Identification of intelectin overexpression in malignant pleural mesothelioma by serial analysis of gene expression (SAGE)

Lung Cancer (2005) 48, 19—29

Identification of intelectin overexpression in malignant pleural mesothelioma by serial analysis of gene expression (SAGE) Anil Walia, Patrice J. Morinb, Colleen D. Houghb, Fulvio Lonardoa, Tsukasa Seyac, Michele Carboned, Harvey I. Passa,∗ a

Thoracic Oncology Program, Karmanos Cancer Institute, and John D. Dingell VA Medical Center, Wayne State University, 3990 John R, Suite 2102, Detroit, MI 48201, USA b Laboratory of Cellular and Molecular Biology, National Institute on Aging (NIH), Baltimore, MD 21224, USA c Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan d Department of Pathology, Cardinal Bernardin Cancer Center, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA Received 26 July 2004 ; received in revised form 18 October 2004; accepted 19 October 2004 KEYWORDS Intelectin; Mesothelioma; SAGE; Glycoprotein; Lectin

Summary Malignant pleural mesothelioma (MPM) is a fatal neoplasm with no acceptable curative approaches. We used serial analysis of gene expression (SAGE) to compare the gene expression pattern of a surgically resected MPM to the autologous normal mesothelium. Intelectin gene overexpression (>139-fold) was found in the tumor. Online SAGE datasets revealed intelectin to be consistently present in mesothelioma(s), ovarian cancer, and colon cancer. Intelectin mRNA expression was found by RT-PCR in 4 of 5 resected MPM tumors, and Intelectin protein expression was confirmed by immunohistochemistry in 28 of 53 MPM tumors, and in 4 of 4 mesothelioma cell lines studied by Western blot. A marked induction in intelectin gene expression was observed among human primary mesothelial cells as a consequence of crocidolite asbestos exposure and simian virus 40 infection. Intelectin overexpression in mesothelioma could have potential screening, and therapeutic implications. © 2004 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

* Corresponding author. Tel.: +1 313 745 8746; fax: +1 313 993 0572. E-mail address: [email protected] (H.I. Pass).

Malignant pleural mesothelioma (MPM) is a fatal tumor that continues to be resistant to various treatment options available at present. It is an aggressive malignancy, which, if untreated, has a median

0169-5002/$ — see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.10.011

20 survival of less than 8 months [1]. Although recent treatment regimens for selected cases (stage I epithelial) have extended the median survival time from less than a year to almost 2 years, it still remains a difficult clinical problem for which new therapeutic options are being evaluated and tested [2,3]. Although the association between the asbestos and mesothelioma has been well established, the multitude of events which characterize mesothelial carcinogenesis remains under investigation and include cytogenetic aberrations with alterations of oncogenes, tumor suppressor genes, and genes associated with growth factors or immune modulation [4,5]. Presently, however, there are no methods to successfully identify patients exposed to asbestos that are at high risk for developing mesothelioma. The aim of this study was to identify differences in the gene expression of MPM tumor compared to normal mesothelium. Serial analysis of gene expression (SAGE) was used to investigate the pattern of differentially expressed gene tags in a human MPM tumor and its matching normal peritoneum. We found elevated expression of intelectin (ITLN1), a novel galactose binding lectin that is usually expressed in heart, colon, small intestine and which has not been previously described in human malignancies. Given the level of intelectin tags expressed in MPM in comparison to mesothelin, we consider it to be a novel finding for mesothelioma and evaluated its expression in a series of MPMs.

2. Results 2.1. Analysis of online SAGE database using the t test The SAGE results obtained on the MPM tumor (RO96) in comparison to its matching peritoneum are shown in Table 1. Our analysis of SAGE databases revealed 38 tags overexpressed and 17 underexpressed in malignant pleural mesothelioma as compared with normal peritoneum. The results in Table 1 verify previous mesothelioma associated findings including the overexpression of cytokeratins and mesothelin [13]. The tag corresponding to a hypothetical protein (AGATCCCAAG) which was subsequently identified as ITLN1 was among the 20 most likely to be overexpressed at a greater than 129-fold increase over normal peritoneum. We used the online SAGE Tag to Gene mapping database survey function (xProfiler) to quantitate tags per million (the virtual Northerns), and found that 14 of the 94 libraries in the online database

A. Wali et al.

Fig. 1 Online SAGE Tag to Gene mapping function (Virtual Northern) showing the distribution of the Hs.50813 (AGATCCCAAG) tag for intelectin within 94 individual libraries. The tags/million column gives a quantitation of the specific tag’s frequency within a specific library, which is reflective of the level of the corresponding transcript. Intelectin expression was found in 14 of the 94 individual libraries composed of 3,934,110 tags representing cumulative online SAGE database.

contained the IntL tag (Hs.50813; Fig. 1). These 14 libraries could be further classified into various categories. The second largest category consisted of four libraries derived from colon epithelium and adenocarcinomas (ADCA) [two from normal colon epithelium NC1 (239 IntL tags/million overall tags), NC2 (282 tags/million), and two from colon adenocarcinomas Tu102 (364 tags/million) and Tu98 (40 tags/million)]. The other category consisted of ovarian serous adenocarcinomas [OVT-6 (23 tags/million), OVT-7 (18 tags/million)]; gastric cancer G234 (15 tags/million) and MCF7 (16 tags/million). Of note, four normal tissue libraries including lung (44 tags/million), liver (14 tags/million), heart (71 tags/million) and muscle (18 tags/million) completed the list.

2.2. RT-PCR analysis of mesotheliomas RT-PCR data confirmed induction of ITLN1 gene expression in R096 tumor versus its peritoneum (Fig. 2a—d), and we also extended this RT-PCR analysis to a small number of MPM tumors in comparison to normal peritoneum. The data shown in Fig. 2 confirmed presence of ITLN1 expression in three out of

Intelectin expression in pleural mesothelioma

Table 1

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Genes elevated more than 20-fold in mesothelioma compared to peritoneum

Tag

Fold meso

Gene

Ovarian cancera

Colon cancera

TCCCCTACAT AGATCCCAAG CCTCCAGCTA TCCCTTTAAG CAGGCCCTGC GCAGGCCAAG TCCCTATTAG CAAACCATCC GAAGCCCCAG TCCCCGTAAT GACATCAAGT TCCCTATAAG CCCCCTGCAG TAGACTAGCA TCCCCGTACT TCCCCGTCAT TGTGGGAAAT TCCCCGACAT AACGCGGCCA CCGTCCAAGG TCCCTGTTAA GCCGGGCCCT TAGCAGCAAT TTTCCCTCAA TGGTTGGTGG GTGCGGAGGA TAAGCTGTGC TTAAACAAAG ACTCCTACTT GCCCCTGCTG GCCCCTCCAG CCTCCAGCAG GCCGGGTGGG CCACCACCCA AGCTGGATGC ATGCTCCCTG CAGTTGTGGT TCCCTAATAA

293 139 112 80 71 61 60 59 59 59 57 52 49 46 39 38 37 37 35 34 34 32 32 31 29 26 26 26 25 25 24 24 24 23 22 21 21 21

Disintegrin-like/metalloprotease (ADAMTS16) Intelectin (ITLN) Keratin 8 (KRT8) Unknown CD3Z antigen, zeta polypeptide (Cd3z) B-factor, properdin (BF) FLJ36644 Keratin 18 (KRT18) Unknown Unknown Keratin 19 (KRT19) Unknown Mesothelin (MSLN) Tetraspan 3 (TSPAN-3) Unknown Unknown Secretory leukocyte protease inhibitor (SLPI) Unknown Macrophage migration inhibitory factor (MIF) Ribosomal protein S16 (RPS16) beta-2-Microglobulin (B2M) vitronectin (VTN) BCG-induced gene in monocytes (BIGM103) Protease, serine, 11 (PRSS11) Plasmolipin (TM4SF11) Serum amyloid A1 (SAA1) Duodenal cytochrome b (FLJ23462) Retinoic acid receptor responder (RARRES1) Uroplakin 1B (UPK1B) Keratin 5 (KRT5) Claudin 15 (CLDN15) Retinoic acid receptor responder (RARRES3) Basigin (BSG) Calbindin 2 (CALB2) Calbindin 2 (CALB2) Galectin 3 binding protein (LGALS3BP) Neuronatin (NNAT) Unknown

++ − + + − + + + + + + + + + + + + + + ++ − − − − − − − − − − − − + − − + − −

+ + ++ − − − + + − + + + − + + + − + + + + − − − − − − − − − − − + − − − − +

a Expression in other tissues is also shown for comparison (−, <15 transcript/100,000; +, 16—150 transcript/100,000; ++, >150 transcript 100,000).

Fig. 2 RT-PCR analysis of intelectin gene expression in MPM: (a) lane 3 (R096 pleura) and lane 4 (R096 tumor); lane 2, water blank and lanes 1and 5 (100 bp DNA ladders); (b) shows amplification of ␤-actin in same samples; (c) shows amplification of intelectin in normal pleura, lane [8], MPM tumors R049 lane [9], R059 lane [10], *R092 positive ITLN1faint band lane [11], R093 lane [12], R096 lane [13], water blank lane [7] and 100 bp DNA ladder in lane [6]; (d) shows amplification of ␤-actin in same samples.

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Fig. 3 Western blot analysis of intelectin protein in MPM: (a) total cellular protein extracts from snap frozen normal pleura (R130), lane 1; MPM tumors (R049, R059, R092, R093, R018 and R083) lanes 2—7; (b) same blot was reprobed with GAPDH as a loading control; (c) SV40 infected mesothelial foci (F1, F3, F4) lanes 8—10; MeT5A lane 11; and four mesothelioma cell lines (H2452, H2595, H2461 and H2714) lanes 12, 13, 14, 15, respectively; (d) shows GAPDH expression in the same blot for normalization. Western blot protocol used is described in detail in the methods section. Overexpression of hIntL was clearly seen in R059 and R018 samples, whereas varying levels were observed in other samples. All mesothelioma cell lines showed higher levels of hIntL compared to foci-1 and -4.

five different MPM tumors using RT-PCR methodology, and the epithelioid histotype of MPM exhibited prominence of ITLN1 expression compared to mixed types.

2.3. Western blot analysis of MPM tumors and cell lines To confirm over expression of ITLN1 at protein level, we analyzed total cellular protein extracts from snap frozen normal pleura, SV40 infected mesothelial foci, MeT5A, and four mesothelioma cell lines (Fig. 3a—d). Overexpression of ITLN1 was clearly seen in R059 sample confirming higher levels of RNA with varying levels observed in other samples. Human mesothelioma cell lines contained ITLN1 and exhibited higher levels of a glycosylated form of ITLN1 protein in cell line H2714 compared to foci-1 and -4.

2.4. Overexpression of intelectin and not ADAMTS 16 in majority of MPM tumors To validate higher levels of ITLN1 expression in a new set of mesotheliomas, we measured gene expression profile of 24 MPM tumors by Affymetrix U133A microarray suite 5.0 as shown in Fig. 4. Gene expression values of ADAMTS 16 (probe set ID 238125 at; Hs.352628) and intelectin (probe set ID 223597 at; Hs.50813) were compared to the levels found in normal peritoneum. The vast majority of MPM samples had higher levels of ITLN1 gene expression in comparison to a few ADAMTS16 overexpressing samples (Fig. 4). This was the rational for focusing on ITLN1 expression rather than ADAMTS 16.

2.5. Immunohistochemical study of MPM Cellular localization of ITLN1 was studied by immunohistochemistry in a multi-tissue block, con-

taining representative tissue sections of resected MPMs. This study revealed that 28/53 MPMs were positive for ITLN1. These included 26 epithelioid, 2 biphasic out of 48 epithelioid, 2 biphasic and 2 sarcomatoid MPMs. Intelectin stained tumor cells within the cytoplasm and/or at the membrane border (Fig. 5), while stromal fibroblasts were negative. ITLN1 was frequently found to be positive within endothelial cells, and rarely within reactive type II pneumocytes, while other cellular components of tumor and normal lung were negative. ITLN1 distribution was also studied in lung cancer, where it was found to be positive only in 2/14 adenocarcinomas, with a membrane pattern, whereas it was negative in 10 squamous cell carcinomas and 6 small cell carcinomas.

2.6. Immunohistochemical analysis of multiple tissue microarrays We have, recently, completed a comprehensive IHC analysis performed on 276 multiple organ tissue microarrays consisting of both normal and deseased tissues (CybrdiTM ). We observed none of the normal lung, kidney, thyroid, brain, uterus, overy, prostate tissues stained positive for the intelectin expression. A very few positive samples for intelectin expression were observed among tumor samples such as 1/2 adenocarcinoma and 1/7 squamous cell carcinoma of lung; 2/4 renal cell carcinoma (clear cell type); 2/5 squamous cell carcinoma of cervix and 1/2 papillary serous adenocarcinoma of ovary. However, a large number of normal and cancerous tissue were found to be positive for intelectin expression among liver (3/5 normal; 4/5 hepatocellular carcinoma); esophagus (4/7 normal; 1/1 carcinoma in situ; 4/6 squamous cell carcinoma); stomach (3/5 normal; 5/7 adenocarcinoma); small intestine (2/5 normal); colon (4/5 normal; 5/9 adenocarcinoma) and pancreatic (2/2 normal; 1/4 adenocarcinoma).

Intelectin expression in pleural mesothelioma

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Fig. 4 Gene expression profile of ADAMTS 16 and Intelectin in 24 malignant pleural mesothelioma cases analyzed by Affymetrix microarray suite 5.0. Higher levels of ITLN1gene expression values were consistenly observed in most of the MPM cases (from #51 to #79) whereas a relatively low level of ADAMTS 16 expression was seen in very few cases compared to normal peritoneums (# 80, 81, and 82), respectively. ITLN1 (filled bars) and ADAMTS 16 (empty bars).

2.7. Effect of asbestos exposure and SV 40 infection on intelectin gene expression We have observed a pronounced two-fold increase in intelectin gene expression between two independent sets of human primary mesothelial cultures treated with crocidolite asbestos for 2 h or infected with SV40 wild-type strain 776 alone for 48 h. The infection of these primary mesothelial cultures with SV40 mutant 883 viral construct had a marginal increase in intelectin gene expression, however, addition of crocidolite asbestos during last 2 h of infection resulted in further increase in intelectin gene expression as shown in Fig. 6. These results are indicative of an important role for both SV 40 infection and asbestos exposure in modulating intelectin gene expression.

3. Discussion In this communication, we provide the first detailed description of the differential gene expres-

sion patterns among the normal pleura and resected malignant pleural mesothelioma tumor with SAGE. We have identified intelectin as a novel finding for mesothelioma using SAGE, confirmed the presence of the intelectin mRNA transcripts by RT-PCR, and verified protein expression by Western blot and immunohistochemical analysis of 53 surgically resected mesotheliomas. These studies were concordant, as overexpression of intelectin was identified in the MPMs studied by each technique. This is the first report describing a comprehensive assessment of this novel lectin to be associated with malignancies of pleural lining of the lung and the gastrointestinal tract. Previously, it was shown to be predominantly expressed in one of the four differentiated cells in the small intestine, ‘‘paneth cells’’, that are derived from the stem cells present in the basal crypt region located just above the paneth cell region [14]. Based upon the high level of intelectin expression seen in normal and adenocarcinoma of colon, it is plausible to speculate that involvement of intelectin with some aspects of the early stages of carcinogenic events associated with the gastrointestinal malignancies.

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Fig. 5 Micrograph showing representative pattern of stain of MPM for ITLN1 by immunohistochemistry: (a) hematoxylin/eosin image of MPM, epithelioid type. Neoplastic cells form papillary clusters, surrounded by desmoplastic stroma; (b) the same case, stained for ITLN1; (e and f) are representative cases from tissue microarray that show ITLN1 positivity localized in the cytoplasm of neoplastic cells (b, d, f), with focal membrane accentuation (e and f), or at the cell membrane (c) while the adjacent stroma is negative (20× magnification).

Intelectin encodes a 40 kDa glycoprotein that recognizes galactofuranose in carbohydrate chains of bacterial cell wall [12]. This novel human galactose-binding lectin was identified and shown to be a new type of Ca 2+ -dependent lectin that has affinity to galactofuranosyl residues [12]. Human intelectin, a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit is composed of a 120kDa homotrimer in which 40-kDa polypeptides are bridged by disulfide bonds [12]. The hIntL gene consists of eight exons on chromosome 1q21.3, and hIntL mRNA is expressed in the heart, small intestine, colon, and thymus [12]. The amino acid sequence of ITLN1 has been shown to be highly homologous (81.5%) with mouse intelectin [14]. In host defense, the lectin-dependent complement activation pathway plays an important role in innate immunity by functioning as opsonins or agglutinins for bacterial infections [15—17]. The animal galactosebinding lectins similar to ITLN1 have also been implicated in cell differentiation [18], apoptosis [19],

recognition of tumor antigens [20], and the uptake of galactosylated glycoproteins such as aged proteins [21]. To make a pathological distinction between malignant pleural mesothelioma and adenocarcinoma of the lung has been quite daunting task given existing set of markers for this purpose, and further studies evaluating ITLN1 as a differential marker between MPM and other malignancies are necessary. Expression profiling for classification of tumors including MPM has been performed. A simple technique based on the expression levels of a small number of genes, was designed to accurately distinguish between genetically disparate tissues using gene expression ratios and rationally chosen thresholds. The study tested the fidelity of ratio-based diagnosis in differentiating MPM and lung cancer in 181 tissue samples (31 MPM and 150 ADCA). Validation of this microarray data and ratio-based diagnosis was performed using calretinin/claudin-7 and VAC(/TACSTD1 ratios, the two ratios correctly identified 23 of 24 samples. Using two and three expres-

Intelectin expression in pleural mesothelioma

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Fig. 6 Role of crocidolite asbestos and SV 40 viral infection on Intelectin gene expression (Affymetrix probe set ID 223597 at; Hs.50813). Primary human mesothelial cultures obtained from two congestive heart failure patients were treated under following experimental conditions: Control Group (A0,G0) represents two untreated primary human mesothelial cultures; Group 1 (A1,G1) are primary mesothelial cultures treated with crocidolite asbestos alone 5 ␮g/cm2 for 2 h; Group 2 (A2,G2) cultures treated with SV 40 small t deletion mutant dl883 alone for 48 h; Group 3 (A3,G3) cultures treated with SV 40 dl883 for 48 h followed by crocidolite asbestos addition during the last 2 h of the experiment; and Group 4 (A4,G4) represents two primary cultures treated with the most common SV 40 strain found in malignant mesothelioma, i.e., SV 40 wild-type (WT) strain 776 for 48 h.

sion ratios, it was found that the differential diagnoses of MPM and lung ADCA were 95% and 99% accurate, respectively [26,27]. Recently, cDNA microarray filters with 4132 clones were used to identify differential gene expression profile among 4 control pleural tissue samples and 16 mesothelioma patient tumor specimens [28]. Use of various normalization and analysis approaches showed significant induction of 166 genes, and down-regulation of 26 genes between these two groups. Expression profiling showed marked up-regulation of genes involved in glucose metabolism, protein translation, and cytoskeletal remodeling pathways. Prominent up-regulated genes included gp96, lung resistancerelated protein, galectin-3-binding protein, laminin receptor, and voltage-dependent anion channels. Our group has, recently, shown that gene expression profiles in malignant pleural mesothelioma can predict time to progression and survival patterns among two separate series of patient populations who underwent cytoreduction at two different cen-

ters here in USA [23]. The 27 gene classifier was able to predict actual time to progression and survival with 95.2% accuracy in one test set where as 76% accuracy was achieved in the separate validation set of MPMs. These data are indicative of pretherapy gene expression analysis that can be beneficial in predicting clinical outcomes of the patients undergoing surgical treatments. Intelectin overexpression has potential screening applications in MPM. Since intelectin is not strongly expressed in normal mesothelium, expression of intelectin could be utilized in conjunction with other markers to confirm presence of mesothelioma, particularly in small biopsy or cytopathological samples. The immmunohistochemical stain pattern observed with intelectin raises the possibility that intelectin may be secreted into pleural space or released into the blood, and assayed from these fluids as a harbinger of MPM development. The finding of intelectin overexpression in a select group of mesothelioma patients and other gas-

26 trointestinal related malignancies affords a potential therapeutic application for this protein. As a galactose-binding lectin, intelectin can be used as an effective target for immunotoxins to treat these patients. Our findings raise the possibility that either antibody-based immunotherapy against intelectin or cell mediated immunotherapy may hold promise as a novel line of defense for a particular set of mesothelioma as well as GI cancer patients. Moreover, a modified natural polysaccharide modified citrus pectin (MCP) has been found to have clinical activity as an anti-cancer agent in a number of Phase I/II clinical trials [22]. MCP alters melanoma progression in vivo, and the mechanism is believed to be through the galactose containing carbohydrate side chains of MCP mimicking or competing with the natural ligand(s) of galectin. One can postulate that this modified pectin may have the capability not only to interfere with galectin3 and bFGF receptor interactions, but also with other carbohydrate-recognition proteins such as intelectin to decrease invasion and impair tumor angiogenesis. We have shown in vitro experiments that both crocidolite asbestos exposure or SV 40 infection of primary mesothelial cells can cause upregulation of intelectin gene expression either alone or in combination. Our results are in direct agreement with other published reports where concentrations of asbestos and asbestos-like fibers have been linked to the alterations in the glycohistochemical properties of alveolar lining cells [29]. Similarly, changes in nuclear glycoproteins especially proteins bearing the terminal N-acetylglucosamine sugar moieties have been shown to be altered as a consequence of SV 40 viral transformation in WI-38 human lung fibroblasts [30]. These studies lend a firm support to our data and might probably explain the contention of intelectin transcriptional regulation being mediated by these two known co-carcinogens in mesothelioma tumorigenesis. To what extent these in vitro studies reflect the in vivo situation in mesothelioma carcinogenic events remains to be elucided and are the subject of future experiments. Our results confirm that SAGE data can be used to identify genes that are differentially expressed between tissues. Intelectin becomes the first novel gene identified by this technique that has been validated in fresh and archived MPM tissue. Further analysis of other unknown SAGE tags should yield valuable information regarding early detection and prognosis of this disease. We have demonstrated that intelectin, one of the tags identified in our SAGE library of mesothelioma, is consistently and highly overexpressed in a proportion of mesotheliomas and gastrointestinal malignancies at the pro-

A. Wali et al. tein level. Thus, further analysis of intelectin gene expression in the pathogenesis of MPM and GI related cancers should provide a valuable insight into future therapeutic interventions for these diseases.

4. Materials and methods 4.1. MPM tumor and matching peritoneum With investigational review board approval, tumor from a 39-year-old male (R096) undergoing extrapleural pneumonectomy for T2NOMO mesothelioma was immediately snap frozen in liquid nitrogen along with uninvolved peritoneum to construct SAGE libraries. Other mesothelioma tumors (R018, R049, R059, R083, R092, R093) as well as normal pleura (R130) from the Karmanos Thoracic Oncology Archives were used for confirmatory analyses.

4.2. SAGE The construction and analysis of the SAGE libraries were performed as previously described (Hough et al. [6]). Briefly, the frozen tumor and normal peritoneum samples (approximately 0.5 g) were lysed in guanidium isothiocyanate and total RNA purified by centrifugation on CsCl. Polyadenylated mRNA was isolated from total RNA using the Messagemaker kit (Life Technologies), and the cDNA was generated using the cDNA Synthesis System (Life Technologies). The cDNA was used to create the SAGE tags, using NlaIII as an anchoring enzyme and BsmFI as a tagging enzyme, as described in the SAGE protocol (Velculescu et al. [7]). Approximately 1500 clones were sequenced for each of the two libraries and the sequence deposited in the CGAP SAGE database (Lal et al. [8]). This sequencing yielded 53,746 tags and 41,121 tags for the peritoneum and malignant mesothelioma samples, respectively.

4.3. Computational analysis of SAGE databases Sequence data were analyzed using the SAGE software to quantify the tags, make comparisons and identify corresponding transcripts [7]. In addition, Online database SAGE data (http://www.ncbi.nlm.nih.gov/SAGE) was also used for virtual gene expression analysis (Lash et al., 2000). Virtual Northern blots were created using a feature available on the online SAGE database. Using this tool, we were able to view the expression levels of selected set of SAGE tags

Intelectin expression in pleural mesothelioma in all of the SAGE libraries. Data were represented as ‘‘virtual Northerns’’ to visualize the levels of gene expression across multiple samples.

4.4. Mesothelial and mesothelioma cell lines The SV40 Tag immortalized mesothelial cell line, MeT5A was obtained from American Type Culture Collection (Rockville, MD) and cultured in LHCMM medium. In addition, three human mesothelial foci immortalized by SV40 infection of human pleural mesothelial cells (Foci 1, 3, and 4) [10] were maintained in RPMI medium supplemented with 10% fetal calf serum. Four MPM patient derived cell lines [H2452, H2595, H2461 and H2714] established in our laboratory and characterized in detail [11] were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 100units/ml of penicillin, 100 ␮g/ml streptomycin, 4 mM l-glutamine, and 10% fetal calf serum. Cells were incubated at 37◦ C in a humidified atmosphere of 5% CO2 in air and were passaged weekly. Two primary human mesothelial cultures obtained from two congestive heart failure patients were treated separately under following experimental conditions: Control Group (A0,G0) consisted of two individual untreated primary human mesothelial cultures; Group 1 (A1,G1) comprised of same primary mesothelial cultures treated with crocidolite asbestos alone 5 ␮g/cm2 for 2 h; Group 2 (A2,G2) were cultures treated with SV 40 small t deletion mutant dl883 alone for 48 h; Group 3 (A3,G3) were cultures treated with SV 40 dl883 for 48 h followed by crocidolite asbestos addition during the last 2 h of the experiment; and Group 4 (A4,G4) consisted of primary cultures treated with the most common SV 40 strain found in malignant mesothelioma, i.e., SV 40 wild-type (WT) strain 776 for 48 h. Affymetrix human genome U133plus microarrays were performed on the RNA preparations obtained from primary human mesothelial cultures treated under different experimental conditions. The arrays were analyzed by GeneSight software package from BioDiscovery Inc. and intectin values were assessed by Microarray suite 5.0 [23].

4.5. Confirmation of intelectin expression with RT-PCR analysis Total RNA was extracted from the MPM tumors, normal human pleural tissues and cell lines using Trizol Reagent (Life Technologies). cDNA was synthesized from 3 ␮g of RNA using the Superscript II kit (Life Technologies) according to the manufacturer’s instructions, with oligo-dT primer (10 pmol) for

27 15 min at 68 ◦ C. The most suitable PCR amplification conditions were determined for intelectin expression using a set of primers by on line primer design program (http://www-genome.wi.mit.edu/cgibin/primer/primer3 www.cgi). PCR primers used in amplification of full length cDNA (962 bp) of intelectin (NCBI GenBank AB036706; Hs.50813) were as following: 5 sense ATG AAC CAA CTC AGC TTC CTG CTG and 3 antisense GGT TCC CTC CCA CAA AAC TC. Primers used in ␤-actin amplification (531 bp) were 5 sense GAT TCC TAT GTG GGC GAC GAG and 3 antisense CCA TCT CTT GCT CGA AGT CC. PCR conditions for both primer sets were as follows: 35 cycles at 94 ◦ C for 60 s, 55 ◦ C for 60 s, and 72 ◦ C for 60 s using Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ). The PCR products were electrophoresed through 1.0% agarose gel. Each expression was normalized to ␤-actin signal as an internal control. Negative controls of each gene would be PCR reactions without template cDNA.

4.6. Expression analysis of ADAMTS 16 and intelectin using Affymetrix U133A probe arrays The RNA target (biotin-labeled RNA fragments) were produced from a new batch of 24 MPM tumor total RNA preparations by first synthesizing double-stranded cDNA followed by an in vitro transcription reaction and a fragmentation reaction. A hybridization cocktail containing the fragmented cRNA, probe array controls (Affymetrix U133A), bovine serum albumin (BSA), and herring sperm DNA was prepared and hybridized to the probe array at 45 ◦ C for 16 h. The hybridized probe array was then washed and bound biotin-labeled cRNA detected with streptavidin phycoerythrin conjugate. A subsequent signal amplification was performed with a biotinylated anti-streptavidin antibody. The washing and staining procedures was automated using the Affymetrix fluidics station. Each probe array was scanned twice (Hewlett-Packard GeneArray Scanner), the images overlayed, and the average intensities of each probe cell compiled using Affymetrix microarray suite 5.0 as described in detail for our previous batch of 21 MPM cases (Affymetrix U95 set) [23].

4.7. Western blot analysis of intelectin Total cellular protein extraction from snap frozen normal pleura, MPM tumors and cell lines ground to a powder under liquid nitrogen was carried out following standard protocols. Cell lysates were pre-

28 pared from these samples by allowing them to incubate on ice for 30 min in 400 ␮l of lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris, pH 8.0, 0.5 mM phenylmethylsulfonylfluoride (PMSF), 1 ␮g/ml each: pepstatin, aprotinin and leupeptin). The lysates were centrifuged at 14,000 rpm at 4 ◦ C for 15 min to remove debris. Supernatant proteins, 50 ␮g from each sample were separated by SDS-12% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Nytran membranes (S&S) by standard procedures. Polyclonal antibodies against human intelectin (kindly provided by author T.S.) were used as per standard procedures and as described in detail [12]. Proteins were visualized by treatment with enhanced chemiluminescence reagent (ECL, duPont-NEN Renaissance System) according to manufacturer’s instructions, followed by exposure to film (Kodak X-Omat).

A. Wali et al. localization of the primary antibody and color development was obtained by incubation with biotinilated anti-rabbit, followed by incubation with streptavidin-coupled horseradish peroxidase, using reagents provided in an avidin-biotin detection kit (Vectastatin kit, Vector Labs, Burlingame, CA). Sections were lightly counter-stained with hematoxylin (Sigma) and cover-slipped.

Acknowledgements This work was supported in part by a grant from the Early Detection Research Network,VA Merit awards to HIP, and an American Lung Association grant and VA Merit award to AW. We thank Julia SluchakCarlsen, Apurvi Desai and Valerie Murphy for technical support.

4.8. MPM tissue microarrays and immunohistochemistry for intelectin

References

MPM tissue microarray was constructed using Beecher Instruments (Silver Spring, MD) as described in detail previously for other tumors [24]. Briefly, samples of formalin-fixed, paraffinembedded resected mesotheliomas were obtained from the archives of the Department of Pathology, Harper University Hospital, Detroit Medical Center, Michigan. Consent was obtained from all patients and the project was approved by the local Institutional Review Boards. All patients had cytoreductive surgery, mediastinal lymph node dissection with staging by the International Mesothelioma Interest Group (IMIG) staging system [25]. The tissue microarray contained 53 MPMs, in addition to control tissues, including normal pleural mesothelium, fibrous pleurisy, kidney and lung samples. Immunohistochemistry was performed using standard Avidin Biotin Complex techniques. In brief, slides were deparaffinized and rehydrated via passage through graded alcohol solutions, according to standard histological techniques. Heat induced epitope retrieval (HIER) was obtained by heating the slides for 20 in a citrate buffer (2 mM Citric acid/10 mM NA Citrate) pH 6 in a steam bath followed by cooling down for 20 at room temperature (RT). Slides were pre-saturated with 0.05% bovine serum albumin in phosphate buffered saline (PBS) (Sigma) for 10 , quenched in 2% solution of hydrogen peroxide for 10 , and pre-incubated with 0.5% non immune goat serum (Sigma) for 10 at RT. The sections were then incubated with a polyclonal anti-ITLN1 antibody at a 1:400 dilution, for 120 min, at room temperature. After three washes in BSA/PBS (5 each)

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