Jurkat phosphorylated substrates

0 5 10 20 50 % CSE

DLD-1

Jurkat

A

B C

Figure 1. Dose-dependent eff ect of CSE on cell viability and metabolic activity in DLD-1 and Jurkat cells. DLD-1 and Jurkat cells were exposed to increasing concentrations CSE for 16 or 3 hours, respectively. (A) Necrotic cell death was evaluated by Sytox green nuclear staining. Fluorescent images were taken at a magnifi cation of 40x. (B) The metabolic activity was quantifi ed using the WST-1 assay. 5 mmol/l H2O2 was used as positive control for inducing necrosis and full suppression of metabolic activity. Data represent means ± SD of at least three independent experiments. *P<0.05 vs. control of corresponding bars.


54

CHAPTER 3

chapter

3chapter

3

were detected (Supplementary Table S2A and B). A schematic representation of all diff erentially phosphorylated peptides can be found in Supplementary Figure S1. Overall, analyses revealed that CSE activates signals associated with inhibition of ligand-induced apoptosis (Figure 2), while cell proliferation pathways and autophagy were induced (Supplementary Figure S1). Individual proteins were analyzed by Western blotting and in most cases confi rmed the kinome analysis (Figure 3A and B, for DLD-1 and Jurkat cells, respectively), with some exceptions described below. Exposure of DLD-1 cells to 20% CSE increased the phosphorylation of STAT5, ERK1/2, Bad and protein levels of Bcl-XL (anti-apoptotic/pro-survival); increased phosphorylation of Akt, GSK3β and protein levels of cyclin D1 (pro-proliferation); and increased phosphorylation of AMPK and lipidation of LC3 (anti-apoptotic/pro-

Figure 2. Provisional signal transduction schemes - impression of kinome profi ling results. Paired cultures of DLD-1 cells and Jurkat cells were challenged with vehicle or 20% CSE followed analysis for kinase activity using peptide arrays. Results were collapsed on selective signal transduction categories and expressed in gray scale (representing the number of peptides phosphorylated as a fraction of all peptides in that category). Vehicle and CSE eff ects were contrasted. The results provide an overview of anti-apoptosis and pro-survival pathways active in DLD-1 cells and Jurkat cells and the eff ects of CSE thereon.


55


chapter

3chapter

3

CSE 0% 20% kD

60

90

44/42

23

30

60

46

36

62

16/14

37

p-Src

p-STAT5

p-ERK1/2

p-Bad

Bcl-XL

p-Akt

p-GSK3β

Cyclin D1

p-AMPK

LC3-I/II

GAPDH

60

90

44/42

23

30

60

46

36

62

16/14

37

p-Src

p-STAT5

p-ERK1/2

p-Bad

Bcl-XL

p-Akt

p-GSK3β

Cyclin D1

p-AMPK

LC3-I/II

GAPDH

CSE 0% 20% kD

BA

Figure 3. Cell signaling pathways activated by CSE. DLD-1 and Jurkat cells were exposed for 30 min to 0% and 20% CSE and expression of phosphorylated forms of Src, STAT5, ERK1/2, Bad, Akt, GSK3β and AMPK, and protein expression of Bcl-XL, cyclin D1 and LC3 in DLD-1 (A) and Jurkat (B) cells was analyzed by Western blotting. GAPDH was used as loading control.

autophagy) (Figure 3A). The CSE-induced phosphorylation of AMPK in DLD-1 was not detected in the kinome analysis, possibly by low basal levels in control cells and/or the fact that only 1 AMPK target peptide was phosphorylated in the kinome array in those cells. In Jurkat cells, 20% CSE decreased phosphorylation of ERK1/2, but increased protein levels of Bcl-XL (anti-apoptotic/pro-survival); increased phosphorylation of Akt, GSK3β and protein levels of cyclin D1 (pro-proliferation); and increased phosphorylation of AMPK (anti-apoptotic/pro-autophagy) (Figure 3B). Together, these data provide novel data on the eff ects of cigarette smoke in two diff erent in vitro models with relevance for IBD and demonstrate that the main action of cigarette smoke is activation of anti-apoptotic and pro-survival pathways.

SMOKE EXERTS CYTOPROTECTIVE EFFECTS UPON PRO-APOPTOTIC STIMULATION

Next, we analyzed whether the CSE-induced anti-apoptotic/pro-survival pathways translate into functional consequences with respect to protection against pro-apoptotic triggers, including anti-Fas, CM and menadione. Anti-Fas-induced caspase-3 activity was dose-dependently inhibited by CSE (10-20%) in both DLD-


56

CHAPTER 3

chapter

3chapter

3

1 (Figure 4A) and Jurkat cells (Figure 4B). Similarly, CM- and menadione-induced caspase-3 activity was dose-dependently inhibited by CSE (5-20%) in DLD-1 cells (Figure 4A). Jurkat cells were not sensitive for CM- and menadione-induced apoptosis. In line with the reduction in caspase-3 activity, CSE also reduced the level of PARP cleavage (marker for late apoptosis) induced by anti-Fas, CM or menadione in DLD-1 and Jurkat cells (only for anti-Fas for the latter) (Figure 4C and D). The CSE inhibitory eff ects were most pronounced for menadione-induced apoptosis. CSE alone did not induce caspase-3 activity. Similar results were obtained when these experiments were performed with other intestinal (HCT116, SW480, Caco-2) or lymphocyte (MOLT-4) cell lines (Supplementary Figure S2 A-D).

DISSECTION OF THE RELATIVE IMPORTANCE OF CSE-INDUCED SIGNALING PATHWAYS IN CYTOPROTECTION Several pro-apoptotic pathways are simultaneously suppressed by CSE. In order to determine which pathways contribute to the anti-apoptotic eff ect of CSE, we blocked the AMPK pathway (by 5’-iodotubercidin), the PI3K survival pathway (by LY294002)

A B

C (c-)PARP 116 kDa 89 kDa

GAPDH 37 kDa

% CSE Con 0 5 10 20 - + + + + anti-Fas

0.9 14.1 9.7 6.1 2.1 cleaved uncleaved

D (c-)PARP 116 kDa 89 kDa

GAPDH 37 kDa

% CSE Con 0 5 10 20 - + + + + anti-Fas

0.1 13.5 6.2 5.6 1.0 cleaved uncleaved

Figure 4. CSE inhibits death receptor- and oxidative stress-induced apoptosis in DLD-1 and Jurkats cells. (A) Caspase-3 activity in total extracts of DLD-1 cells exposed to anti-Fas, CM or menadione in combination with increasing concentrations CSE. (B) Caspase-3 activity in total extracts of Jurkat cells exposed to anti-Fas in combination with increasing concentrations CSE. Maximal caspase-3 activity is presented as 100%. Data represent means ± SD of at least three independent experiments. *P<0.05 vs. control. (C,D) Western blot analysis of PARP showing that CSE inhibits anti-Fas-induced cleavage to the 89 kDa form in DLD-1 (C) and Jurkat (D) cells. GAPDH was used as loading control.


57


chapter

3chapter

3A B

C

Figure 5. The anti-apoptotic eff ect of CSE is not solely dependent of the AMPK, PI3K or ERK1/2 pathway. DLD-1 and Jurkat cells were exposed to anti-Fas and CSE with or without (A) the AMPK inhibitor 5’-iodotubercidin, (B) the PI3-kinase inhibitor LY294002 (C) the ERK1/2 inhibitor U01260. Caspase-3 activity was presented as fold-change compared to control; control values were set at one. Data represent means ± SD of at least three independent experiments. *P<0.05 vs. anti-Fas treated cells, #P<0.05 vs. cells co-treated with anti-Fas and inhibitor, $P<0.05 vs. anti-Fas/CSE co-treated cells.

or the ERK1/2 pathway (by U01260) in anti-Fas/CSE co-treated DLD-1 and Jurkat cells (Figure 5). The CSE-induced reduction in caspase-3 activity in anti-Fas-treated Jurkat cells was signifi cantly reversed by the AMPK-inhibitor (Figure 5A). A similar trend was observed for DLD-1 cells, but was not statistically signifi cant. Inhibition of the PI3K and ERK1/2 pathway signifi cantly increased the caspase-3 activity in anti-Fas/CSE co-treated cells (Figure 5B and C), however, this was also observed in anti-Fas treated cells. This implies that the PI3K or ERK1/2 pathways are already activated by anti-Fas and a potential additive eff ect of CSE cannot with certainty be deduced from these inhibitor experiments (Figure 5B and C). Taken together, these data show that the anti-apoptotic eff ect of CSE is mediated through multiple pathways and that inhibition of the AMPK pathway may provide a degree of specifi city between the epithelial compartment and T-cell compartment respectively. The putative


58

CHAPTER 3

chapter

3chapter

3

A B

C D

E

Figure 6. Nicotine, CO and acrolein contribute to the anti-apoptotic eff ects of cigarette smoke. Caspase-3 activity in DLD-1 cells after co-treatment with anti-Fas, CM or menadione and increasing concentrations of (A) the water-soluble fraction of CSE, (B) Nicotine (0-10 µM), (C) 100% CO-saturated medium, (D) CORM-2 or (E) acrolein (0-100 µM). Data represent means ± SD of at least three independent experiments. *P<0.05 vs. control, #P<0.05 vs. CORM-treated control bars.

involvement of the ERK1/2 or the PI3K pathway in the CSE-mediated anti-apoptotic eff ect cannot be established, since those pathways are already activated by anti-Fas.

EFFECTS OF SMOKE CONSTITUENTS

Finally, we aimed to determine which compounds in cigarette smoke contribute to the anti-apoptotic eff ect of CSE. In a setup that can be compared to a “water pipe”, we fi rst passed the smoke of 2 research cigarettes through 25 ml of water, after which it was passed through 25 ml medium. The anti-apoptotic eff ect completely shifted to the “water-trap” indicating that the protective compound(s) were effi ciently and


59


chapter

3chapter

3

completely solubilized in water (Figure 6A). Cigarette smoke compounds that are highly water-soluble are nicotine and acrolein, but may also include gases like CO. These compounds were further evaluated for their anti-apoptotic properties on anti-Fas-treated DLD-1 cells. Nicotine decreased anti-Fas induced caspase-3 activity with 31 ± 9.9% (10 µM; Figure 6B). Incubation with CO-saturated medium yielded a reduction of 46 ± 2.5% (Figure 6C). CM- and menadione-induced apoptosis were also inhibited by CO-saturated medium. CORM2 and acrolein dose-dependently decreased caspase-3 activity up to 59 ± 1.9% (200 µM; Figure 6D) and 69 ± 3.5% (100 µM; Figure 6E), respectively. Tested concentrations had no eff ect on the metabolic activity of DLD-1 cells, nor did it induced necrosis (data not shown). We conclude that cigarette smoke contains multiple water-soluble components exhibiting a cytoprotective action on intestinal epithelial cells and T-lymphocytes, possibly explaining the multitude of cytoprotective pathways induced.

DISCUSSION

In this study, we show that cigarette smoke induces phosphorylation of kinases that are involved in anti-apoptotic (STAT5B/ERK1/2) and pro-survival pathways (AMPK/Akt/PI3K) in intestinal epithelial cells and T-lymphocytes. This was accompanied by enhanced levels of anti-apoptotic Bcl-XL/Bcl-2 proteins, pro-survival proteins GSK3β, cyclin D1 and lipidation of the autophagy marker LC3. Cigarette smoke enhanced the metabolic activity of intestinal epithelial cells and T-lymphocytes and suppressed anti-Fas-, cytokine- and oxidative stress-induced apoptosis. The smoke components nicotine, CO and acrolein all suppressed apoptosis to variable degrees and inhibition of single pro-survival kinase pathways did not block the protective eff ect of cigarette smoke. Our data show that cigarette smoke induces a plethora of pro-survival pathways and protects intestinal epithelial cells and T-lymphocytes against infl ammation-induced apoptosis. Cigarette smoke may aggravate CD by enhancing the resistance of T-lymphocytes to apoptosis, while it may ameliorate UC by preventing excessive damage to the intestinal epithelial layer (Figure 7).The kinome profi ling combined with the Western blot analysis of protein phosphorylation revealed a smoke-dependent stimulation of pro-survival pathways (AMPK/Akt/PI3K and GSK3β/cyclin D1/LC3) and anti-apoptotic pathways (STAT5B/ERK1/2 and Bcl-XL/Bcl-2). These observations correspond well to previous studies, where eff ects of cigarette smoke were investigated in COPD and lung cancer using micro array analysis and/or proteomic approaches17,18. Similar kinase pathways were found to be aff ected by smoke, supporting a common role by which cigarette smoke


60

CHAPTER 3

chapter

3chapter

3

Figure 7. Proposed model for the dichotomal eff ects of cigarette smoke on Crohn’s disease and ulcerative colitis explained by inhibition of apoptosis. Upper panel: Normal colon with mild apoptosis activity and mixed infi ltrate restricted to the top of villi. Crypt architecture is preserved. The crypts are parallel to each other and extend from the luminal surface to the muscularis mucosae. Upper left panel: Ulcerative colitis with high apoptosis rates in the epithelium. Severe diff use crypt atrophy and diff use chronic infl ammation with mixed infi ltrate. Crypt distortion includes crypt branching and loss of parallelism. Lower left panel: Crohn’s disease with diff use transmucosal chronic infl ammation including lamina propria infi ltrate, mainly T-lymphocytes. Abnormal crypt architecture. Upper right panel: Ulcerative colitis after smoking with less apoptosis activity in the epithelium. Restoration of crypt architecture and less mucosal infi ltrate. Lower right panel: Crohn’s disease after smoking with severe transmucosal chronic infl ammation and T-lymphocyte infi ltrate. Less apoptosis of T-lymphocytes shifting toward excessive proliferation.


61


chapter

3chapter

3

acts throughout the human body. Cigarette smoke seems to act, at least partly, through the AMPK pathway as AMPK inhibition reversed the CSE-cytoprotective eff ect in Jurkat cells and a similar trend was observed for DLD-1 cells. A potential role of the PI3K and/or ERK pathway in the anti-apoptotic eff ect of smoke under infl ammatory conditions remains obscure, as these are already activated by anti-Fas. However, key factors in these pathways are activated by CSE in the absence of anti-Fas and therefore may provide a basal protection to apoptosis. Maintenance of the colonic epithelium is the result of a delicate balance between cell proliferation at the base and cell death (apoptosis) at the surface epithelium. Apoptosis must therefore be highly regulated and minor perpetuations in epithelial apoptosis rates may disturb the epithelial barrier and allow luminal pathogens to pass the epithelium. The ongoing exposure of pathogens triggers the immune system and causes infl ammation of the colon and further increases epithelial cell damage. UC is characterized by high epithelial cell apoptosis rates19 and impaired barrier function that lead to increased intestinal permeability. In fact, an increased intestinal permeability to harmful substances may be one of the initiating factors in the development of UC. Several studies report that smoking may act to tighten the gut and confer its benefi cial eff ect upon UC by reducing intestinal permeability20,21. Here we show that smoking inhibits apoptosis in colonic epithelial cells and increases the metabolic state of these cells, thereby improving epithelial survival and barrier function.Apoptosis also controls the life span of invading immune cells during infl ammation and thereby modulates infl ammatory responses via limiting expansion of activated T-cells. This process is of key importance in the intestinal mucosa, as it limits the size of activated T-cell compartment and thereby prevents uncontrolled expansion of the infl ammatory response leading to tissue damage. The resistance of mucosal T-cells to undergo apoptosis is strongly implicated in the pathogenesis of IBD. T-cells from CD patients have an intrinsic defect in activation-dependent apoptosis, which leads to an inappropriate cellular expansion and an ongoing infl ammatory reaction22. This defect in apoptosis has been attributed to an imbalance between two mitochondrial proteins, the anti-apoptotic Bcl-2/Bcl-XL and the pro-apoptotic Bax23,24. Here, we show that exposure to cigarette smoke enhances the levels of Bcl-2/Bcl-XL and thereby protects T-cells against infl ammation-induced apoptosis. This indicates that the detrimental eff ect of smoking on CD pathogenesis could be due to a further enhanced resistance of T-cells against apoptosis. Smoke exposure has been shown to increase T-cell populations. Tanigawa et al. reported that cigarette


62

CHAPTER 3

chapter

3chapter

3

smokers have enhanced numbers of circulating T-lymphocytes25. Also, Verschuere et al. reported a signifi cant increase and recruitment of various immune cell types into murine Peyer’s patches such as CD4+ T-cells, CD8+ T-cells and regulatory T-cells after chronic smoke exposure, although this was not associated with tissue damage26. The induction of apoptosis in T-cells is thought to be a key therapeutic action of several immunosuppressive drugs and anti-TNF agents are used in the treatment of CD27-29. Therefore smoking might impair the eff ect of these drugs, however, clinical studies are not conclusive in this respect. In rheumatoid arthritis (RA), another autoimmune disease characterized by resistance to apoptosis and smoke-related disease development30,31, a specifi c genetic variant of PTPN22 was found to be associated with RA and heavy smoking32,33. As PTPN22 is involved in T-cell receptor signaling, it further supports the importance of T-cell signaling in autoimmune diseases like IBD. While this cigarette smoke-induced inhibition of apoptosis is aggravating CD, it is protective against UC. Although smoking ameliorates UC, we have to stress that smoking leads to many health problems that results in oxidative stress-induced cell damage. For example, smoking is known to be an independent risk factor for carcinogenesis in Barrett’s esophagus and colorectal neoplasia34-36. As a correlation exists between infl ammation and the risk of gastrointestinal cancer37,38, one could speculate about the role of cigarette smoking in infl ammation-associated carcinogenesis in the colon. However, no data is available about an increased risk of colon cancer neither in smoking CD patients with colonic disease nor in smoking UC patients who are in remission upon smoking. In order to pinpoint potential active mediators responsible for the eff ects smoking exerts, we analyzed the anti-apoptotic eff ect of purifi ed and water-soluble components from cigarette smoke in T-lymphocytes and intestinal epithelial cells. We observed signifi cant cytoprotection for all components tested, suggesting the involvement of various cytoprotective pathways. Nicotine is the most well studied compound of cigarette smoke and may be benefi cial by increasing mucin synthesis39, gut permeability20 and by decreasing pro-infl ammatory cytokines40 mainly through its action on the nicotinic acetylcholine receptor on immune cells41. In this report, we show a signifi cant cytoprotective action of nicotine at concentrations similar to concentrations found in blood of smokers42. However, confl icting data of therapeutic eff ects of nicotine exists and other compounds may contribute more to the cytoprotective action of smoke. Recent studies on acrolein are in line with our results as it was shown to exert anti-infl ammatory actions through multiple signal transduction pathways, including mitogen-activated protein kinase (MAPK)


63


chapter

3chapter

3

pathways43, inhibition of NF-κB and by activating Nrf2, thereby inducing anti-infl ammatory genes such as HO-144. Although several studies indicate that acrolein induces apoptosis and necrosis in various cell types45, acrolein was also found to inhibit apoptosis by infl uencing redox-sensitive caspase signaling cascades46, which is consistent with our fi ndings. Studies on CO/CORMs exposure seem to be more consistent in proving a potent anti-infl ammatory action, as it was shown to protect cells through anti-infl ammatory, anti-proliferative and anti-apoptotic signaling in several models of colitis47,48. These protective eff ects of CO involve inhibition of T-cell activation and proliferation, suppressing infl ammation supposedly by downregulating of pro-infl ammatory cytokines and modulation of numerous cellular targets including MAPKs. Depending on stimulus and timing, their activity is diff erentially regulated by CO49,50. Here, we show a cytoprotective action of CO and CORMs against infl ammation-induced apoptosis, which is consistent with other studies on CO/CORMs51. Cigarette smoke constituents exert both immunosuppressive and immunogenic eff ects and we show a general anti-apoptotic eff ect that could explain the dichotomy of cigarette smoke on CD and UC. It is important to emphasize that components in cigarette smoke do not suppress or enhance infl ammation by a single specifi c mechanism, but by a combination of protein/kinase pathways that collectively result in anti- (UC) or pro- (CD) infl ammatory responses. Further studies on smoke components are needed in order to provide novel strategies to maximize the benefi cial eff ects of smoking in UC and minimize the deleterious eff ects on CD and general health.

ACKNOWLEDGEMENTS

The authors would like to thank C. Haven (Illustrator Medbeeld) for assistance on the preparation of Figure 7.

REFERENCES

1. Nikolaus, S and Schreiber, S. Diagnostics of infl ammatory bowel disease. Gastroenterology 2007;133:1670-1689.

2. Jostins, L, Ripke, S, Weersma, RK, Duerr, RH, McGovern, DP, Hui, KY and Cho, JH. Host-microbe interactions have shaped the genetic architecture of infl ammatory bowel disease. Nature 2012;491:119-124.

3. Budarf, ML, Labbe, C, David, G and Rioux, JD. GWA studies: rewriting the story of IBD. Trends Genet 2009;25:137-146.


64

CHAPTER 3

chapter

3chapter

3

4. Harries, AD, Baird, A and Rhodes, J. Non-smoking: a feature of ulcerative colitis. Br Med J (Clin Res Ed) 1982;284:706.

5. Somerville, KW, Logan, RF, Edmond, M and Langman, MJ. Smoking and Crohn’s disease. Br Med J (Clin Res Ed) 1984;289:954-956.

6. Birrenbach, T and Bocker, U. Infl ammatory bowel disease and smoking: a review of epidemiology, pathophysiology, and therapeutic implications. Infl amm Bowel Dis 2004;10:848-859.

7. Cosnes, J. Tobacco and IBD: relevance in the understanding of disease mechanisms and clinical practice. Best Pract Res Clin Gastroenterol 2004;18:481-496.

8. van der Heide, F, Dijkstra, A, Weersma, RK, Albersnagel, FA, van der Logt, EM, Faber, KN and Dijkstra, G. Eff ects of active and passive smoking on disease course of Crohn’s disease and ulcerative colitis. Infl amm Bowel Dis 2009;15:1199-1207.

9. Karban, A and Eliakim, R. Eff ect of smoking on infl ammatory bowel disease: Is it disease or organ specifi c? World J Gastroenterol 2007;13:2150-2152.

10. Blokzijl, H, Vander Borght, S, Bok, LI, Libbrecht, L, Geuken, M, van den Heuvel, FA and Faber, KN. Decreased P-glycoprotein (P-gp/MDR1) expression in infl amed human intestinal epithelium is independent of PXR protein levels. Infl amm Bowel Dis 2007;13:710-720.

11. van der Toorn, M, Rezayat, D, Kauff man, HF, Bakker, SJ, Gans, RO, Koeter, GH and Slebos, DJ. Lipid-soluble components in cigarette smoke induce mitochondrial production of reactive oxygen species in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2009;297:L109-14.

12. Blokzijl, H, van Steenpaal, A, Vander Borght, S, Bok, LI, Libbrecht, L, Tamminga, M and Faber, KN. Up-regulation and cytoprotective role of epithelial multidrug resistance-associated protein 1 in infl ammatory bowel disease. J Biol Chem 2008;283:35630-35637.

13. Conde de la Rosa, L, Schoemaker, MH, Vrenken, TE, Buist-Homan, M, Havinga, R, Jansen, PL and Moshage, H. Superoxide anions and hydrogen peroxide induce hepatocyte death by diff erent mechanisms: involvement of JNK and ERK MAP kinases. J Hepatol 2006;44:918-929.

14. Diks, SH, Kok, K, O’Toole, T, Hommes, DW, van Dijken, P, Joore, J and Peppelenbosch, MP. Kinome profi ling for studying lipopolysaccharide signal transduction in human peripheral blood mononuclear cells. J Biol Chem 2004;279:49206-49213.

15. Diks, SH, Parikh, K, van der Sijde, M, Joore, J, Ritsema, T and Peppelenbosch, MP. Evidence for a minimal eukaryotic phosphoproteome? PLoS One 2007;2:e777.

16. Schoemaker, MH, Ros, JE, Homan, M, Trautwein, C, Liston, P, Poelstra, K and Moshage, H. Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-kappaB-regulated inhibitor of apoptosis protein 2 (cIAP2) prevents apoptosis. J Hepatol 2002;36:742-750.

17. Mercer, BA and D’Armiento, JM. Emerging role of MAP kinase pathways as therapeutic targets in COPD. Int J Chron Obstruct Pulmon Dis 2006;1:137-150.


65


chapter

3chapter

3

18. Wen, J, Fu, JH, Zhang, W and Guo, M. Lung carcinoma signaling pathways activated by smoking. Chin J Cancer 2011;30:551-558.

19. Iwamoto, M, Koji, T, Makiyama, K, Kobayashi, N and Nakane, PK. Apoptosis of crypt epithelial cells in ulcerative colitis. J Pathol 1996;180:152-159.

20. McGilligan, VE, Wallace, JM, Heavey, PM, Ridley, DL and Rowland, IR. Hypothesis about mechanisms through which nicotine might exert its eff ect on the interdependence of infl ammation and gut barrier function in ulcerative colitis. Infl amm Bowel Dis 2007;13:108-115.

21. Prytz, H, Benoni, C and Tagesson, C. Does smoking tighten the gut? Scand J Gastroenterol 1989;24:1084-1088.

22. Peppelenbosch, MP and van Deventer, SJ. T cell apoptosis and infl ammatory bowel disease. Gut 2004;53:1556-1558.

23. Ina, K, Itoh, J, Fukushima, K, Kusugami, K, Yamaguchi, T, Kyokane, K and Fiocchi, C. Resistance of Crohn’s disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance. J Immunol 1999;163:1081-1090.

24. Itoh, J, de La Motte, C, Strong, SA, Levine, AD and Fiocchi, C. Decreased Bax expression by mucosal T cells favours resistance to apoptosis in Crohn’s disease. Gut 2001;49:35-41.

25. Tanigawa, T, Araki, S, Nakata, A and Sakurai, S. Increase in the helper inducer (CD4+CD29+) T lymphocytes in smokers. Ind Health 1998;36:78-81.

26. Verschuere, S, Allais, L, Bracke, KR, Lippens, S, De Smet, R, Vandenabeele, P and Cuvelier, CA. Cigarette smoke and the terminal ileum: increased autophagy in murine follicle-associated epithelium and Peyer’s patches. Histochem Cell Biol 2012;137:293-301.

27. Van den Brande, JM, Braat, H, van den Brink, GR, Versteeg, HH, Bauer, CA, Hoedemaeker, I and van Deventer, SJ. Infl iximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease. Gastroenterology 2003;124:1774-1785.

28. van Deventer, SJ. Transmembrane TNF-alpha, induction of apoptosis, and the effi cacy of TNF-targeting therapies in Crohn’s disease. Gastroenterology 2001;121:1242-1246.

29. Tiede, I, Fritz, G, Strand, S, Poppe, D, Dvorsky, R, Strand, D and Neurath, MF. CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest 2003;111:1133-1145.

30. Costenbader, KH, Feskanich, D, Mandl, LA and Karlson, EW. Smoking intensity, duration, and cessation, and the risk of rheumatoid arthritis in women. Am J Med 2006;119:503.-e1-9.

31. Hutchinson, D, Shepstone, L, Moots, R, Lear, JT and Lynch, MP. Heavy cigarette smoking is strongly associated with rheumatoid arthritis (RA), particularly in patients without a family history of RA. Ann Rheum Dis 2001;60:223-227.

32. Costenbader, KH, Chang, SC, De Vivo, I, Plenge, R and Karlson, EW. Genetic polymorphisms in PTPN22, PADI-4, and CTLA-4 and risk for rheumatoid arthritis in two longitudinal


66

CHAPTER 3

chapter

3chapter

3

cohort studies: evidence of gene-environment interactions with heavy cigarette smoking. Arthritis Res Ther 2008;10:R52.

33. Begovich, AB, Carlton, VE, Honigberg, LA, Schrodi, SJ, Chokkalingam, AP, Alexander, HC and Gregersen, PK. A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J Hum Genet 2004;75:330-337.

34. Tsoi, KK, Pau, CY, Wu, WK, Chan, FK, Griffi ths, S and Sung, JJ. Cigarette smoking and the risk of colorectal cancer: a meta-analysis of prospective cohort studies. Clin Gastroenterol Hepatol 2009;7:682-688.e1-5.

35. Raimondi, S, Botteri, E, Iodice, S, Lowenfels, AB and Maisonneuve, P. Gene-smoking interaction on colorectal adenoma and cancer risk: review and meta-analysis. Mutat Res 2009;670:6-14.

36. Coleman, HG, Bhat, S, Johnston, BT, McManus, D, Gavin, AT and Murray, LJ. Tobacco smoking increases the risk of high-grade dysplasia and cancer among patients with Barrett’s esophagus. Gastroenterology 2012;142:233-240.

37. Ullman, TA and Itzkowitz, SH. Intestinal infl ammation and cancer. Gastroenterology 2011;140:1807-1816.

38. Velayos, FS, Loftus, EV,Jr, Jess, T, Harmsen, WS, Bida, J, Zinsmeister, AR and Sandborn, WJ. Predictive and protective factors associated with colorectal cancer in ulcerative colitis: A case-control study. Gastroenterology 2006;130:1941-1949.

39. Zijlstra, FJ, Srivastava, ED, Rhodes, M, van Dijk, AP, Fogg, F, Samson, HJ and Williams, GT. Eff ect of nicotine on rectal mucus and mucosal eicosanoids. Gut 1994;35:247-251.

40. Sher, ME, Bank, S, Greenberg, R, Sardinha, TC, Weissman, S, Bailey, B and Wexner, SD. The infl uence of cigarette smoking on cytokine levels in patients with infl ammatory bowel disease. Infl amm Bowel Dis 1999;5:73-78.

41. Wang, H, Yu, M, Ochani, M, Amella, CA, Tanovic, M, Susarla, S and Tracey, KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of infl ammation. Nature 2003;421:384-388.

42. Benowitz, NL, Hukkanen, J and Jacob, P,3rd. Nicotine chemistry, metabolism, kinetics and biomarkers. Handb Exp Pharmacol 2009;(192):29-60. doi:29-60.

43. Randall, MJ, Spiess, PC, Hristova, M, Hondal, RJ and van der Vliet, A. Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1. Redox Biol 2013;1:265-275.

44. Spiess, PC, Kasahara, D, Habibovic, A, Hristova, M, Randall, MJ, Poynter, ME and van der Vliet, A. Acrolein exposure suppresses antigen-induced pulmonary infl ammation. Respir Res 2013;14:107.

45. Kern, JC and Kehrer, JP. Acrolein-induced cell death: a caspase-infl uenced decision between apoptosis and oncosis/necrosis. Chem Biol Interact 2002;139:79-95.

46. Hristova, M, Heuvelmans, S and van der Vliet, A. GSH-dependent regulation of Fas-


67


chapter

3chapter

3

mediated caspase-8 activation by acrolein. FEBS Lett 2007;581:361-367.

47. Takagi, T, Naito, Y, Mizushima, K, Akagiri, S, Suzuki, T, Hirata, I and Yoshikawa, T. Inhalation of carbon monoxide ameliorates TNBS-induced colitis in mice through the inhibition of TNF-alpha expression. Dig Dis Sci 2010;55:2797-2804.

48. Sheikh, SZ, Hegazi, RA, Kobayashi, T, Onyiah, JC, Russo, SM, Matsuoka, K and Plevy, SE. An anti-infl ammatory role for carbon monoxide and heme oxygenase-1 in chronic Th2-mediated murine colitis. J Immunol 2011;186:5506-5513.

49. Otterbein, LE, Bach, FH, Alam, J, Soares, M, Tao Lu, H, Wysk, M and Choi, AM. Carbon monoxide has anti-infl ammatory eff ects involving the mitogen-activated protein kinase pathway. Nat Med 2000;6:422-428.

50. Morse, D, Pischke, SE, Zhou, Z, Davis, RJ, Flavell, RA, Loop, T and Choi, AM. Suppression of infl ammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1. J Biol Chem 2003;278:36993-36998.

51. Pae, HO, Oh, GS, Choi, BM, Chae, SC, Kim, YM, Chung, KR and Chung, HT. Carbon monoxide produced by heme oxygenase-1 suppresses T cell proliferation via inhibition of IL-2 production. J Immunol 2004;172:4744-4751.


68

CHAPTER 3

chapter

3chapter

3

SUPPLEMENTARY FIGURES

Antibody Dilution Source

Poly(ADP-ribose) polymerase (PARP) 1:1000 Cell Signaling (#9542)

p-SrcTyr416 1:1000 Cell Signaling (#2101)

p-STAT5Tyr694/Tyr699 1:1000 Cell Signaling (#9359)

p-AktSer473 1:1000 Cell Signaling (#9271)

p-GSK3β Ser9 1:1000 Cell Signaling (#9336)

p-BadSer112 1:1000 Cell Signaling (#9291)

LC-3 1:1000 Cell Signaling (#2775)

p-AMPKThr172 1:1000 Biosource (#44-1150G)

p-ERK1/2Thr202/Tyr204 1:1000 New England Biolabs (#9106)

cyclin D1 1:1000 Santa Cruz (#sc-246)

Bcl-XL 1:1000 Santa Cruz (#sc-634)

Supplementary Table S1. Antibodies used in this study.

Supplementary Table S2A and S2B.

On request ([email protected]).


69


chapter

3chapter

3

Supplementary Figure S1. Provisional signal transduction schemes - impression of kinome profi ling results. Paired cultures of DLD-1 cells and Jurkat cells were challenged with vehicle or 20% CSE followed analysis for kinase activity using peptide arrays. Results were collapsed on eff ective signal transduction categories and expressed in gray scale (representing the number of peptides phosphorylated as a fraction of all peptides in that category). Vehicle and CSE eff ects were contrasted. The results provide an overview of the signaling pathways active in DLD-1 cells and Jurkat cells and the eff ects of CSE thereon.


70

CHAPTER 3

chapter

3chapter

3

A B

C D

Supplementary Figure S2. Caspase-3 activity in total extracts of (A) HTC116 cells, (B) Caco-2 cells, (C) SW480 cells and (D) MOLT-4 cells exposed to anti-Fas, CM or menadione in combination with increasing concentrations CSE. Maximal caspase-3 activity is presented as 100%. Data represent means ± SD of at least three independent experiments. *P<0.05 vs. control.


university of groningen gene-environment interactions in ... · pdf filecigarettes (lexington,...

Documents