br Introduction Receptor tyrosine kinases RTKs are criticall

Introduction Receptor tyrosine kinases (RTKs) are critically involved in the development and progression of human cancers and are therefore useful targets for anti-cancer therapies [1]. The Eph receptors represent the largest subfamily of receptor protein kinases and interact with ligands called ephrins. Based on the sequence relationships and structures, ephrin ligands are divided into two subgroups, ephrinA and ephrinB. The ephrinA (A1–A5) subgroup is anchored to the cell membrane by a glycosylphosphatidylinositol linkage, whereas ephrinB (B1–B3) subgroups are transmembrane proteins [1], [2], [3]. Eph family receptors can also be divided into two subgroups based on the relatedness of their extracellular domain sequences and on their ability to bind to the two subgroups of ephrins. The EphA (A1–A8) interacts with ephrinA ligands, whereas the EphB (B1–B6) interacts preferentially with ephrinB ligands [4]. As an exception, EphA4 can bind both ephrinA and most of ephrinB type ligands [5]. EphrinB2 is the sole ligand for EphB4 and ligand–receptor binding leads to protein clustering followed by receptor activation [6], [7], [8]. Binding and activation of the EphB4/ephrinB2 system requires cell-to-cell interaction rather than long-range communication; EphB4/ephrinB2 mediates bidirectional signaling cascades [9]. This implies that the ephrin system is being activated in a paracrine manner [10], provides positive (attractive) and negative (repulsive) positional guidance cues to EphB/ephrinB expressing SKL2001 and regulate adhesive, migratory and invasive cellular functions [10], [11], [12]. Involvement of EphB4 has been described in several biological functions, such as embryonic development, including pattern formation, cell aggregation and migration, segmentation, neural development, angiogenesis and vascular hierarchical remodeling [13], [14], [15], [16]. EphrinB2, the transmembrane ligand of EphB4, also participates in vascular remodeling via reverse signaling [16]. Dysregulation of the EphB receptor and ephrinB ligand could promote tumor progression via controlling cell shape, proliferation, differentiation and migration [17], [18]. EphB4 and ephrinB2 mediate the enhanced proliferation, migration and metastatic potential of tumor cells [18], [19], [20], [21], [22], [23]. In addition, elevated expression and activity of Eph receptors have been correlated with the growth of solid tumors [20]. Preliminary studies have identified expression of EphB4 receptors in several human tumors including hematologic malignancies, glioblastoma and carcinoma of the lung, breast and colon. The cognate ligand ephrinB2 has been expressed in the leukemias, primary and metastaic melanomas compared to benign melanocytic nevi and cancer of the gastro-intestine, lung, liver and kidney [19], [24], [25], [26], [27], [28], [29], [30]. Coexpression of ephrinB2 and EphB4 was found in the ovarian carcinoma [31], colon carcinoma [32] and cancer cell lines and tumors of small cell lung carcinoma [33], neuroblastoma [34] and uterine endometrial carcinoma [27]. To understand the clinical significance of EphB4/ephrinB2, we studied the expression and localization of EphB4 receptor and its corresponding ligand ephrinB2 against clinical backgrounds in uterine cervical cancers.
Materials and methods Prior informed consent for the following studies was obtained from all patients and the study was approved by the Research Committee for Human Subjects, Gifu University School of Medicine. Sixty two patients ranging from 35 to 74 years of age with uterine cervical cancers (19 stage I cases, 27 stage II cases, 11 stage III cases and 5 stage IV and 15 cases of adenocarcinoma, 47 cases of squamous cell carcinoma, 25 cases with lymph node metastasis and other 37 cases without lymph node metastasis) underwent surgical resection, which produced macroscopically disease-free status, at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, between March 1997 and October 2003. None of the patients had received any pre-operative therapy. All patients from stage IA2 underwent radical hysterectomy. The tissues of uterine cervical cancers were obtained immediately after surgery. The tissues for RNA isolation were snap-frozen and stored at −80 °C, and those for immunohistochemistry were fixed with 10% formalin and embedded in paraffin wax. The clinical backgrounds of uterine cervical cancer were evaluated by International Federation of Gynecology and Obstetrics (FIGO) classification [35]. Tumor sizes in stage Ib and II were determined by measurement of the maximum longitudinal diameter of the excised tumors. Several microscopic sections from each extirpated tumor with the approximate maximum longitudinal diameter were stained with hematoxylin and eosin. All surgically removed specimens were histologically examined to demonstrate pelvic lymph node metastasis as an advancement indicator.