Laminin α2 Chain-Positive Vessels and Epidermal Growth Factor in Lung Neuroendocrine Carcinoma
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.041310
ISSN1525-2191
AutoresDomenico Vitolo, Luciano Ciocci, G.P. Deriu, Silvia Spinelli, Stefania Cortese, Laura Masuelli, Stefania Morrone, Mary Jo Filice, Giorgio F. Coloni, Pier Giorgio Natali, Carlo Baroni,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoCapillaries expressing the laminin α2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin α2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin α2 chain rather than through the laminin β1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the α6β1 integrin, which is one of the laminin α2 chain receptors. EGF up-regulated α6β1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-α6 integrin chain monoclonal antibody. These data indicate that laminin α2 chain and α6β1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin α2 chain-positive vessels may favor metastasis of EGF-dependent tumors. Capillaries expressing the laminin α2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin α2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin α2 chain rather than through the laminin β1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the α6β1 integrin, which is one of the laminin α2 chain receptors. EGF up-regulated α6β1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-α6 integrin chain monoclonal antibody. These data indicate that laminin α2 chain and α6β1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin α2 chain-positive vessels may favor metastasis of EGF-dependent tumors. Metastasis is the leading cause of death in cancer patients and involves a complex multistep process including detachment of tumor cells from the primary cancer, invasion of surrounding tissue, entry into the circulatory system, reinvasion, and proliferation at a distant secondary site. A wide variety of factors contributing to the spread of tumor cells includes cytokines, hormones, growth factors, cell adhesion molecules, and extracellular matrix proteins (ECMPs) such as laminins. Laminins are a family of α-β-γ heterotrimeric ECMPs, commonly present in basement membranes of the epithelium and endothelium. These molecules promote a number of functions in normal and neoplastic tissues including cell adhesion and migration via integrins, cell proliferation, differentiation, and cell shape.1Patarroyo M Tryggvason K Virtanen I Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (290) Google Scholar More than 12 isoforms are presently known and can be distinguished by their arrangements of α, β, and γ subunits, physical properties, and tissue and cell distribution; they are differentially recognized by several integrins.1Patarroyo M Tryggvason K Virtanen I Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (290) Google Scholar, 2Sorokin LM Paush F Frieser M Kroger S Ohage E Deutzmann R Developmental regulation of the laminin α5 chain suggests a role in epithelial and endothelial cell maturation.Dev Biol. 1997; 189: 285-300Crossref PubMed Scopus (214) Google Scholar, 3Kikkawa Y Sanzen N Sekiguchi K Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells—laminin-10/11 mediates cell adhesion through integrin α3β1.J Biol Chem. 1998; 25: 15854-15859Crossref Scopus (188) Google Scholar, 4Tani T Lehto VP Virtanen I Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion.Exp Cell Res. 1999; 248: 115-121Crossref PubMed Scopus (56) Google Scholar, 5Yamamoto H Itoh F Iku S Hosokawa M Imai K Expression of the gamma(2) chain of laminin-5 at the invasive front is associated with recurrence and poor prognosis in human esophageal squamous cell carcinoma.Clin Cancer Res. 2001; 7: 896-900PubMed Google Scholar, 6Lohi J Laminin-5 in the progression of carcinomas.Int J Cancer. 2001; 94: 763-767Crossref PubMed Scopus (109) Google Scholar Some epithelial laminin isoforms provide specific contributions to promote local tumor invasion, as reported for laminin-10 in lung carcinomas,3Kikkawa Y Sanzen N Sekiguchi K Isolation and characterization of laminin-10/11 secreted by human lung carcinoma cells—laminin-10/11 mediates cell adhesion through integrin α3β1.J Biol Chem. 1998; 25: 15854-15859Crossref Scopus (188) Google Scholar laminin γ2 chain in esophageal carcinomas,5Yamamoto H Itoh F Iku S Hosokawa M Imai K Expression of the gamma(2) chain of laminin-5 at the invasive front is associated with recurrence and poor prognosis in human esophageal squamous cell carcinoma.Clin Cancer Res. 2001; 7: 896-900PubMed Google Scholar and laminin-5 in other human solid tumors.6Lohi J Laminin-5 in the progression of carcinomas.Int J Cancer. 2001; 94: 763-767Crossref PubMed Scopus (109) Google Scholar Laminin α2 chain represents the α2 chain of laminin-21Patarroyo M Tryggvason K Virtanen I Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (290) Google Scholar; in normal human tissues the distribution of the laminin α2 chain is restricted to the sarcolemma, nerve sheaths, placenta, and basement membranes of small vessels of the central nervous system. Moreover, in reactive nonneoplastic conditions, a proportion of capillaries of granulation tissue consists of endothelial cells and basement membranes positive for the laminin α2 chain.1Patarroyo M Tryggvason K Virtanen I Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (290) Google Scholar In neoplastic conditions this ECMP is expressed in hemangiomas. α1β1, α2β1, α3β1, α6β1, α6β4, and α7β1 represent the integrin receptors for this ECMP.4Tani T Lehto VP Virtanen I Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion.Exp Cell Res. 1999; 248: 115-121Crossref PubMed Scopus (56) Google Scholar, 7Leivo I Engvall E Merosin, a protein specific for basement membranes of Schwann cells, striate muscle and trophoblast, is expressed late in nerve and muscle development.Proc Natl Acad Sci USA. 1988; 85: 1544-1561Crossref PubMed Scopus (322) Google Scholar, 8Leivo I Engvall E Laurila P Miettinen M Distribution of merosin, a laminin-related tissue-specific basement membrane protein, in human Schwann cell neoplasms.Lab Invest. 1989; 61: 426-432PubMed Google Scholar, 9Ehrig K Leivo I Agraves WS Ruoslahti E Engvall E Merosin, a tissue specific basement membrane protein, is a laminin-like protein.Proc Natl Acad Sci USA. 1990; 87: 3264-3268Crossref PubMed Scopus (319) Google Scholar, 10Damsky CH Fitzgeraald ML Fischer SJ Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway in vivo.J Clin Invest. 1992; 89: 210-222Crossref PubMed Scopus (604) Google Scholar, 11Vachon PH Loechel F Xu H Wewer UM Engvall E Merosin and laminin in myogenesis; specific requirement for merosin in myotube stability and survival.J Cell Biol. 1996; 134: 1483-1497Crossref PubMed Scopus (193) Google Scholar, 12Miyagoe-Suzuki Y Nakagawa M Takeda S Merosin and congenital muscular dystrophy.Microsc Res Tech. 2000; 48: 181-191Crossref PubMed Scopus (94) Google Scholar Furthermore, we have previously demonstrated in glioblastoma multiforme that solid glomeruloid endothelial cell proliferations consist of endothelial cells and basement membranes that, respectively, produce and contain the laminin α2 chain.13Vitolo D Paradiso P Uccini S Ruco LP Baroni CD Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread.Histopathology. 1996; 28: 521-528Crossref PubMed Scopus (51) Google Scholar The restriction of the laminin α2 chain expression to endothelial sprout-like structures, such as solid endothelial cell proliferations, and to early developing capillary-like structures, such as those of granulation tissue,1Patarroyo M Tryggvason K Virtanen I Laminin isoforms in tumor invasion, angiogenesis and metastasis.Semin Cancer Biol. 2002; 12: 197-207Crossref PubMed Scopus (290) Google Scholar, 14Vitolo D Ciocci L Cicerone E Rossi C Tiboni F Ferrauti P Gallo A Baroni CD Laminin α2 chain (merosin M chain) distribution and VEGF, FGF2 and TGFβ1 gene expression in angiogenesis of supraglottis, lung and breast carcinomas.J Pathol. 2001; 195: 197-208Crossref PubMed Scopus (11) Google Scholar suggested that the up-regulation of this ECMP might be related to early phases of development of new vessels and, therefore, it might be considered as a marker of early angiogenesis.13Vitolo D Paradiso P Uccini S Ruco LP Baroni CD Expression of adhesion molecules and extracellular matrix proteins in glioblastomas: relation to angiogenesis and spread.Histopathology. 1996; 28: 521-528Crossref PubMed Scopus (51) Google Scholar, 14Vitolo D Ciocci L Cicerone E Rossi C Tiboni F Ferrauti P Gallo A Baroni CD Laminin α2 chain (merosin M chain) distribution and VEGF, FGF2 and TGFβ1 gene expression in angiogenesis of supraglottis, lung and breast carcinomas.J Pathol. 2001; 195: 197-208Crossref PubMed Scopus (11) Google Scholar We have also supported this hypothesis by providing evidence that, on early endothelial single cell cultures, gene and protein expression of the laminin α2 chain was stronger and present in a greater number of cells than for the laminin β1 chain.14Vitolo D Ciocci L Cicerone E Rossi C Tiboni F Ferrauti P Gallo A Baroni CD Laminin α2 chain (merosin M chain) distribution and VEGF, FGF2 and TGFβ1 gene expression in angiogenesis of supraglottis, lung and breast carcinomas.J Pathol. 2001; 195: 197-208Crossref PubMed Scopus (11) Google Scholar Furthermore, we have reported that in supraglottis carcinomas, laminin α2 chain-positive vessels were distributed either in the stroma or in neoplastic parenchyma, in close contact with neoplastic cells producing vascular endothelial growth factor (VEGF); moreover, in breast and non-small cell lung carcinomas these vessels were predominantly distributed in the stroma where mononuclear cells produce VEGF, fibroblast growth factor-2 (FGF2), and transforming growth factor-β1. These data suggested again that laminin α2 chain-positive vessels may represent early developing vascular structures in human solid tumors.14Vitolo D Ciocci L Cicerone E Rossi C Tiboni F Ferrauti P Gallo A Baroni CD Laminin α2 chain (merosin M chain) distribution and VEGF, FGF2 and TGFβ1 gene expression in angiogenesis of supraglottis, lung and breast carcinomas.J Pathol. 2001; 195: 197-208Crossref PubMed Scopus (11) Google Scholar It has been reported that angiopoietin 2 and VEGF increase permeability of the endothelium of previously existing and newly formed vessels during angiogenesis15Gupta MK Qin RY Mechanism and its regulation of tumor-induced angiogenesis.World J Gastroenterol. 2003; 9: 1144-1155Crossref PubMed Scopus (194) Google Scholar; moreover, remodeling of ECMPs in basement membranes of vessels is observed during angiogenesis, and penetration of newly-formed vessels occurs during tumor invasion and metastasis. In this regard, the metastatic tendency of several human solid tumors has been directly related to the number of cells producing VEGF rather than to the number of vessels present in the neoplastic tissue.16Decaussin M Sartelet H Robert C Moro D Clara ZC Brambilla C Brambilla E Expression of vascular endothelial growth factor (VEGF) and its two receptors (VEGF-R1-FLT1 and VEGF-R2-KDR) in non-small cell lung carcinomas: correlation with angiogenesis and survival.J Pathol. 1999; 188: 369-377Crossref PubMed Scopus (212) Google Scholar, 17Vesweber D Molecular mechanisms that control endothelial cell contacts.J Pathol. 2000; 190: 281-291Crossref PubMed Scopus (113) Google Scholar Therefore, we investigated whether and how laminin α2 chain expression during angiogenesis may favor transendothelial migration of neoplastic cells and, possibly, metastasis. To address in vitro studies on the prognostic significance of laminin α2 chain-positive vessels we have evaluated ex vivo, at tissue level, the presence of these vessels in lung small cell carcinomas (SCCs) and lung large cell neuroendocrine carcinomas (LCNCs), which represent a human solid tumor with a rate of metastasis that is higher than other carcinomas.18Colby TV Koss MN Travis WD Tumors of the lower respiratory tract.in: Atlas of Tumor Pathology, third series, fascicle 13. Armed Forces Institute of Pathology, Washington DC1995: 235-315Google Scholar, 19Mills SE Gaffey M Frierson HF Tumors of the upper aerodigestive tract and ear.in: Atlas of Tumor Pathology, third series, fascicle 26. Armed Forces Institute of Pathology, Washington DC2000: 45-117Google Scholar, 20Roset PP Oberman HA Tumors of the mammary gland.in: Atlas of Tumor Pathology, third series, fascicle 7. Armed Forces Institute of Pathology, Washington DC1993: 157-244Google Scholar Moreover, we have performed test migration assays on a lung neuroendocrine carcinoma cell line, named AE-2, and on a breast carcinoma cell line from the American Type Culture Collection, Rockville, MD, named MDA-MB231, through a purified laminin α2 chain and other ECMPs present in vascular basement membranes to establish their role in transendothelial migration of neoplastic cells and, therefore, in metastasis. Four atypical carcinoid tumors (Table 1), five SCCs, and four LCNCs (Table 2) were collected at surgery during frozen section procedures. Small samples from these tumors were immediately embedded in OCT compound, snap-frozen in liquid nitrogen to avoid RNA degradation, and stored at −80°C. Tumors were classified and graded according to the 1999 World Health Organization nomenclature.21Travis TW Colby V Corrin B Shimosato Y Brambilla E In Collaboration with Sobin LH and Pathologists in 14 Countries.Histological Typing of Lung and Pleural Tumors. 3rd ed. Springer-Verlag, Germany, Berlin1999Crossref Google ScholarTable 1Laminin α2 Chain-Positive Vessels in Atypical Carcinoid of the LungCaseVDFVIIIRAα2α5α6β1β4FibronectinTenascinLaminin β1 chainLaminin α2 chain1SV151000100100010010010010023PV85700100100010010010010050Neoplastic cells0502100000002SV10100010010007010010010010PV9080010030010010010010030Neoplastic cells0205100000003SV101000100100010010010010010PV90700100100090100100100100Neoplastic cells040410000004SV1000100100100010010010025PV1008001008508510010010025Neoplastic cells050610000000The results are expressed as percentage of positive vessels of 200 vessels counted.VD, vascular distribution; SV, stromal vessels; PV, parenchymal vessels. Open table in a new tab Table 2Laminin α2 Chain-Positive Vessels in Large Cell Neuroendocrine Carcinomas (LCNCs) and in Small Cell Carcinomas (SCCs) of the LungCaseVDFVIIIRAα2α5α6β1β4FibronectinTenascinLaminin β1 chainLaminin α2 chain1 LCNCSV20100020100010010010010025PV8085010100010010010010040Neoplastic cells05015100000002 LCNCSV20100020100010010010010020PV8085010100010010010010035Neoplastic cells05015100000003 LCNCSV201000201000100100100100100PV80850101000100100100100100Neoplastic cells05025100000004 LCNCSV251000100100010010010010010PV75750100100010010010010030Neoplastic cells07020100000005 SCCSV101000100100010010010010030PV90750100100010010010010080Neoplastic cells06015100000006 SSCSV101000100100010010010010010PV901000100100010010010010050Neoplastic cells04025100000007 SSCSV35100010010001001001001000PV6530030100010010010010030Neoplastic cells05060100000008 SSCSV20100010010002510010010060PV808007010000100100100100Neoplastic cells06025100000009 SSCSV301000100100010010010010070PV7020030100020100100100100Neoplastic cells0502010000000The results are expressed as percentage of positive vessels of 200 vessels counted.VD, vascular distribution; SV, stromal vessels; PV, parenchymal vessels. Open table in a new tab The results are expressed as percentage of positive vessels of 200 vessels counted. VD, vascular distribution; SV, stromal vessels; PV, parenchymal vessels. The results are expressed as percentage of positive vessels of 200 vessels counted. VD, vascular distribution; SV, stromal vessels; PV, parenchymal vessels. Five-μm-thick cryostat sections were cut from neoplastic tissue. The sections were fixed in acetone, preincubated with normal serum to prevent nonspecific binding, and incubated with optimal dilutions of the following monoclonal antibodies specific for FVIIIRA, CD31 (Dako A/S, Glostrup, Denmark), fibronectin (clone120.5, IgG1mouse), tenascin (cloneT2, IgG1mouse), α2 (CD49b), α5 (CD49e), α6 (CD49f) (from Immunotech, Marseilles, France), laminin α2 chain (merosin M chain) (clone5H2, IgG1 mouse), and laminin clone 4E10 IgG1 κ mouse (MAB1921), which detects a conformational epitope of B1 heterodimer (β1 chain) (Chemicon International Inc., Temecula, CA). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Negative control sections were obtained after incubation with nonimmune isotype Ig of the same class of each antibody and by omission of the primary antibody. Slides were counterstained with hematoxylin and mounted for microscopic examination. The expression of the antigens was evaluated independently by two investigators. The number and distribution of tumor vessels were evaluated on contiguous sections and immunostained using immunohistochemistry with monoclonal antibodies specific for endothelial markers such as von Willebrand factor, FVIIIRA, CD31, and endothelial integrin chains such as α4 (CD49d), α5 (CD49e), α6 (CD49f), and β4 (from Immunotech). Moreover, distribution of tumor vessels was cross-checked using monoclonal antibodies specific for fibronectin (clone120.5, IgG1mouse), tenascin (cloneT2, IgG1mouse) (from Immunotech), laminin α2 chain (merosin M chain, clone5H2, IgG1 mouse), and laminin (β1chain, clone 4E10 IgG1 κ mouse) MAB1921 (from Chemicon International Inc.), which are known to be ECMPs present in endothelial basement membranes. Therefore, vascularity in neoplastic tissues was expressed as the relative number of vessels distributed in the stroma and parenchyma of a total of 200 vessels counted in randomly chosen fields at ×400 magnification. EAHY endothelial cells22Meri S Mattila P Renkonen R Regulation of CD59 expression on the human endothelial cell line EA.hy 926.Eur J Immunol. 1993; 23: 2511-2516Crossref PubMed Scopus (32) Google Scholar (1 × 106/ml) were maintained in 250-ml plastic tissue culture flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), sodium bicarbonate solution, l-glutamine, HAT, and penicillin-streptomycin and were incubated in 5% CO2 and 95% air at 37°C. AE-2 is the name given to a neuroendocrine lung carcinoma cell line that was kindly provided by Dr. Pier Giorgio Natali (Istituto Regina Elena, Rome, Italy). This cell line consists of epithelial cells measuring 20 to 35 μm in size at their greatest dimension and present a high nucleus:cytoplasm ratio. The AE-2 cell line was maintained in 250-ml plastic tissue culture flasks in RPMI supplemented with 10% FCS, and incubated in 5% CO2 and 95% air at 37°C. The AE-2 cells are characterized by a weak adhesion to plastic; therefore they float in the medium either as single cells or as clusters resembling rosette-like structures (Figure 3). The cells may be suspended in the medium using a light trypsin treatment. MDA-MB231 and MDA-MB435 cell lines were obtained from American Type Culture Collection, Rockville, MD. Cells were maintained in DMEM containing 10% FCS. Cells were lysed using RIPA buffer (1% Triton X-100, 0.5% deoxycholic acid, 0.05% sodium dodecyl sulfate, 200 mmol/L NaCl, 25 mmol/L Tris, pH 7.4, 1 mmol/L phenylmethyl sulfonyl fluoride, 1 mmol/L NaOV4). AE-2 and MDA-MB231 cell lines were maintained in RPMI supplemented with 10% FCS, washed twice in phosphate-buffered saline (PBS), and then suspended at 1 × 106 cells/ml in RPMI medium alone. AE-2 and MDA-MB231 cell suspensions were immediately used to prepare cytosmears using a Shandon Cytospin 3. The cytosmears were dried overnight at room temperature and then fixed in acetone, preincubated with normal serum to prevent nonspecific binding, and incubated with optimal dilutions of monoclonal antibodies specific for cytokeratin (clone MNF116), NCAM (neuronal cell adhesion molecule), chromogranin A, CD31 (from Dako A/S), fibro-nectin (clone120.5, IgG1 mouse), tenascin (cloneT2, IgG1mouse), α2 (CD49b), α3 (CD49c), α4 (CD49d), α5 (CD49e), α6 (CD49f) (from Immunotech), and laminin α2 chain (merosin M chain, clone 5H2, IgG1), laminin (β1chain, clone 4E10, IgG1 κ mouse, catalogue number MAB1921; Chemicon International Inc.). The immunoreaction products were developed using the avidin-biotin-peroxidase complex method. Negative controls were obtained after incubation with nonimmune Ig of the same isotype class of each specific antibody and by omission of the primary antibody. The slides were counterstained with hematoxylin and mounted for microscopic examination. The expression of antigens was evaluated independently by two investigators (Figure 3). For Western blotting analysis, 100 μg of protein lysate derived from MDA-MB435 (control), AE-2, and MDA-MB231 cell lines were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as previously described.23Bei R Masuelli L Moriconi E Visco V Moretti A Kraus MH Muraro R Immune responses to all ErbB family receptors detectable in serum of cancer patients.Oncogene. 1999; 18: 1267-1275Crossref PubMed Scopus (64) Google Scholar After electrophoresis, the proteins were transferred to a nitrocellulose membrane at 40 V for 1 hour. Membranes were treated with specific polyclonal antibodies (1:100) specific for EGFR, ErbB2, ErbB3, and ErbB4, purchased from Santa Cruz Biotechnology, Santa Cruz, CA. After washing, membranes were incubated with horseradish peroxidase conjugated with goat anti-rabbit serum (Santa Cruz Biotechnology). Bound antibody was visualized by a Supersignal West Pico chemiluminescence kit (Pierce, Rockford, IL) (Figure 4). AE-2 cell suspensions (1 × 106/ml) were maintained in 250-ml plastic tissue culture flasks in RPMI supplemented with 50 ng/ml EGF or with 10% FCS (control), and incubated in 5% CO2 and 95% air at 37°C. Ten-ml samples of EGF-treated and of non-EGF-treated AE-2 cells were harvested after 30 and 90 minutes in culture, were centrifuged and the AE-2 cell pellets were homogenized by using 1 ml of the RNA Fast RNA isolation system (Molecular System Co., San Diego, CA). The amount of the RNA yield was measured by optical density reading, and only RNA samples showing an A260/A280 ratio from 1.8 to 2.0 were used to obtain cDNA. cDNA samples were generated by reverse transcription of 10 μg of total RNA in a solution containing 10 mmol/L Tris-HCl (pH 8.3 at room temperature), 1.5 mmol/L MgCe2, 100 μg/ml of bovine serum albumin, fraction V, a mixture of four dNTPs, at a concentration of 2.5 mmol/L each, oligo (dT) primers (5 μg/ml), 20 U of placental RNase inhibitor, 100 U of Moloney murine leukemia virus reverse transcriptase and H2O up to a 20-μl final total volume. The number of 40 cycles of PCR was assessed to synthesize the optimal yield product using 3 μl of the cDNA solutions, obtained from the RT products, and optimal concentration of forward and reverse oligonucleotide primers as reported by NCBI on-line service primer information: RH12420-EGFR (140 bp): 5′: TCG,GTG,TAA,ACG,TTG,CAA,AA and 3′:GAC,CAC,GGA,GGA,TAG,TAT,GAG,C; GDB:181407-Erb-B2 (148 bp): 5′: TCC,GTT,TCC,TGC,AGC, AGT,CTC,CGC,A and 3′: AGA,GAG,CCA,GCC,CTC,TGA,CGT,CCA,T; STS-M34309-Erb-B3 (218 bp): 5′: AAT,TCT,TAT,GGT,ATG,TAG,CCA,GC and 3′:TTG,ACA,GTC,TGA,TGG,GAA,AC; RH68995-Erb-B4 (214 bp): 5′: ACC,TGG,CAG,ATA,CTC,AGA,AAT,G and 3′: CAT,AGT,CCC,TGG,ATA,CCG,TTG; M10277-human β-actin (225 bp): 5′: AGC,ACA,GAG,CCT,CGC,CTT,TG and 3′: CGC,CCA,CAT,AGG,AAT,CCT,TC. PCR assays for β-actin were performed as a control to assess the cDNA yield obtained from each RNA sample enrolled in the present study. The 50-μl PCR solution contained optimal MgCl2 (from 1 to 2.5 mmol/L) and dNTP concentrations, previously tested dilutions of both up- and downstream oligonucleotide primers and 0.3 U of TaqDNA polymerase (no. 801.0046; Perkin-Elmer-Cetus). The size of the PCR products was evaluated by electrophoresis on a 2.5% agarose gel (Figure 5). As previously reported,14Vitolo D Ciocci L Cicerone E Rossi C Tiboni F Ferrauti P Gallo A Baroni CD Laminin α2 chain (merosin M chain) distribution and VEGF, FGF2 and TGFβ1 gene expression in angiogenesis of supraglottis, lung and breast carcinomas.J Pathol. 2001; 195: 197-208Crossref PubMed Scopus (11) Google Scholar aliquots of cells (1 × 106/ml) were harvested in three different assays and then transferred onto tissue chamber slides with nonsupplemented DMEM, supplemented DMEM, supplemented DMEM and 10 ng/ml of VEGF, supplemented DMEM and 10 ng/ml of FGF2, supplemented DMEM and 10 ng/ml of VEGF and FGF2. Tissue chamber slides were harvested after 1, 3, 18, 24, 48, and 96 hours in culture for immunocytochemical analysis. The proportion of adherent cells immunostained for the laminin α2 chain, laminin β1 chain, and FVIIIRA was determined by counting 200 cells at ×400 magnification in randomly chosen fields. According to the results of this kinetic study, after 18 hours in culture adhering EAHY cells were at the same time confluent, laminin α2 chain-positive and laminin β1 chain-negative. Therefore, this time of culture was chosen to obtain EAHY-coated filters in Transwell chambers to perform test migration assays on the AE-2 line (Figure 6). Confluent EAHY cells were trypsinized, washed, suspended with supplemented DMEM with 10 ng/ml of VEGF at the concentration of 1.5 × 106 cells per ml and transferred into polycarbonate tubes. Therefore, these endothelial cells were treated for different time periods (3, 12, 18, and 24 hours) with 10 ng/ml of VEGF, harvested, and then aliquots were incubated with an appropriate dilution of anti-laminin α2 chain monoclonal antibody (merosin M chain), clone 5H2-IgG1, laminin (β1chain; clone 4E10 IgG1 κ mouse, catalogue number MAB1921; Chemicon International Inc.). After washing, cells were stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgG and fluorescence was analyzed by a FACScalibur cytofluorimeter (BD Biosciences, San Jose, CA) following the observation of 10,000 events using CellQuest software. Four independent assays were performed as described.24Pouliot N Nice EC Burgess AW Laminin-10 mediate basal and EGF-stimulate motility of human colon carcinoma cells via alpha (3) beta (1) and alpha (6) beta (4) integrins.Exp Cell Res. 2001; 266: 1-10Crossref PubMed Scopus (55) Google Scholar In brief, blind well chemotaxis chambers (Boyden chamber) with 13-mm diameter polyvinylpyrrolidone-free polycarbonate filters, 8-μm pore size, were used. The filters were coated with human laminin-purified protein (catalog no. AG56P, Chemicon International Inc.), which consists of a mixture of β1 chains from human laminins, principally laminin-10. This preparation is immunologically and biologically identical to intact human laminins. Moreover, these polyvinylpyrrolidone-free filters were coated with purified laminin α2 chain (catalogue no. CC085, Chemicon International Inc.), and purified human fibronectin (catalogue no. F1904, Chemicon International Inc.). AE-2 and MDA-MB231 cell line cells were lightly trypsinized, washed, resuspended in serum-free DMEM at the proper concentration of 0.5 × 106/ml and used as follows: 200-μl volumes of each cell suspension, untreated and pretreated with 50 ng/ml EGF from Sigma-Aldrich Co. (St. Louis, MO), and were loaded in the upper compartment of the Boyden chambers. In addition, 50 ng/ml of EGF were used as a chemoattractant in those assays in which EGF pretreated AE-2 and MDA-MB231 were used, and placed in the lower compartment of the Boyden chambers. After 3 hours of incubation at 37°C in 5% CO2, the cells were removed from the upper side of the filters; the filters were then fixed and stained with Diff-Quick. The results of four independent experiments are reported as the mean ± SD of the numbers of migrated AE-2 and MDA-MB231 cells counted in 10 high-power fields
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