ABSTRACT
Transcription factor 21 (TCF21) has been identified as a candidate tumor suppressor at 6q23-q24 that is epigenetically inactivated in many types of human cancers. We recently found that TCF21 methylation level was significantly increased in clear cell renal cell carcinoma (ccRCC). The purpose of this study was to investigate the prognostic impact of TCF21 expression in ccRCC and analyze the relationship between TCF21 expression and methylation level. We used real-time PCR and immunohistochemical staining to detect the expression of TCF21, and used methylation specific-PCR (MS-PCR) to determine the methylation status of TCF21 in ccRCC samples and cell line 786-O. The results showed that TCF21 expression level in ccRCC samples was significantly lower than in normal adjacent tissue samples (NAT samples). The Kaplan-Meier survival analysis demonstrated that TCF21 was a significant prognosticator of cancer-specific survival (p=0.001). Furthermore, the DNA demethylating agent 5'-azacytidine restored part of TCF21 expression by suppressing TCF21 methylation in 786-O. The methylation level of TCF21 in ccRCC samples was much higher than in NAT samples. These results suggest that the expression of TCF21 was an independent prognostic factor for poor survival in patients with ccRCC. Aberrant methylation was an important reason for the down-regulation the expression of TCF21, and may be associated with tumorigenesis in ccRCC.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/physiology , Carcinoma, Renal Cell/mortality , Kidney Neoplasms/mortality , Basic Helix-Loop-Helix Transcription Factors/analysis , Basic Helix-Loop-Helix Transcription Factors/genetics , Carcinoma, Renal Cell/chemistry , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , DNA Methylation , Down-Regulation , Humans , Kidney Neoplasms/chemistry , Kidney Neoplasms/pathologyABSTRACT
The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1-7 days of differentiation), mid-stage EBs groups (9-15 days of differentiation), maturing EBs groups (17-45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.