1 MEMOREC Stoffel GmbH, Stöckheimer Weg 1, D-50829 Köln, Germany and
2 INBIFO GmbH, Fuggerstr. 3, D-51149 Köln, Germany
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Abstract |
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Abbreviations: C/EBP, CAAT/enhancer binding protein; CS, cigarette smoke; GADD, growth arrest and DNA damage inducible; HO-1, haem oxygenase-1; HSP, heat shock protein; IL, interleukin; MT, metallothionein; ONOO-, peroxynitrite; PBS, phosphate-buffered saline; TNF, tumour necrosis factor
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Introduction |
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The DNA damaging activity of smoke-bubbled PBS is expressed mainly by the appearance of single-strand breaks in the DNA of exposed cells. Mechanistic studies have shown that hydroxyl radicals produced by Fenton chemistry (4) are involved mainly in inducing this type of DNA damage (58). However, reactive oxygen species resulting from Fenton chemistry obviously do not participate to a very great extent in the alteration of the gene expression pattern observed in smoke-bubbled PBS-treated cells. This was at least deduced from experiments in which inhibitors of hydroxyl radical formation via the Fenton reaction were found to be unable to suppress the CS-related expression of stress genes such as haem oxygenase-1 (ho-1) and c-fos (7,8). Instead, investigations on biologically active compounds present in smoke-bubbled PBS revealed that scavengers of nitric oxide and superoxide, i.e. oxy-haemoglobin and superoxide dismutase, each specifically inhibited c-fos expression in smoke-bubbled PBS-treated cells, suggesting that peroxynitrite (ONOO-) formation is involved in CS-dependent cell signalling (9). As the calculated peroxynitrite concentration potentially formed by CS in aqueous solution is not sufficient to explain the effects seen with smoke-bubbled PBS, CS-related aldehydes, such as formaldehyde, acetaldehyde and acrolein, were identified in further experiments as strong depletors of intracellular glutathione (GSH) (10). This is a prerequisite for enabling peroxynitrite to interfere with specific target molecules resulting in the activation of stress signal transduction and stress gene expression in CS-treated cells.
Beyond its genotoxic and stress gene-inducing activities, CS has been shown to induce pro-inflammatory effects, e.g. as reflected by the augmented release of interleukin (IL)-8 in vitro and in vivo (11). This response was found to be dependent on the activation of the pro-inflammatory transcription factor NF-B (12), which has been shown recently to be regulated in an I
B
-independent but thioredoxin-dependent manner in smoke-bubbled PBS-treated cells in vitro (13).
Since its recent invention, DNA microarray techniques have revolutionized nearly all disciplines of molecular biology, medicine and pharmacology (for review, see ref. 14). In fact, this technique has successfully been used to investigate the spectrum of gene expression changes as a cellular reaction to known toxicants (15).
Here we report on gene expression profiling studies in Swiss 3T3 cells exposed for up to 24 h to subcytotoxic concentrations of smoke-bubbled PBS through microarray glass chips carrying 513 cDNA probes, which correspond to genes known to be involved in antioxidant response, cell cycle regulation, inflammation, DNA repair, transcriptional activation and apoptosis. The results obtained show a detailed, time-dependent up and down regulated expression of genes covering an extensive antioxidant, cell cycle-regulatory and inflammatory/immune-regulatory response.
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Materials and methods |
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Cell culture and treatment
Swiss albino 3T3 (ATCC CCL 92) mouse fibroblast cells were cultured in 15 cm tissue culture dishes (Greiner, Frickenhausen, Germany), in 20 ml of Dulbecco's modified Eagle medium (DMEM) supplemented with 4% sodium hydrogen carbonate, L-glutamine, streptomycin, penicillin and 10% fetal calf serum (FCS) (Invitrogen, Groningen, The Netherlands).
Growth-arrested (0.5% FCS; 48 h) 3T3 cells were used in these experiments. Prior to treatment, the cells were washed with serum-free DMEM and immediately exposed for the indicated incubation times to serum-free culture medium containing 0.03 puffs/ml smoke-bubbled PBS. Untreated (control) cells were handled analogously but received equal amounts of CS-free PBS instead of smoke-bubbled PBS.
RNA isolation
Total RNA was isolated from exposed and control cells (2x107 each) by standard methods using a commercially available RNA isolation kit (Qiagen, Hilden, Germany). The quality of the RNA was checked for integrity, genomic DNA impurity, 28S/18S ratio and OD280/260 ratio.
Gene expression studies
Northern hybridization.
Sixteen micrograms of RNA per sample was analysed by routine methods including denaturing RNA gel electrophoresis, blotting, hybridization and autoradiography (16). 32P-Labelled fragments of the murine ho-1, st2/st2l andas internal controlthe ß-actin gene served as hybridization probes.
cDNA-array production.
Defined 200400 bp fragments of selected cDNAs were generated by RTPCR (SuperscriptTM II, Invitrogen) using sequence-specific primers and RNA derived from appropriate murine tissue and cell lines. A list of all genes including their accession numbers is available on the Internet (see supplementary material under http://www.memorec.com). Each fragment was cloned into pGEM®-T Vector (Promega, Mannheim, Germany) and sequence-verified. Correct annotation of the genes was verified by automized blast search using the Unigene and SwissProt databases. Inserts were amplified (Taq PCR Master Mix, Qiagen) using vector sequence-derived primers with the sense primer carrying a 5'-amino-modification. PCR fragments were purified (QIAquick 96 PCR BioRobot Kit, Qiagen), checked on an agarose gel and diluted to a concentration of 100 ng/µl. Amplified inserts were transferred to a 384 well plate and spotted four times each on treated glass slides with a customized ink jet spotter. Two nanolitres of each probe were spotted by dispensing 20 drops of 100 pl each. Probes were re-hydrated for 2 h in a humidified chamber and blocked (17).
Sample labelling and hybridization.
One hundred micrograms of total RNA was combined with a control RNA consisting of an in vitro-transcribed Escherichia coli genomic DNA fragment carrying a 30 nt poly(A)+-tail (CR1), and the mRNA was isolated (Oligotex mRNA Mini Kit, Qiagen). The resulting mRNA was diluted to 17 µl and combined with 2 µl of a second control RNA, a mixture of three different transcripts CR2, CR3 and CR4. The mRNA was then reverse-transcribed by adding it to a mix consisting of 8 µl 5x first-strand buffer (Invitrogen), 2 µl Primer-Mix [oligo(dT) and randomeres] (MEMOREC, Köln, Germany), 2 µl low C dNTPs (10 mM dATP, 10 mM dGTP, 10 mM dTTP, 4 mM dCTP), 2 µl FluoroLinkTM Cy3/5-dCTP (Amersham Pharmacia Biotech, Freiburg, Germany), 4 µl 0.1 M DTT and 1 µl RNasin (2040 U) (Promega). One microlitre (200 U) of SSII enzyme was added, incubated at 42°C for 30 min followed by the addition of a further 1 µl of SSII enzyme and incubated under the same conditions as detailed above. A 0.5 µl sample of RNaseH (Invitrogen) was added and incubated at 37°C for 20 min to hydrolyse RNA. Cy3- and Cy5-labelled samples were combined and cleaned up using QIAquickTM (Qiagen). Eluents were diluted to a volume of 50 µl. Fifty microlitres of 2x hybridization solution (MEMOREC) pre-warmed to 42°C was added. Hybridization was performed according to manufacturer's guidelines (MEMOREC) using a GeneTACTM hybridization station (Perkin Elmer, Langen, Germany).
Data analysis of array hybridizations.
Image capture and signal quantification of hybridized PIQORTM cDNA arrays were done with the ScanArray3000 (GSI Lumonics, Watertown, MA) and ImaGene software version 4.1 (BioDiscovery, Los Angeles, CA). For each spot, the local signal was measured inside a fixed circle of 350 µm diameter, and the background was measured outside the circle within specified rings 40 µm distant to the signal and 40 µm wide. Signal and background was taken to be the average of pixels between defined low and high percentages of maximum intensity with percentage parameter settings for low/high being 0/97% for signal and 0/80% for background. Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Cy5/Cy3. The ratios were normalized to the median of all ratios by using only those spots for which the fluorescent intensity in one of the two channels was three times the negative control. Subsequently, the mean of the ratios of four corresponding spots representing the same cDNA was computed. The negative control for each array was computed as the mean of the signal intensity of two spots representing herring sperm and two spots representing spotting buffer only. Only genes displaying a net signal intensity 3-fold higher in the control or treatment sample than in the negative control were used for further analysis.
Protein expression studies
A 1 ml aliquot of cell culture medium obtained from CS-treated or control Swiss 3T3 cells was assayed by `sandwich' ELISA techniques for KC, IL-6, TGF-ß1, IL-1, IL-1ß, tumour necrosis factor (TNF)-
and IL-12 (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions and quantitatively analysed (ELISA reader `340 ATTC'; SLT Labinstruments GmbH, Crailsheim, Germany) using a 4-parameter logistic curve-fit for the standard curve (`easyWIN fittin V6.0a', TECAN, Crailsheim, Germany).
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Results and discussion |
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Based on these observations, the experiment to evaluate the kinetics of differential gene expression detection in CS-treated cells was designed along the following lines: serum-starved Swiss 3T3 cells were exposed to 0 (untreated control) or 0.03 puffs/ml smoke-bubbled PBS for 0.5, 1, 2, 4, 8 and 24 h and labelled cDNA samples resulting from harvested RNA were hybridized to 513 cDNA probes on glass chips as outlined in the Materials and methods. The cDNA probes used in this study were designed to be gene-specific, had a uniform length of between 200 and 400 nt and were selected from different functional categories including genes known to be involved in antioxidant response, cell cycle-regulation, inflammation, DNA repair, transcriptional activation and apoptosis.
For quantification, fluorescence derived from microarrays hybridized in parallel to control ('Cy3'-labelled) and exposed ('Cy5'-labelled) cDNA samples were `overlayed' and analysed (see the Materials and methods). As an example, Figure 1A shows the `overlay' of a microarray hybridized with cDNA samples originating from 8 h incubations highlighting the hybridization products for ho-1, hsp105, sarp1 and c-maf. Red fluorescence identifies up regulated genes, while green fluorescence reflects genes that become suppressed in the presence of smoke-bubbled PBS. The expression factorevaluated from quadruplicate hybridizationwas determined as a mean value according to the ratios of the Cy5/Cy3 signal intensities (Figure 1B
). More than 4-fold increases or decreases of the gene expression ratio were regarded as relevant. For comparison, the results obtained during microarray hybridization and northern blotting of the two strongest up regulated genes, i.e. ho-1 and st2/st2l, are shown in Figure 2
.
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Cluster analysis, using a hierarchical clustering algorithm method to group genes on the basis of similarity in the pattern with which their expression varied over all experiments (18), revealed three subgroups (see supplementary material under http://www.memorec.com): cluster 1 reflects genes becoming up regulated early (02 h) during exposure. This cluster contains mainly genes that encode regulatory proteins such as JunB, growth arrest and DNA damage inducible (GADD)34, CAAT/enhancer binding protein ß (C/EBPß) and Id3. Cluster 2 comprises genes that are activated at later time points (28 h) of exposure. This group also includes genes known to be controlled by these regulatory proteins, e.g. genes coding for HO-1, metallothionein 1/2 (MT1/2) and heat shock proteins (HSPs). Genes that are down regulated during exposure are displayed in the third cluster.
Previously, we have presented evidence that peroxynitrite is a stress-inducing compound formed by CS in aqueous solution (9). This finding is corroborated by the emergence of five genes which are up regulated >5-fold, i.e. ho-1, gadd34, gadd45, mt1 and mt2, and which have been shown to be used by cells to combat stress arising from peroxynitrite (1921).
The antioxidant response
The antioxidant response of smoke-bubbled PBS-exposed cells is governed by the paramount expression of ho-1, which, after 8 h of exposure, becomes up regulated 84-fold in comparison with control cells (Table I, Figures 2 and 3
). HO-1 is supposed to counteract oxidative stress by converting haem moieties in a first, rate-limiting step to biliverdin and, finally, in the presence of biliverdin reductase, to bilirubin (22). Both biliverdin and bilirubin are efficient antioxidants (22,23). Another possible antioxidant mechanism of HO-1 could involve the destruction of haem itself. Haem and haem proteins have been shown by several investigators to be instrumental in exacerbating oxidative injury (24,25).
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The CS-dependent induction of ho-1 may be achieved potentially, at least in part, via activation of transcription factors binding to AP-1 and C/EBP recognition elements, which are both integral parts of the murine ho-1 promoter (29). junB, which encodes a potential dimerization partner of AP-1 molecules (30) and which has been identified as a target in redox regulation (31) becomes activated strongly early during exposure (Figure 4). The same holds true for the gene encoding C/EBP (32), which has been shown to become immensely expressed during oxidative lung injury (33), and other transcription factors as revealed by our `cluster analysis' (18) (see supplementary material). These transcriptional activators may collaborate with other transcription factors, e.g. regulatory proteins binding to the cadmium response element, which has been shown to be involved in ho-1 expression in human U937 cells exposed to aqueous extracts of CS (34).
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The strong expression of the antioxidant genes ho-1, mt1/2, hsp105 and hsp90 generally reflects the pro-oxidative quality of aqueous extracts of CS. In addition to HO-1-derived bilirubin, MT proteins have been identified as efficient scavengers of reactive oxygen and nitrogen species including peroxynitrite (20,37). HSPs are involved in refolding processes of (oxidatively) damaged proteins (38) and HSP90, because of its numerous cysteine residues, has been especially described as a modulator of the redox status of the cytosol (39).
The inflammatory/immune-regulatory response
A special feature of this microarray analysis is represented by the strong up regulation of genes known to be involved in inflammation- and immune regulation-related processes. In fact, the gene encoding ST2/ST2L, an orphan receptor exhibiting strong homology to the IL-1 receptor (40), was found to be expressed >20-fold during these studies (Table I, Figure 2B
). ST2/ST2L is expressed as a soluble (ST2) form and a membrane-bound form (ST2L). Both forms originate from a single gene by differential splicing, which is regulated by the use of alternate promoters (41) with st2 being the variant predominantly expressed in fibroblasts directed by an AP-1 site-containing promoter (42).
In order to discriminate between st2 and st2l expression in CS-treated 3T3 cells, northern blotting was performed using DNA sequences inherited by st2 and st2l mRNA for probing, which would give rise to mRNA species of 2.7 and
5.0 kb, respectively (42). Although there was an apparent increase in radioactively labelled probe DNA hybridizing to RNA species of molecular weights higher and lower than
2.7 kb after 8 h of exposure (Figure 2B
), this was clearly identified as an overload effect by fine-tuned radio-scanning of the blot (data not shown), indicating that it is st2, and not st2l, which is expressed by CS in 3T3 cells. Intriguingly, st2 expression has been observed in a murine in vivo inflammation model following UVB irradiation and in BALB/c3T3 cells following treatment with pro-inflammatory stimuli such as TNF, IL-1
, IL-1ß and phorbol ester (43). In order to investigate whether the increase in st2 expression maximally after 8 h of exposure (Figure 2B
) might be preceded by the expression of pro-inflammatory cytokines via an autocrine loop mechanism, ELISA techniques were used to determine cytokine concentrations in the cell culture medium of CS-exposed cells. Although small but significant amounts of the pro-inflammatory IL-6 were detectable in exposed cells, it is probably not involved in st2 expression because it was only detectable at later exposure times (data not shown). In addition, other pro-inflammatory cytokines, i.e. IL-1
, IL-1ß, IL-12 and TNF-
were not detected in the medium of CS-treated cells leaving the question open which trigger induces st2 in these cells. However, it is of note that recent findings show that ST2 harbours strong anti-inflammatory properties (44), thus potentially attenuating a CS-elicited pro-inflammatory response.
The increase of another inflammation-related protein, KC, in the medium of CS-treated cells by cytokine ELISA analysis confirms (on the protein level) the significant, up to 5.1-fold up regulation of the corresponding gene as detected by microarray hybridization (Figure 5). An early event in inflammatory processes is represented by the infiltration of neutrophils, a process which in the human situation is regulated partly by chemoattractant cytokines including IL-8 and GRO
(45), the latter being the human homologue of KC. In fact, KC has been shown to become attached to human neutrophils via binding to the IL-8 receptor (46), thus potentially substituting for IL-8 in mice, where no direct equivalent of IL-8 exists. In this context, it is of special note that studies in vitro and in vivo have demonstrated that IL-8 is released from human bronchial epithelial cells and is significantly increased in the bronchoalveolar lavage from smokers (11).
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Recently, induction of id3 transcription has been attributed to TGF-ß signalling (48). However, although this cytokine was detected in low but significant amounts in the medium of CS-exposed cells, it was only found in samples derived from cells exposed to smoke-bubbled PBS for 24 h (data not shown) and therefore has to be excluded as the main source of id3 activation in CS-treated cells. In another report, expression of id3 was linked to T-cell receptor ligation involving the Ras-extracellular signal-related kinase (ERK) signalling pathway (51), which is also triggered by smoke-bubbled PBS in 3T3 cells (S.Gebel, C.Knörr and T.Müller, in preparation).
The genotoxic/cell cycle-regulatory response
Up regulation of genes belonging to the family of gadd genes reflects the genotoxic component of the CS-evoked transcriptional response in 3T3 cells. Of the three gadd genes investigated, gadd34 and gadd45 become strongly induced already after 2 h of exposure (gadd34, 5.2-fold; gadd45, 3.5-fold) before they are maximally expressed after 4 h of exposure (Table I). In contrast, gadd153 is only slightly activated, which may be due to the enhanced expression of the transcriptional repressor ATF3 (Table I
), which has been shown to suppress gadd153 activation (52).
Expression of gadd34 has been implicated in apoptosis induction (53,54) while activation of gadd45 appears to exert pleiotropic effects including cell cycle arrest at G2/M, induction of DNA repair genes and control of genomic stability (55). Cells exposed to 0.03 puffs/ml smoke-bubbled PBS do not show any sign of cytotoxicity, but remain viable and become strictly arrested at G2/M (8), thus corresponding to the effects described for GADD45, while the relevance of the GADD34-mediated apoptotic signal remains to be elucidated for CS-treated cells.
Suppressed genes
According to the appraisal factor applied, only two genes, sarp1 and c-maf, were shown to be down regulated >4-fold during CS-exposure after 8 h of exposure (Table I, Figure 6
).
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C-Maf is a transcription factor binding to AP-1-like and cAMP response element (CRE)-like consensus sequences (57). Although c-maf is widely expressed in adult and embryonic tissues, only a few target genes are known which are transcriptionally regulated by c-Maf. In the context of this study, however, it seems notable that the tumour suppressor gene p53 is activated by c-Maf via binding to an AP-1 consensus sequence (58). As this sequence element is also used by AP-1-related proteins to positively regulate p53 transcription (58), it might be speculated that by down regulating c-maf activity, AP-1-related transcription factors such as JunB and c-Fos (8) might be privileged in regulating p53 transcription in CS-treated cells.
In addition to sarp1 and c-maf, borderline effects of gene suppression were observed for two other genes in CS-treated cells, i.e. sip1 (3.9-fold), a transcriptional repressor interacting with Smad proteins (59) and tweak (3.8-fold), a member of the TNF ligand family inducing angiogenesis and proliferation of endothelial cells (60).
Conclusions
Using microarray techniques, we have shown in kinetic terms the fine-tuned stress response in Swiss 3T3 cells exposed to non-toxic doses of aqueous solutions of CS. Beyond the expected antioxidative and genotoxic/cell cycle-regulatory response as reflected by the up regulation of genes encoding `leading proteins' under pro-oxidative conditions, i.e. enzymes and protein factors such as HO-1, MTs and HSPs, the identification of genes not yet linked to a CS-evoked stress response is particularly intriguing. Although the data presented here necessarily await confirmation in in vivo investigations, the finding that inflammation- and immune regulation-related genes such as kc and id3 become strongly expressed in CS-treated cells in vitro may nevertheless add to our understanding the mechanism of the development of CS-related disorders. For example, inflammatory processes as a consequence of the modulation of pro- and anti-inflammatory genes may increase the burden of reactive oxygen species, which has been associated with multiple steps in CS-related carcinogenesis.
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Acknowledgments |
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References |
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