Differentially expressed genes in asbestos-induced tumorigenic human bronchial epithelial cells: implication for mechanism
Yong L. Zhao,
Chang Q. Piao,
Li J. Wu1,
Masao Suzuki and
Tom K. Hei2
Center for Radiological Research, College of Physicians & Surgeons of Columbia University, VC11-218, 630 West 168th Street, New York, NY 10032, USA
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Abstract
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Although exposure to asbestos fibers is associated with the development of lung cancer, the underlying mechanism(s) remains unclear. Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, we previously showed that UICC chrysotiles can malignantly transform these cells in a stepwise fashion before they become tumorigenic in nude mice. In the present study we used cDNA expression arrays to screen differentially expressed genes among the tumorigenic cells. A total of 15 genes were identified, 11 of which were further confirmed by northern blot. Expression levels of these genes were then determined among transformed BEP2D cells at different stages of the neoplastic process, including non-tumorigenic cells that were resistant to serum-induced terminal differentiation, early and late passage transformed BEP2D cells, five representative tumor cell lines and fused tumorigeniccontrol cell lines which were no longer tumorigenic. A consistent 2- to 3-fold down-regulation of the DCC (deleted in colon cancer), Ku70 and heat shock protein 27 genes were detected in all the independently generated tumor cell lines while expression levels in early transformants as well as in the fusion cell lines remained normal. In contrast, all the tumor cell lines examined demonstrated 2- to 4-fold overexpression of the insulin receptor and its signal transduction genes. Differential expression of these genes was completely restored in the fusion cell lines examined. No alteration in c-jun or EGF receptor expression was found in any of the cell lines. Our data suggest that activation of the insulin receptor pathway and inactivation of DCC and Ku70 may cooperate in malignant transformation of BEP2D cells induced by asbestos.
Abbreviations: AP-1, activator protein-1; DCC, deleted in colon cancer; DNA-PK, DNA-dependent protein kinase; ERK, extracellular signal-related kinases; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; HSP27, heat shock protein 27; MAPK, mitogen-activated protein kinase; NF
B, nuclear factor
B; RTK, receptor tyrosine kinases; SV40, simian virus 40.
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Introduction
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Although occupational exposure to asbestos fibers is associated with development of pulmonary fibrosis, bronchogenic carcinoma and mesothelioma (1), the mechanism(s) of fiber carcinogenesis is not clear. There is evidence suggesting that asbestos-induced reactive oxygen species and/or growth factors may be involved in the genotoxic/carcinogenic process (2,3). The observation that antioxidant enzymes, such as catalase and superoxide dismutase, can protect cells against the cytotoxic and mutagenic effects of asbestos provides further evidence for the role of oxygen radicals in fiber toxicology (4,5). Asbestos has been shown to trigger many signaling events via activation of nuclear factor
B (NF
B) and other early response proto-oncogenes, including c-fos and c-jun (6,7), which in turn increase the DNA-binding activity of the transcription factor activator protein-1 (AP-1) and lead to increased cellular proliferation and morphological transformation of tracheal epithelial cells (8,9).
Previous studies from this laboratory have shown that asbestos is a potent gene and chromosomal mutagen and induces predominantly multilocus deletions (10,11). While chromosomal deletions in malignant mesotheliomas are relatively common events (12,13), there is no evidence that mutations in several common suppressor genes, such as Rb, p53 and Wilm's tumor, play any causal role in asbestos-associated cancers (1416). These findings suggest that loss and/or inactivation of multiple tumor suppressor genes are a possible mechanism of fiber carcinogenesis (17). It is likely that oncogenes and other, as yet unidentified, tumor suppressor genes may act in concert to mediate asbestos-induced bronchogenic cancers and mesotheliomas.
For a better understanding of the molecular mechanism of bronchial carcinogenesis induced by asbestos, it would be ideal to use a human bronchial epithelial cell line to study the various asbestos-induced genetic alterations leading to malignancy. Up to the present moment, no primary human cell model is available for this area of study because the frequency of human cell transformation has been estimated to be ~1015, an incidence too low to be reproduced in any laboratory setting (18). Treatment of normal human mesothelial cells with amosite asbestos has been shown to extend the proliferative lifespan of four of 16 independently derived primary cultures (19). However, these cells eventually all senesce and enter crisis. Using human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells, our laboratory recently showed that a single, 7 day treatment with a 4 µg/cm2 dose of chrysotile induced neoplastic transformation of these cells in a stepwise fashion at a frequency of ~107 (20). Transformed cells progress through a series of stepwise changes, including altered growth pattern, resistance to serum-induced terminal differentiation and agar-positive growth, before becoming tumorigenic and producing subcutaneous tumors upon inoculation into athymic nude mice (20,21). However, control BEP2D cells are anchorage dependent and non-tumorigenic even in late passage (20). Since BEP2D cells express both E6 and E7 viral proteins, these data suggest that abnormal p53 and Rb functions are insufficient for tumorigenic conversion in these cells and additional genetic factors and cellular events are required. There is recent evidence that simian virus 40 (SV40), which also inactivates Rb and p53 functions in infected cells, is found in >60% of human malignant mesotheliomas (22). These data suggest that asbestos and SV40 could potentially act as co-carcinogens in asbestos-mediated malignancies and provide further support for the suitability of BEP2D cells in mechanistic studies of fiber carcinogenesis.
Identification of alterations in gene expression profiles in asbestos-induced transformed cells at various stages of the neoplastic process will lead to a better understanding of the mechanism of fiber carcinogenesis. A recently developed technique, cDNA expression array, allows the large scale comparison of multiple genes in a single hybridization. The assay has the advantage of providing rapid and immediate information on the genes of interest as well as the functions of their proteins. In this study we have used cDNA expression array to compare the expression of 588 known cellular genes in cell lines derived from control and tumorigenic BEP2D cells induced by asbestos fibers. The hybridization signals were further screened by northern blotting, using cell lines derived from different transformation stages (20). In addition, fusion cell lines between fiber-induced tumorigenic and control BEP2D cells were generated to determine the dominant or recessive nature of the neoplastic phenotype. We show herein that all fusion clones were no longer tumorigenic when subsequently inoculated into nude mice. Furthermore, there was a consistent 2- to 3-fold down-regulation of DNA-dependent protein kinase (DNA-PK) regulatory subunit Ku70 and DCC (deleted in colorectal cancer) gene expression, as well as a 2-fold increase in expression of the insulin receptor gene among tumorigenic BEP2D cells. These changes in expression levels were completely abrogated in all the fusion cell lines tested. Our data suggest that deregulation of these genes may play an important role in fiber carcinogenesis.
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Materials and methods
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Cell lines
Human papillomavirus (HPV18)-immortalized human bronchial epithelial (BEP2D) cells were used in this study (23). These cells are anchorage dependent and non-tumorigenic in nude mice even in late passages (20). Tumorigenic cells were previously derived by treatment of exponentially growing BEP2D cells with a 4 µg/cm2 dose of chrysotile fibers for 7 days. Tumors >1 cm in diameter were resected from nude mice and used to establish independently generated cell lines. The human epithelial origin of the cell lines established was determined using isozyme analyses as well as immunohistochemical staining for human cytokeratin (20,23).
Previous studies from this laboratory have demonstrated that transformed BEP2D cells arise through a series of sequential stages (18,20). In order to study the alterations in gene expression during the course of the transformation process, BEP2D cells at different stages of the neoplastic process were used in northern blot analyses. These included transformed/non-tumorigenic cell lines derived from a serum-resistant colony after fiber treatment, early passage (1 week after fiber treatment) and late passage (just before inoculation into nude mice) transformed cells, five representative tumor cell lines and four independently cloned, non-tumorigenic fusion cell lines. All cultures were maintained in serum-free LHC-8 medium supplemented with growth factors as described previously (18,20,23).
Cell fusion
Tumorigenic BEP2D cells induced by asbestos treatment were fused with control cells using polyethylene glycol. Briefly, 5x106 tumorigenic cells infected with a pRC/CMV expression vector containing a neo gene were fused with an equal number of control BEP2D cells containing a pBabe plasmid which was resistant to puromycin using 1 ml of pre-warmed 50% PEG 1500. The polyethylene glycol was added drop-wise over a 1 min period to initiate the cell fusion process followed by addition of 20 ml of culture medium over 6 min with constant agitation. The resultant fusion cells were then selected in medium containing both G418 and puromycin over a 12 day period, expanded in culture and re-inoculated into nude mice for tumorigenic expression as described (18,20). Controls involving the fusion of tumortumor and controlcontrol cells were similarly conducted.
Tumorigenicity in nude mice
Male Nu/Nu mice from Harlem SpragueDawley were given a whole body 4 Gy dose of
-irradiation 24 h before tumor inoculation. Each animal was anesthetized lightly with isofurane (Anaquest, Madison, WI) and injected s.c. with either 56x106 fusion or control cells in 0.2 ml of saline at two different sites, one in the inter-scapular area and the other in the lower back. Animals were maintained under sterile condition for ~78 months and palpated for tumor appearance once a week. Control animals were inoculated with either BEP2D cells or with previously established fiber-induced tumorigenic cells. Animals were killed as soon as tumor nodules attained ~0.50.8 cm in size.
Isolation of total RNA and poly(A)+ mRNA
Total RNAs were extracted from exponentially growing cultures using Trizol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Poly(A)+ mRNAs were then enriched using an oligotex mRNA kit (Qiagen, Valencia, CA). Typically, 34 µg of mRNA can be isolated from 100 µg of total RNA using this system. Total RNA and mRNA concentrations were assessed by absorbency at 260 nm using a UV spectrophotometer (Perkin Elmer). The quality of total RNA and mRNA was determined by running on a denaturing formaldehyde gel. For cDNA array, mRNAs were treated with DNase I (Genhunter, Nashville, TN) at 37°C for 30 min in order to eliminate any contamination with genomic DNA.
cDNA expression array and hybridization
The 588 human cDNAs of known genes were spotted in duplicate on a nylon membrane (Clontech Laboratories, Palo Alto, CA). 32P-labeled cDNAs were prepared by reverse transcription using 1 µg mRNA from control and tumorigenic BEP2D cells in the presence of gene-specific primers and [
-32P]dATP. The probes were then purified using a spin column to remove unincorporated nucleotides. The radioactivity of the probes used for hybridization was adjusted to 1x106 c.p.m./ml hybridization solution. After pre-hybridization at 68°C for at least 30 min, hybridization was performed at 68°C in a rolling bottle overnight, followed by washing under stringent conditions as recommended by the manufacturer. The hybridization signals were analyzed by autoradiography and further quantified by phosphorimaging (ImageQuant software). Expression levels of the housekeeping genes of human ß-actin and glyceraldehyde 3-phosphate dehydrogenase (G3PDH) were used as standards for normalizing the expression levels of other genes.
Northern blotting
For northern blotting, 2.5 µg mRNA was denatured and separated on a 1% denaturing agarose formaldehyde gel. The mRNAs were then transferred to a nylon membrane (Millipore, Bedford, MA) by downward capillary blotting in 20x SSC (3 M NaCl, 0.3 M sodium citrate·2H2O, pH 7.0) followed by UV crosslinking. Specific probes were generated by labeling of PCR-amplified cDNA fragments with [
-32P]dCTP using a random primed DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The membranes were pre-hybridized for 30 min and then hybridized with cDNA probe in ExpressHyb hybridization solution (Clontech) for 812 h at 68°C. The blots were washed twice in 2x SSC, 0.1% SDS at room temperature for 15 min followed by washing twice in 0.2x SSC, 0.1% SDS at 55°C for 15 min. The membranes were exposed to Kodak BioMax film at 70°C for 1272 h. The band intensities were evaluated by phosphorimaging and normalized to the ß-actin expression level.
Probes for northern blots
All probes were acquired by PCR amplification of gene fragments. The primers were designed by a GenBank database search. The sequence and length of primers (S, sense; A, antisense) are listed in Table I
. The amplified products were excised from agarose gels and the DNAs were extracted with a gel extraction kit (Qiagen) according to the user manual.
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Results
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Fusion of tumor and control BEP2D cells abrogates the malignant phenotype
Table II
shows the results of a representative fusion study. The tumorigenic AsbTB2A cell line was a highly malignant derivative of BEP2D cells obtained by treatment with chrysotile asbestos as described (20). It was evident that the tumorigenic phenotype of TB2A cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. A total of five representative, independently derived fusion clones were tested for their tumorigenic potential in nude mice and none produced any tumors. Furthermore, concurrent fusion of tumor cells with tumor cells resulted in tumorigenic hybrids whereas fusion among wild-type BEP2D cells resulted in non-tumorigenic hybrid clones.
Gene expression profile by cDNA array
32P-labeled cDNA probes were generated from each poly(A)+ mRNA of control and asbestos-induced tumorigenic BEP2D cells and separately hybridized to identical cDNA arrays which contained 588 known genes representing different functional categories and nine housekeeping genes as positive controls. After exposure to X-ray film with a Biomax MS intensifying screen, the patterns of expression of the 588 known genes were examined. The hybridization signals were also quantified by phosphorimaging. By comparing the hybridization signals normalized to the average intensities of the ß-actin and G3PDH genes, a total of 15 genes were identified to be differentially expressed in asbestos-induced tumorigenic cells relative to controls. Representative genes are marked in Figure 1
(arrow). The ß-actin and G3PDH genes, which were initially chosen as positive controls for hybridization, were, as expected, highly and steadily expressed in these two cell lines. Arrays were hybridized with probes from mRNAs of control and tumorigenic cells. After exposure and quantification, they were stripped and rehybridized with new probes from different batches of the same mRNAs from control and tumorigenic cells. Similar results were obtained from the two rounds of independent hybridization, suggesting that the identified changes in gene expression were consistent and reproducible.

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Fig. 1. Gene expression profile of control and tumorigenic BEP2D cells. 32P-labeled cDNAs were synthesized by reverse transcription using 1 µg mRNA from control and representative tumorigenic BEP2D cells induced by asbestos fiber treatment and hybridized to the Atlas cDNA arrays. After stringent washing, the blots were exposed to Kodak MS film for ~2 days at 70°C and quantified by phosphorimaging. Arrows, representative cDNA spots that are differentially expressed in tumorigenic cells relative to control: 1, DCC; 2, HSP27; 3, NF B (p50/105); 4, IR.
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Northern blot analysis of cells from different stages of the neoplastic process
The 15 genes whose expression levels were detected by cDNA array to be different from control BEP2D cells were further cross-checked by northern blot using mRNA obtained from various cell lines, including serum-resistant transformed cells, early and late passage transformed cells, five representative tumor cell lines and four fusion cell lines. Eleven of the 15 genes were confirmed to be differentially expressed in all tumorigenic cells compared with controls (Figure 2
and Table III
). Expression levels of three genes, DCC, Ku70 and heat shock protein 27 (HSP27), were found to be lowered by 2- to 3-fold in all five tumor cell lines and restored to control level among the fusion cells (Table III
). In contrast, expression levels of these genes among serum-resistant transformed cells and in early and late passage cells were comparable with the controls. Eight genes, including the insulin receptor (IR), src homolog 2 adaptor (SHB), Grb2, ERK2, c-fos, NF
B (p50/105), Ets-like gene and cdc2-related kinase (PISSLRE) were found to be overexpressed in all five tumor cell lines at levels ranging from 2- to 5-fold of controls. Overexpression of these genes, however, was abrogated in all fusion cell lines examined. Furthermore, no change in expression levels of these genes was identified in either serum-resistant or in early and late passage transformed cells with the notable exception of the IR and c-fos genes (Table III
).

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Fig. 2. Northern blot results of differentially expressed genes found in the cDNA array. Aliquots of 2.5 µg mRNA were isolated from control BEP2D cells (lane1), non-tumorigenic serum-resistant transformed cells (lane 2), early passage cells (1 week post-asbestos treatment), late passage cells (just before inoculation into nude mice), five representative tumor cell lines (lanes 59) and a representative non-tumorigenic fusion cell clone (lane 10). The other fusion cell lines gave similar results. The mRNA blots were hybridized to 32P-labeled human cDNA probes. After stripping, the membranes were rehybridized to human ß-actin probe.
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Discussion
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Although asbestos fiber has been known to be a cancer-causing agent for centuries, its carcinogenic mechanism remains unclear. There are indications that as many as five interrelated mechanisms may be involved, depending on fiber type, fiber dimension and type of tumor induced (17,24). The observation that asbestos is mutagenic and induces primarily multilocus deletions (10,11) provides a mechanistic basis for the loss of putative tumor suppressor functions in asbestos-induced tumors. This finding is consistent with the cytogenetic abnormalities, mainly deletions, identified in many asbestos-induced mesotheliomas (12,13), as well as our present cell fusion studies, in which the tumorigenic phenotype of TB2A cells could be completely suppressed by fusion with non-tumorigenic BEP2D cells. In order to find the gene(s) involved, we used cDNA expression array to screen the expression profiles of 588 known genes in control and asbestos-induced tumorigenic BEP2D cells. To alleviate concerns about false positive signals, northern blots were used to cross-check the message level. Our finding of 25% false positives is consistent with other recent reports (25,26).
Asbestos fibers have been shown to trigger a number of signaling cascades involving mitogen-activated protein kinase (MAPK) which may be initiated through receptor-mediated events (27). The insulin receptor is a member of a large family of receptor tyrosine kinases (RTK) possessing intrinsic cytoplasmic enzymatic activity, which are essential components of signal transduction pathways that affect cell proliferation, differentiation and migration (28). There is evidence that activation of RTK such as the epidermal growth factor receptor can in turn activate numerous intracellular signaling cascades via the MAPK pathway, including extracellular signal-related kinases (ERK), which leads to activation of the transcription factor c-fos and N-terminal kinases (JNK and SAPK), which in turn activates c-jun (2,7). The proteins encoded by these stress genes could dimerize with the transcription factor AP-1 and promote the G1
S phase transition (8,29). In the present study, expression of the insulin receptor was up-regulated by ~1.5-fold in both the early and late passage transformed cells but by ~2.2-fold in all tumor cell lines examined. This increase in insulin receptor expression could, perhaps, account for the increased expression of other downstream genes such as SHB, Grb2, Erk2, the Ets-like gene and c-fos found in all tumor cell lines (28). The insulin receptor has been shown to be a potential oncogene for mammary epithelial cells (30,31), overexpression of which could enhance growth and the transformed phenotype in cultured cells. However, there is evidence that insulin receptor activation alone is insufficient to induce malignant transformation in epithelial cell lines (31). This is consistent with our present finding that a 6-fold overexpression of its downstream signal, c-fos, while conferring a serum-resistant phenotype, fails to induce malignant characteristics.
NF
B is an early response gene induced by a variety of stimuli, including oxidative stress (33,34). Several lines of evidence have shown that asbestos can activate NF
B and its translocation to the nucleus, which is involved in its tumor growth, metastasis and anti-apoptosis effects (6). In the present study we found that NF
B (p50) was overexpressed by 1.7-fold in late passage transformed BEP2D cells and by 3-fold in all five tumor cell lines examined. This finding is consistent with the observation that asbestos induces a dose-dependent protracted increase in NF
B DNA-binding activity in tracheal and lung epithelial cells (34,35). The observations that antioxidants or overexpression of the NF
B inhibitor I
B decrease NF
B gene expression and inhibit tumorigenicity (36) provide circumstantial evidence in support of a role of NF
B overexpression in fiber carcinogenesis. The possibility that NF
B might be activated via the insulin receptor by transcriptional and anti-apoptotic modulation in asbestos-treated BEP2D cells is further supported by the recent finding that the anti-apoptotic function of insulin requires NF
B activation (37).
Among the tumorigenic cells, we found consistently reduced expression of DCC and the DNA-PK subunit Ku70, but not in any of the early or late passage transformed BEP2D cells. This finding is consistent with the observation that DCC, a candidate tumor suppressor, is frequently deleted not only in colorectal cancer, but also in a number of other malignancies as well (38,39). Ku70, as a regulatory subunit of DNA-PK, is involved in the repair of DNA double-strand breaks by stabilizing broken DNA ends and activating kinase activity. Down-regulation of Ku70 renders cells more sensitive to oxidative stress and increases chromosome instability as a result of DNA recombination errors, which leads to an increased propensity for malignant transformation both in vitro and in vivo (40,41). These data, together with our present results, suggest that down-regulation of DCC and Ku70 may contribute to the malignant progression of BEP2D cells induced by asbestos.
Finally, our results show that cDNA expression array is a useful and direct method in the identification of differentially expressed genes between samples of the same cell model. Based on our current findings, it is possible that asbestos activates the insulin receptor gene in BEP2D cells which in turn induces overexpression of c-fos via the ERK signaling pathway, as well as NF
B, which renders the cells resistant to apoptosis (28,37). An increased rate of cell proliferation is required for the fixation of genetic alterations and confers a proliferative advantage to preneoplastic population. However, insulin receptor activation in itself is insufficient for malignant transformation. Reduced expression of DCC and Ku70 might collaborate with insulin receptor activation in malignant progression by promoting genomic instability and increased susceptibility to oxidative damage. Evidence for this line of thought comes from the cell fusion studies, which indicate that loss of tumorigenic potential is accompanied by re-expression of these genes at the control levels. While our present findings provide some clues to the mechanism of fiber carcinogenesis, additional studies are needed to determine how these multiple pathways are integrated in the target epithelium.
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Note added in proof
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Since the submission of this manuscript, a paper by Sindhu et al. (Carcinogenesis, 21, 10231029, 2000) reported up-regulation of several early response genes including c-myc, ilk, EGFR, c-jun and fra-1 among rat mesothelioma induced by an i.p. injection of South African crocidolites as well as in pre-cancerous tissues when analyzed using cDNA expression arrays. In contrast, we detected no changes in the expression level of these genes in our present study. The discrepancy may be due to differences in response among different target tissues since there is ample evidence to suggest that bronchial epithelium expresses different growth-related genes in response to asbestos treatment as compared with mesothelial cells. Furthermore, there are also differences in species-specific responses as well.
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Notes
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1 Present address: Department of Ion Beam Bioengineering, Chinese Academy of Sciences, Hefei, China 
2 To whom correspondence should be addressedEmail: tkh1{at}columbia.edu 
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Acknowledgments
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The authors thank Dr Yu-xin Yin for helpful discussions. This work was supported by NIH grants ES 07890 and ES 05786.
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Received March 27, 2000;
revised June 22, 2000;
accepted July 5, 2000.