Identification of a Cis-acting element in the rat ?-fetoprotein gene and its specific binding proteins in F9 cells during retinoic acid-induced differentiation

Abstract

Mouse F9 embryonic teratocarcinoma stem cells can be induced to differentiate into visceral endoderm. Following retinoic acid (RA) treatment, alpha-fetoprotein (AFP), a differentiation marker, is expressed and secreted. The mechanism by which RA regulates AFP expression during differentiation is not clear. The relatively late induction of AFP indicates that the AFP gene may not be a primary target of RA activity during F9 cell differentiation. In this study, a CAT reporter plasmid containing the rat AFP 5'-regulatory region (-7040 to +7) adjacent to the CAT gene (pAFPCAT) was stably transfected into F9 cells and used to delineate a cis-acting element which associates with AFP gene activation. Similar spatial and temporal expression patterns between the transcriptional activity of the recombinant AFP gene and the endogenous AFP gene demonstrate that this stably transfected F9 system can be used to dissect both cis-elements and trans-acting factors responsible for RA-induced AFP expression. Using a series of deletion mutants of the pAFPCAT, the region between -2611 to -1855 was found to be important in AFP-induction. Subsequent analysis identified a functional sequence (-1905 to -1891, 5'-ACTAAAATGGAGACT-3') that differentially binds nuclear proteins from undifferentiated and differentiated F9 cells. This sequence, designed as differentiation-associated sequence (DAS) for its unique binding of a nuclear protein (DAP-II) that appears during RA-induced F9 differentiation, acts as a regulatory protein factor in AFP gene activation.

Endogenous anticancer mechanism differentiation

“…RA induces cellular differentiation, as initially shown in cell lines derived from embryonal carcinoma (113), through the upregulation of genes that promote differentiation, like AFP (114), and downregulation of pluripotency-associated ones like Oct4 or telomerase (115). RA signaling can also lead to cell cycle arrest at the G1 stage through the dowregulation of cyclin D1 by inducing protein degradation and reducing mRNA synthesis, with consequent reduction of the phosphorylation of retinoblastoma (Rb) protein, leading to an inactivation of E2F and deficient upregulation of cyclin E and CDKs (111).…”

Novel and emerging targeted-based cancer therapy agents and methods

“…By inducing CSCs differentiation, cells will become more susceptible to anti-tumor therapy, and lose their ability to rebuild the tumor later. As described 37 years ago, retinoic acid (RA) is an appropriate molecule that induces cellular differentiation in embryonal carcinoma cell lines [135] through the upregulation of genes that promotes differentiation, like α-fetoprotein [136,137] and downregulation of pluripotency-associated ones like Oct4 or telomerase [138].…”