Department of Experimental Toxicology, Medical Institute of Environmental Hygiene at the Heinrich Heine University, Auf'm Hennekamp 50,40225 Düsseldorf, Germany
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Abstract |
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Abbreviations: EGFR, epidermal growth factor receptor; Fra-1, Fos-related antigen-1; IGF II, insulin-like growth factor II; RPM, rat pleural mesothelial; SSH, suppression subtractive hybridization; TGF-, transforming growth factor alpha.
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Introduction |
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Malignant mesothelioma in humans is almost exclusively caused by asbestos or fibrous dusts with particular physical properties (2). Inhaled fibers penetrate the lung tissue and accumulate in the pleural space (3). In the mesothelial cell layer lining the pleural cavity, fibrosis and hyperplasia are induced. After a long period of latency, tumors can occur in this area.
The mechanisms of mesothelioma tumorigenesis are currently investigated using different in vitro experimental systems. Tumor cell lines of humans and rodents have been analyzed for the expression of growth factors and the corresponding receptors that may contribute to tumor formation and to tumor progression. Comparison of cell lines from tumors induced by asbestos with spontaneously immortalized cell lines can provide useful information that can be correlated with fiber-specific tumorigenesis (4). Autocrine growth loops consisting of transforming growth factor alpha (TGF-) and the epidermal growth factor receptor (EGFR), as well as insulin-like growth factor II (IGF II) and the corresponding receptor, have been shown to be relevant only in cell lines from asbestos-induced tumors but not in spontaneously transformed cell lines (4,5).
Early cellular responses to fibers that may lead to the induction of tumorigenesis are investigated using rat pleural mesothelial (RPM) cells treated with several kinds of fibrous and non-fibrous dusts. Cell proliferation and apoptosis are induced simultaneously by the application of fibers (6).
Several mechanisms of signal transduction, including mitogen-activated protein kinases (MAPKs) resulting in activation of the AP-1 transcription factor, are involved in these cellular reactions (7). However, the initiating steps of fibercell interaction leading to the induction of signal transduction cascades are still not understood. Signal transduction pathways in response to fiber stress, as well as the autocrine regulation observed in mesothelial cell lines, have not been demonstrated in vivo. Such information, however, is important to identify general principles of fiber-induced carcinogenesis.
The aim of this study was to identify cellular reactions in vivo associated with crocidolite fiber-induced carcinogenesis. Analysis of signal transduction pathways affected by fiber stress may help us to understand the processes that lead to dysregulation of the cell cycle and, hence, carcinogenesis. Moreover, fibercell interactions that lead to the induction of these processes should be monitored with this approach.
A second aim of this study was to evaluate the in vivo system of intraperitoneal (i.p.) injection in rats (8,9) to investigate fiber-induced carcinogenesis. Using this experimental model system, we demonstrated specific mRNA patterns in different stages of mesothelioma carcinogenesis.
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Materials and methods |
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To provide pretumorous tissue samples for the array assay, another animal experiment was performed. Conditions (animal strain, substances and dosages) were as described above, but animals were killed 52 weeks after treatment. Pathological examination did not reveal any tumors at this time point. Control animals were treated with carrier solution and killed at the same time.
RNA preparation
Total RNA was prepared using TRIzol reagent (Life Technologies, Rockville, MD) according to the manufacturer's advice, homogenizing tissues directly with the reagent. Poly(A)+ mRNA was isolated using oligo(dT)-coated latex particles (Oligotex mRNA kit; Qiagen, Hilden, Germany) according to the manufacturer's protocol.
Suppression subtractive hybridization
Suppression subtractive hybridization (12) was performed using a PCR-Select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA). Two micrograms of poly(A)+ mRNA, pooled from equal amounts of mRNA from tumors from six animals, was used to produce the tester cDNA. Two micrograms of poly(A)+ mRNA, pooled from equal amounts of mRNA from omenta of seven control animals, was used to produce the driver cDNA. After ligation of the adapters and a denaturation step, the tester cDNA fragments were allowed to hybridize with an excess of driver cDNA not bearing adapters. cDNA was then amplified by PCR using adapter sequences as primer targets.
PCR products, thus enriched in mRNA sequences upregulated in tumors, were cloned directly by blunt-end ligation into pCR-TRAP vector (GenHunter, Nashville, TN) according to the manufacturer's instructions. Colonies growing on selection medium were checked for DNA inserts by colony PCR, as recommended.
DNA sequencing
Plasmid DNA from positive clones was prepared from 100 ml overnight cultures using the QIAfilter Plasmid Midi Kit (Qiagen). DNA sequences of plasmid inserts were determined using an ALFexpress DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden). Sequencing reactions were performed with a Thermo Sequenase fluorescent labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) using fluorescently labeled primers (Lseq and Rseq from the cloning kit).
Sequences were analyzed for similarities to known sequences in the DNA databases available at the National Center for Biotechnology Information (Bethesda, MD). The BLAST search option was used via the World Wide Web.
Array assay
The Atlas cDNA expression array (Clontech) was used according to the manufacturer's protocol. Two identical nylon membranes loaded with 588 different human cDNA samples were subjected to parallel hybridization with test cDNA and control cDNA. To prepare hybridization probes representing the mRNA status of pretumorous asbestos-treated omenta and of control omenta, equal amounts of mRNA from seven treated animals and five control animals, respectively, were pooled before cDNA synthesis. Poly(A)+ mRNA (0.2 µg) was subjected to cDNA synthesis in the presence of [-32P]dATP using the array kit. Hybridization was performed as recommended in the kit protocol. Hybridization signals were visualized by placing the membranes against X-ray film. The signals obtained from the two independent hybridizations were compared to select cDNAs that differed in expression.
Dot blot hybridization
Plasmid DNA (5 µg) was spotted on to two separate sheets of nylon membrane (Hybond-N+; Amersham Pharmacia Biotech) according to the manufacturer's alkaline blotting procedure. Aliquots of cDNA (100 ng) were labeled with the Ready To Go labeling kit (Amersham Pharmacia Biotech) using [-32P]dCTP. Probes from driver and tester cDNA with similar specific radioactivity were used for separate stringent hybridization of the membranes, according to the hybridization protocol provided with the membrane.
Semiquantitative RTPCR
For reverse transcription, 500 ng of total RNA were subjected to reverse transcription primed by oligo(dT) with AMV reverse transcriptase (Promega, Madison, WI) according to the recommendations of the manufacturer in a total volume of 50 µl. One microliter of a 1 in 10 dilution of cDNA was subjected to PCR using Taq DNA polymerase (Roche, Mannheim, Germany). To ensure analysis of PCR products in the logarithmic phase of amplification, several parallel reactions with increasing cycle numbers were performed. As a control for cDNA synthesis, cDNA of constitutively expressed genes gapdh and ß-act (encoding glyceraldehyde-3-phosphate-dehydrogenase and ß-actin, respectively) was amplified. PCR conditions were as follows: a hot start after 5 min at 94°C; n cycles consisting of (i) denaturing for 30 s at 94°C; (ii) annealing for 30 s (see below for temperature); (iii) elongation for 75 s at 72°C. The number of cycles (n), annealing temperature, fragment length and primer sequences are listed in Table I. Significant differences in band intensity in ethidium bromide-stained agarose gels were assessed as differences in mRNA expression.
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Primary RPM cells were prepared from Wistar rats, between 8 and 10 weeks of age, as described by Jaurand et al. (14). The cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum, hydrocortisone (100 ng/ml), insulin (2.5 µg/ml) and selenium (2.5 ng/ml). For in vitro experiments, cells were used after passaging one to three times. Only cells from one preparation were used for parallel experiments.
The rat mesothelioma cell line RZ 328 was established from a tumor induced by crocidolite treatment in the animal experiment described above (10) and was kindly donated by Prof. Dr Füzesi (Institute of Pathology, University of Aachen, Germany). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. All cell culture media, sera and supplements were purchased from Sigma (St Louis, MO).
Cell treatment
Cells were grown until confluence was reached. They were shifted to low serum conditions (1% fetal bovine serum) 16 h before treatment. Asbestos fibers were suspended in phosphate-buffered saline by sonication. The fiber suspension was added to cells at the concentrations indicated.
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Results |
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Genes upregulated in mesothelioma
In the first set of experiments, the endpoint of tumor development was analyzed. Suppression subtractive hybridization (SSH) was used to compare tumors induced by i.p. administration of crocidolite asbestos with control tissue. cDNA from control tissues was used as a driver, and cDNA from tumors as tester. With this experimental design, mRNA sequences upregulated in tumors were enriched. In total, 93 plasmid clones were obtained. To eliminate false positives and cloned non-coding sequences, comparative dot blot hybridization and DNA sequence analysis were performed (data not shown). Five cDNA clones showed clear differences in hybridization signals and were of considerable insert length. A search of databases revealed identities to the sequences of genes for rat osteopontin (opn), mouse zyxin (zyx) and rat branched chain aminotransferase (bcat). Two clones (numbers 35 and 49) had ORFs with no homology to sequences in the available databases, but of considerable length. These data are summarized in Table IIA.
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The results are listed in Table IIB. Genes clearly induced at the mRNA level in the omentum of fiber-treated animals compared with non-treated tissues were c-myc, and the ilk and egfr genes, encoding integrin-linked kinase and epidermal growth factor receptor, respectively. The genes encoding monomeric proteins of the transcription factor AP-1, c-Jun and Fos-related antigen-1 (fra-1), revealed enhanced signal strength. The difference in signal strength was greater for fra-1 than for c-jun (data not shown), so only the former gene was investigated further. No genes with a noticeable reduction of mRNA expression in pretumorous tissues were found.
Expression of fra-1, c-myc, ilk and egfr in tumors
To investigate whether the expression of these genes can also be detected in late stages of tumor development, RTPCR was performed with mRNA from tumors. As shown in Figure 2, all genes identified by the array assay were expressed in tumors induced by asbestos (lanes 1), whereas no expression was detectable in non-tumorous omenta (lanes 4). For comparison, RTPCR analysis was also performed with mRNA from tumors induced by nickel powder and benzo[a]pyrene in the same experimental system and that are histologically identical to the tumors induced by asbestos (15). In tumors induced by benzo[a]pyrene, the expression of all genes was weakly detectable (lanes 3). In nickel-induced tumors, egfr and ilk expression was detected but not fra-1 and c-myc expression (lanes 2). These data demonstrate that genes expressed in non-tumorous tissue in response to asbestos are also expressed in the asbestos-induced tumors. To some extent, the upregulation of these genes occurs in tumors induced by other environmental carcinogens and may, therefore, be a feature of mesothelioma.
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Discussion |
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A significant increase in c-myc mRNA was detected in pretumorous tissues from asbestos-treated animals, as well as in the late stages of tumor development. Enhanced c-myc expression was observed in mesothelial cells treated in vitro with crocidolite for 48 h. The gene for cytosolic branched chain amino acid transferase (bcat) was also upregulated in fiber-induced mesothelioma. This gene was first described in mice as a target for Myc regulation in c-myc-based tumors (16,17). Gene disruption experiments with the homologous bcat gene in yeast have revealed correlation with cell cycle regulation at the G1S transition (18).
In untransformed cells, Myc expression has been shown to be dependent on mitogenic stimuli and to be required for cell proliferation and to prevent differentiation (19). The MycMax heterodimer can bind DNA target sequences regulating genes involved in both cell proliferation and apoptosis (20,21). These two effects occur simultaneously when mesothelial cells are treated with asbestos fibers (22). Our data suggest that, in response to asbestos treatment, cell proliferation and/or apoptosis may be mediated by the c-myc proto- oncogene. Similar data come from human tissues investigated at the protein level (23). A significant increase in signal strength in the mesothelioma tissues, compared with basal expression in non-neoplastic mesothelia, has been observed. However, Goodglick et al. (24) did not find an increase in c-myc mRNA in northern blot analysis with murine mesothelial cell lines derived from different stages of asbestos-induced tumors and reactive tissues. These differing results regarding the expression of the proto-oncogene c-myc may demonstrate a species-specific reaction against asbestos in mouse. On the other hand, these differences may be due to the different experimental approaches.
The results from the array assay demonstrated clearly an induction of AP-1 mRNA in vivo in fiber-treated tissues. Besides c-jun, the fra-1 gene exhibited enhanced expression at the mRNA level in response to fibers (Table II, Figure 3
). The upregulation of this gene in tumors and in fiber-treated cells suggest a role in asbestos-induced carcinogenesis.
Fra-1 forms heterodimers with Jun, generating the AP-1 transcription factor (25). The DNA binding activity and specificity of the Fra-1-containing AP-1 seem to be the same as those of the Fos-containing transcription factor. However, a potent Fos transactivation domain is absent in the smaller Fra-1 protein which, thus, may have a distinct effect. These two characteristics may result in the Fra-1-containing AP-1 having a different specificity from other AP-1 transcription factors.
The dose-dependent induction of c-jun and c-fos at the transcriptional level in response to crocidolite and chrysotile asbestos in RPM cells has been reported by Heintz et al. (26). In hamster tracheal epithelial (HTE) cells, in contrast to c-jun, induction of c-fos was not detected after treatment with asbestos. The authors suggested that in HTE cells Fra-1 may be the partner for Jun, generating an AP-1 transcription factor that mediates the proliferative effects of fibers (26).
Our results with RPM cells showed that fra-1 transcription increased, in a dose-dependent manner, after treatment with crocidolite asbestos (Figure 3b). These results indicate that, in asbestos-treated RPM cells, an additional set of genes is activated by the Fra-1-containing AP-1.
The transcription of AP-1 genes in RPM cells in response to fiber treatment is induced by a protein kinase C (PKC)-dependent signal transduction cascade including MAPKs and the extracellular signal-regulated kinases ERK-1 and -2 (27). Interestingly, autophosphorylation of the EGFR, an event activating the ERK cascade, is induced by asbestos fibers (7). In our experiments, enhanced egfr expression was detected in vivo in pretumorous tissues from asbestos-treated rats, as well as in asbestos-induced tumors. However, there was no increase in egfr expression at the mRNA level in asbestos-treated rat mesothelial cells. Thus, egfr may not be an early responsive gene. The upregulation in the pretumorous tissues after 52 weeks may result from enhanced receptor turnover during persisting fiber stress. In a human mesothelial cell line, expression and accumulation of the EGFR protein occur in vitro after treatment with asbestos fibers (28). However, this upregulation was observed by immunostaining predominantly in singular cells that were in contact with very long fibers (60 µm) or phagocytosing them. In our in vitro experiments analyzing total mRNA from the whole cell population treated with fibers of various length, such an upregulation in individual cells may not be detectable at the mRNA level. Together with the data from mesothelioma cell cultures (4) and from asbestos-treated RPM cells (28), our data suggest that EGFR has a role in fiber-induced mesothelioma development.
A group of genes upregulated by asbestos in the differential RNA analyses is characterized by its connection to integrin-mediated signal transduction. For opn, the gene encoding osteopontin, an extracellular glycoprotein, there were higher levels of mRNA in mesothelioma than in non-tumorous tissue. Osteopontin is known to be expressed most prominently in osteoblasts, but enhanced expression has been found in epithelial tissues and in human carcinomas of different origin (29,30). The interaction of osteopontin with several integrins has been reported (31,32). A change in opn mRNA expression may suggest signal transduction from this extracellular protein, via integrins, to the intracellular space.
Two genes coding for proteins directly linked to the intracellular part of the focal adhesion contact exhibited elevated mRNA expression. The gene for zyxin (zyx) was upregulated in mesothelioma compared with control tissues. Zyxin, a low-abundance phosphoprotein, was discovered by characterization of a rabbit serum that stained subcellular adhesion plaques (33). It consists of three distinct domains: a proline-rich N-terminal region, three double zinc fingers and a central nuclear export signal sequence (34). Both terminal domains interact with structural proteins like -actinin and various signal transduction proteins. Depending on the stimulus received via the focal adhesion complex, zyxin either acts as a nuclear shuttle protein or mediates reactions through its influence on cytoskeleton assembly (35).
Integrin-linked protein kinase, another protein directly connected with the focal adhesion contact, was upregulated at the mRNA level in vivo in pretumorous asbestos-treated tissues and in mesothelioma (Table II, Figure 2
). This enzyme is an intracellular binding partner of integrin ß1 (36). In epithelial cell lines, integrin-linked kinase increases the expression of certain cyclines. Overexpression of this protein results in overriding of the adhesion-dependent regulation of the cell cycle progression through G1 and into S phase, leading to anchorage-independent growth of these cells (37).
Thus, there is upregulation of three independent genes (opn, zyx and ilk) that are linked to transduction of signal from extracellular matrix to the cytoplasm in reaction to asbestos treatment. These results indicate an involvement of integrin-linked signal transduction in asbestos-induced carcinogenesis. Moreover, extracellular matrix proteins seem to be the primary targets of fiber action.
From the results described here, we cannot completely rule out that granular dusts may also have effects on the regulation of the signal transduction genes identified in this study. However, the in vitro findings in RPM cells for egfr and AP-1 indicate that these effects are specific for carcinogenic fibers (27,28). Moreover, we recently demonstrated at the protein level that Fra-1 can be induced in RPM cells by crocidolite asbestos and man-made fibers (MMVF 21) but not by granulous TiO2 (38). The physical and chemical properties that are relevant for mRNA upregulation must be investigated further using different fibrous and non-fibrous dusts.
In summary, Fra-1, c-myc and EGFR may be involved in asbestos-induced mesothelioma carcinogenesis in vivo. Our results also suggest that integrin-linked signal transduction pathways and extracellular matrix proteins may be possible targets of fiber effects.
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Acknowledgments |
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Notes |
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References |
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