Immunodetection was performed with a chemoluminescence system (ECL, GE Healthcare, Freiburg, Germany)


Immunodetection was performed with a chemoluminescence system (ECL, GE Healthcare, Freiburg, Germany). main tumor tissues for our analysis. For expression analysis we employed RT-PCR and Western blotting. DNA-Methylation of the L1CAM promoter was decided after bisulfite conversation and DNA sequencing. Tumor tissues were examined by immunohistochemical (IHC) staining. Results We demonstrate that the treatment of L1CAM low/unfavorable expressing EC cell lines with 5-Azacytidine (5-AzaC) or knock-down of DNMT1 (DNA methyltransferase 1) as well as the HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) up-regulated L1CAM at the mRNA and protein level. The L1CAM gene has two promoter regions with two unique CpG islands. We observed that the expression of L1CAM Monodansylcadaverine correlated with hypermethylation in promoter 1 and 5-AzaC treatment affected the DNA-methylation pattern in this region. The CT-X antigens NY-ESO-1, MAGE-A3 and MAGE-A4 were also strongly up-regulated by 5-AzaC Rabbit Polyclonal to ARNT or knock-down of DNMT1 but did not respond to treatment with TSA. Main EC tumor tissues showed a variable methylation pattern of the L1CAM promoter. No striking differences in promoter methylation were observed between tumor areas with L1CAM expression and those without expression. Conclusions L1CAM expression correlated with methylation of the L1CAM promoter in EC cell lines. In unfavorable cell lines L1CAM expression is usually up-regulated by epigenetic mechanism. Although genes localized on Xq28 are often re-expressed by human tumors, L1CAM and CT-X antigens show unique regulation in response to HADC inhibitors and 5-AzaC. Background The L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in brain development. Work in the past has shown that L1CAM is also overexpressed in many human tumors [1,2]. It was shown that L1CAM augments cell motility, invasion and metastasis formation [1-3]. Generally, its expression in a variety of tumors is usually associated with a poor prognosis [4-7]. L1CAM is usually absent in normal endometrium [8]. In endometrial Monodansylcadaverine carcinomas (ECs), expression is usually absent in most of the indolent endometrioid type EC (type 1 tumor) but present in the more malignant forms of serous-papillary and obvious cell carcinoma (type 2 tumors) [8]. In addition, ECs often occur as a mixed-type, i.e. they are composed of a mixture of endometrioid and serous/obvious cells components that can be morphologically distinguished. Importantly, the expression of L1CAM is also mixed and L1CAM staining of IHC sections can be used to identify even minor components of serous/obvious cell components [8]. The regulation of L1CAM expression at the transcriptional and/or epigenetic level is not well understood. The L1CAM gene is located at chromosome Xq28 and spans about 26?kb with 29 exons, whereof 28 are protein coding exons [9]. The full-length open reading frame consists of 3,825?bp encoding for any 1,275 amino acid polypeptide [9]. During the past years L1CAM was shown to be subject of epigenetic regulation. Kuwajima exhibited that histone deacetylase inhibitors like butyrate and TSA can upregulate both mRNA and protein levels of the cell adhesion molecules Mel-CAM and L1CAM in B16-BL6 melanoma cells [10]. Another statement investigated the methylation status at the L1CAM promoter and found an inverse correlation of DNA methylation and protein expression in both colorectal malignancy (CRC) cell lines and CRC patients [11]. Treatment with the demethylating agent 5-AzaC induced L1CAM mRNA/protein expression in two L1CAM unfavorable CRC cell lines, whereas levels of two L1CAM positive CRC cell lines did not change [11]. However, these findings have neither been confirmed nor extended to other tumor entities. On Xq28, L1CAM colocalizes with CT-X Monodansylcadaverine antigens such as the MAGE-A family and NY-ESO-1 that are frequently overexpressed in human tumors. A recent study in prostate malignancy has recognized Xq28 as one of 35 domains in the prostate malignancy genome that undergo activation due to long-range epigenetic remodelling [12]. In the present study we wished to clarify i) whether L1CAM expression in ECs entails epigenetic mechanisms in cell lines and main tumor tissues and ii) whether L1CAM and the CT-X genes, all encoded in the same locus around the X-chromosome, bear some similarity in their epigenetic regulation. Methods Cell lines and cell culture The EC cell lines were managed in DMEM/F12 medium or RPMI-1640 (PAA Laboratories, Pasching, Austria) Monodansylcadaverine supplemented with 10% fetal calf serum as explained before [8,13,14]. Chemicals and Monodansylcadaverine antibodies Antibodies to the ectodomain of L1CAM (monoclonal antibody (mAb) L1-11A, a subclone of UJ127.11) and L1-9.3 were described before [15,16]. Antibodies for detection in Western blot were as follows: GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany), Acetyl-H3 (9765, New England Biolabs), MAGE-A4 (WH4103M1, Sigma-Aldrich, Taufkirchen, Germany), MAGE-A3 (NBP1-02506, Novus Biologicals, Littleton, USA) and Ny-ESO-1 (Invitrogen, Eggenstein, Germany). 5-AzaC, TSA and VA were obtained.