The erythropoietin-producing hepatoma amplified sequence (Eph) family is the largest subfamily of receptor tyrosine kinases (RTKs). The Ephs (receptors) bind to specific cell-bound ligands, called ephrins. The binding of this ligand-receptor system is dependent on cell-cell interactions. The ephrin-Eph system is important in embryologic development and differentiation of the nervous and vascular systems. In the current study, the authors hypothesized that ephrins may play a role in the growth and development of colon carcinoma and may be expressed differentially in normal and malignant colonic tissues.
Reverse transcriptase-polymerase chain reaction (RT-PCR), Northern Blot analyses, and immunohistochemistry were used to examine 11 colon carcinoma cell lines and 20 human colon carcinoma specimens with adjacent uninvolved mucosa for the expression of EphB and ephrin-B family members.
EphB2, EphB3, and EphB4 mRNA expression and ephrin-B2 mRNA expression was detected in all the cell lines and colon carcinoma specimens examined. Immunohistochemical analysis showed that ephrin-B2 had higher expression in the colon carcinoma specimens studied than in adjacent normal mucosa. Ephrin-B2 and EphB4 most frequently were expressed on the luminal surface of colon carcinoma epithelium.
The Eph family is the largest family of receptor protein tyrosine kinases, containing 14 distinct receptors for which at least 8 ligands have been identified.1, 2 Ligands for the Eph kinases, called ephrins, are anchored on plasma membranes through either a glycosyl phosphatidylinositol linkage (ephrin-A) or a transmembrane domain (ephrin-B).1 The Ephs (receptors) and their ligands (ephrins) can be divided into two subclasses, A and B, on the basis of sequence homology, structure, and binding affinity.1
Ephrins and Ephs are essential for embryonic development.3 However, to our knowledge, few studies to date have investigated the role of these molecules in the adult. In recent years, ephrins and Ephs have been found to be expressed in numerous human tumors.4–8 This ligand-receptor system has been implicated in tumor development in melanoma, glioblastoma, lung carcinoma, and breast carcinoma.4, 5, 9 However, to our knowledge little is known regarding the expression and role of the ephrin-Eph system in colon carcinoma. In the current study, we investigated the expression patterns of ephrin-B and EphB mRNA in a series of human colon carcinoma cell lines and colorectal carcinoma and normal tissue specimens.
MATERIALS AND METHODS
Colon carcinoma specimens and adjacent mucosa were obtained from the pathology suite immediately after resection and were either frozen in optimum cutting temperature (OCT) solution (Miles Inc., Elkhart, IN) and stored at −70 °C or snap frozen in liquid nitrogen. Specimens were procured under a protocol approved by the Institutional Clinical Research Committee.
Cells and Cell Culture
HT-29, NCI-H747, LOVO, SW480, and SW620 human colon carcinoma cells and human umbilical vein endothelial cells (HUVECs) were purchased from American TypeCulture Collection (Rockville, MD). KM12L1, KM12L3, KM12L4, KM20, KM12C, and KM12SM human colon carcinoma cells, SG and L3.6pl human pancreatic carcinoma cells, and SN12C and SN12PM6 human renal carcinoma cells were provided by Dr. I. J. Fidler (The University of Texas M. D. Anderson Cancer Center, Houston, TX). All cell lines were cultured in minimal essential medium supplemented with 10% fetal bovine serum, penicillin-streptomycin, vitamins, sodium pyruvate, L-glutamine, and nonessential amino acids (Life Technologies, Inc., Grand Island, NY) at 37 °C in 5% CO2 and 95% air.10 HUVECs were cultured on 0.5% gelatin (Sigma Chemical Company, St. Louis, MO) in modified minimal essential medium supplemented with 15% fetal bovine serum, 10 ng/mL recombinant human basic fibroblast growth factor, penicillin-streptomycin, vitamins, sodium pyruvate, L-glutamine, and nonessential amino acids (Life Technologies, Inc.) at 37 °C in 5% CO2 and 95% air. Cells were verified to be free of Mycoplasma, reovirus type 3, pneumonia virus of mice, mouse adenovirus, murine hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (Microbiological Associates, Bethesda, MD).
Isolation of mRNA and Northern Blot Analysis
Polyadenylated mRNA was extracted from 107 to 108 subconfluent tumor cells growing in culture by using a FastTrack mRNA isolation kit (Invitrogen Corporation, San Diego, CA). Northern blot analysis was performed as previously described.11 After prehybridization, the membranes were probed for ephrin-B2 full-length cDNA (a generous gift from Dr. Renping Zhou,12 Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ), EphB4 (the probe was a purified polymerase chain reaction [PCR] product), and glyceraldehyde-phosphate dehydrogenase (internal control; American TypeCulture Collection). Each cDNA probe was purified by agarose gel electrophoresis, recovered by using a QIAEX gel extraction kit (QIAGEN Inc., Chatsworth, CA), and radiolabeled by random primer technique with a commercially available kit (Amersham Life Science Inc., Arlington Heights, IL). Nylon filters were washed at 65 °C with 30 mmol/L NaCl, 3 mmol/L sodium citrate (pH 7.2), and 0.1% sodium dodecyl sulfate. Autoradiography then was performed.
Isolation of mRNA from Tumors and Reverse Transcriptase-PCR
mRNA was extracted from tumor tissue and uninvolved mucosa by homogenizing tissue (approximately 0.5 g) in Trizol reagent (Life Technologies, Inc.) using power a homogenizer for 30 seconds followed by mRNA extraction according to the manufacturer's protocol. For reverse transcriptase-PCR (RT-PCR), 3 μg of total RNA was used for cDNA synthesis with avian myeloblastosis virus RT (Life Technologies, Inc.) in a final volume of 20 μL. The reaction mixture included 0.5 M Tris-HCl (pH 8.0), 0.5 M KCl, 0.05 M MgCl2, 2.5 mM dNTP, 40 U of RNase inhibitor (Boehringer Mannheim, Indianapolis, IN), 50 U of RT, and 0.5 μg of random primer. The cDNA synthesis reaction was performed for 1 hour at 37 °C. A portion of the reaction mixture (5 μL) was subjected to PCR amplification in a reaction mixture (50 μL) that contained 1 μmol/L of each of 2 primers (sense and antisense), 1.5 mmol/L of MgCl2, 0.2 mmol/L of each of 4 deoxynucleotides, and 2.5 U of Taq polymerase (Promega, Madison, WI). PCR amplification of ephrin-B ligands and EPH-B receptors was performed under the following conditions: 95 °C for 5 minutes, 35 cycles of 30 second denaturing at 95 °C, 30 seconds of annealing at 55 °C, and 5 minutes of extension at 72 °C. PCR products were analyzed by electrophoresis of 20 μL of each PCR reaction mixture in a 3% agarose gel, and bands were visualized by ethidium bromide staining. The following primers were used6: ephrin-B1: sense primer: 5′-GGAGGCAGACAACACTGTCA-3′ and antisense primer: 5′-GAACAATGCCACCTTGGAGT-3′; ephrin-B2: sense primer: 5′-GCAAGTTCTGCTGGATCAAC-3′ and antisense primer: 5′-AGGATGTTGTTCCCCGAATG-3′; ephrin-B3: sense primer: 5′-CTGAAATGCCCATGGAAAGA-3′ and antisense primer: 5′-ACGCCCAGCAAGAGCAGCGC-3′; EphB1: sense primer: 5′-GAGATGGACAGCTCCAGAGG-3′ and antisense primer: 5′-CCAGCATGAGCTGGTGTAGA-3′; EphB2: sense primer: 5′-AAAATTGAGCAGGTGATCGG-3′ and antisense primer: 5′-TCACAGGTGTGCTCTTGGTC-3′; EphB3: sense primer: 5′-AGCAACCTGGTCTGCAAAGT-3′ and antisense primer: 5′-TCCATAGCTCATGACCTCCC-3′; and EphB4: sense primer: 5′-GTCTGACTTTGGCCTTTCCC-3′ and antisense primer: 5′-TGACATCACCTCCCACATCA-3′.
Immunohistochemical Analysis of Frozen Tissue Specimens
Tumors frozen in OCT solution were cut into 8–10-μm thick sections, mounted on positively charged Superfrost slides (Fisher Scientific, Houston, TX), and air-dried for 30 minutes. Tissues were fixed in cold acetone for 5 minutes, 1:1 acetone/chloroform for 5 minutes, and acetone for 5 minutes and then washed with phosphate-buffered saline (PBS) 3 times for 3 minutes each time. Specimens then were incubated with 3% hydrogen peroxide in PBS for 12 minutes at room temperature to block endogenous peroxidase. Sections were washed 3 times for 3 minutes each with PBS (pH 7.5) and incubated for 20 minutes at room temperature in a protein-blocking solution comprised of PBS supplemented with 1% normal goat serum and 5% normal horse serum. The primary antibodies directed against ephrin-B2 and EphB4 (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1:100 in protein blocking solution, applied to the sections, and incubated overnight at 4 °C.
Sections then were rinsed 3 times for 3 minutes each in PBS and incubated for 10 minutes in protein-blocking solution before the addition of peroxidase-conjugated secondary antibody. The secondary antibody used for ephrin-B2 and EphB4 staining (peroxidase goat antirabbit immunoglobulin [Ig] G) was diluted 1:100 in protein-blocking solution. After incubation with the secondary antibody for 1 hour at room temperature, the samples were washed and incubated with stable diaminobenzidine substrate (Research Genetics, Huntsville, AL). Staining was monitored under a bright-field microscope, and the reaction was stopped by washing with distilled water. The sections were counterstained with Gill No. 3 hematoxylin solution (Sigma Chemical Co.) and mounted with Universal Mount (Research Genetics) for 15 seconds. Control specimens were treated with a similar procedure except that the primary antibody was omitted.
Immunofluorescent Staining for Ephrin-B2 and EphB4
Frozen sections were stained for ephrin-B2 and EphB4 according to the same protocol as described above with the following modifications. After the sections were incubated overnight at 4 °C with the primary antibody (ephrin-B2 and EphB4; 1:100 dilution), washed, and incubated with protein-blocking solution, they were incubated for 1 hour at room temperature with a secondary antibody (Alexa 594 goat antirabbit IgG (H + L); Molecular Probes, Eugene, OR) and mounted with DAPI fluorescence mounting media (Vector Laboratories, Burlingame, CA).
RT-PCR Analyses of Ephrin-B and EphB mRNA Expression in Normal Cells, Human Colon Carcinoma Cell Lines, and Colon Carcinoma Specimens
RT-PCR analyses were used to investigate the expression of ephrin-Bs (ephrin-B1, ephrin-B2, and ephrin-B3) and EphBs (EphB1, EphB2, EphB3, and EphB4) mRNA in HUVECs, eight colon carcinoma cell lines, five colon carcinoma specimens, and five specimens of uninvolved adjacent mucosa. Expression of ephrin-B2 and EphB2, EphB3, and EphB4 was demonstrated in HUVECs and all colon carcinoma cell lines, colon carcinoma specimens, and specimens of uninvolved mucosa. In contrast, expression of ephrin-B1 was detected in HUVECs and all colon carcinoma cell lines studied but was not detectable in tumors or mucosa from the specimens (Table 1). Because of the ubiquitous expression of ephrin-B2 and EphB4 in colon tissues, we focused our subsequent investigations on the expression of these two proteins.
Table 1. Expression of Ephrin-Bs and EphBs in Colon Carcinoma Cell Lines and Tissue Specimens
Determination of Ephrin-B2 and EphB4 mRNA Expression in Colon Carcinoma and Other Malignant Cell Lines
Northern blot analysis was performed to determine the expression of ephrin-B2 and EphB4 in eight colon carcinoma cell lines. We also examined pancreatic and renal carcinoma cell lines for ephrin-B2 and EphB4 expression to determine the relative frequency of the expression of these factors in other tumor systems (Fig. 1). All the colon carcinoma cell lines examined expressed ephrin-B2 and EphB4. In contrast, the expression of ephrin-B2 was lower in the pancreatic carcinoma cell lines studied and was barely detectable in the renal carcinoma cell lines studied.
Immunohistochemical and Immunofluorescent Staining of Surgical Specimens for Ephrin-B2 and EphB4
Immunohistochemical analysis and immunofluorescent staining were performed on 20 colon carcinoma specimens with adjacent uninvolved mucosa. Immunohistochemical analysis and immunofluorescent staining revealed higher expression of ephrin-B2 and EphB4 protein in all colon carcinoma specimens studied compared with normal mucosa (Fig. 2). This finding held true when two immunohistochemical techniques (immunofluorescence and immunoperoxidase staining) were used.
The Eph family receptor kinases and their ligands have been implicated in the development of the nervous and vascular systems.13, 14 Ephs (receptors) and ephrins (ligands) have complementary expression in many tissues during embryogenesis.2 Several studies utilizing ephrin and Eph knockout mice demonstrated that embryonic vascular development was deficient in ephrin-B2, EphB2, and EphB3 knockout mice.14, 15 Cell-bound ephrins induce endothelial cell tube formation, induce capillary sprouting in vitro, and promote the attachment of endothelial cells to extracellular matrix components.14, 16 Interaction between EphBs and ephrin-Bs may initiate bidirectional signaling. When ephrin-Bs bind EphBs, the ephrin-Bs activate the EphB receptors, and the cells expressing ephrin-B also demonstrate an increase in intracellular signaling. In contrast, ephrin-A does not appear to have a cytoplasmic domain, but still participates in extracellular signaling.17 These observations demonstrate the importance of cell-cell interactions rather than the ephrin system being activated in a paracrine manner.18
The binding of ephrins to Ephs requires cell-to-cell contact; soluble forms of ephrins cannot activate their receptor.19 In the current study, we found coexpression of ephrin-Bs and EphBs in all colon carcinoma specimens examined with RT-PCR and Northern blot analyses. In other studies, the expression of EphBs and ephrin-Bs was detected in melanoma, and high levels of ephrin-B2 mRNA expression were found in both advanced primary malignant melanomas and metastatic cell lines and tissues.8 Tang et al. found coexpression of EphBs and ephrin-Bs in small cell lung carcinoma cell lines and tumor specimens6 and also reported high expression of EphB6, ephrin-B2, and ephrin-B3 mRNA in low-stage neuroblastoma.7 However, to our knowledge the exact role of Ephs and ephrins in these tumor systems remains undefined.
Recently, several studies have shown that high expression of ephrins may be associated with increased potential for tumor growth, tumorigenicity, and metastasis.4, 8 Using immunohistochemical analysis and immunofluorescent staining, we found the expression of ephrin-B2 and EphB4 to be greater in all colon carcinoma specimens studied compared with adjacent normal mucosa. The data from the current study suggest that ephrin-Bs and EphBs are overexpressed in malignant colonic epithelium and suggest a role for these proteins in the progression of colon carcinoma.
The authors thank Stephanie Deming, Department of Scientific Publications, and Joan Small, Department of Surgical Oncology, at the University of Texas M. D. Anderson Cancer Center for editorial assistance.