Retinoic acid causes cell growth arrest and an increase in p27 in F9 wild type but not in F9 retinoic acid receptor beta2 knockout cells

We have previously shown that an F9 teratocarcinoma retinoic acid receptor beta(2) (RARbeta(2)) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARalpha(-/-), and F9 RARgamma(-/-) cell lines do growth arrest in response to RA. To examine the role of RARbeta(2) in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARbeta(2)(-/-) cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARbeta(2)(-/-) cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARbeta(2)(-/-) cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARbeta(2)(-/-) cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARbeta(2)(-/-) cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARbeta(2) is associated with the process of carcinogenesis and RARbeta(2) can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARbeta(2) mediates these growth inhibitory effects of RA in F9 cells.     

Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.     

Overexpression of the cellular retinoic acid binding protein-I (CRABP- I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells

Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA) causes their irreversible differentiation into extraembryonic endoderm. To elucidate the role of the cellular retinoic acid binding protein-I (CRABP-I) in this differentiation process, we have generated several different stably transfected F9 stem cell lines expressing either elevated or reduced levels of functional CRABP-I protein. Stably transfected lines expressing elevated levels of CRABP-I exhibit an 80-90% reduction in the RA induced expression of retinoic acid receptor (RAR) beta, laminin B1, and collagen type IV (alpha 1) mRNAs at low exogenous RA concentrations, but this reduction is eliminated at higher RA concentrations. Thus, greater expression of CRABP-I reduces the potency of RA in this differentiation system. Moreover, transfection of a CRABP-I expression vector into F9 cells resulted in five- and threefold decreases in the activation of the laminin B1 RARE (retinoic acid response element) and the RAR beta RARE, respectively, as measured from RARE/CAT expression vectors in transient transfection assays. These results support the idea that CRABP-I sequesters RA within the cell and thereby prevents RA from acting to regulate differentiation specific gene expression. Our data suggest a mechanism whereby the level of CRABP-I can regulate responsiveness to RA during development.     

Expression of c-kit protooncogene is stimulated by cAMP in differentiated F9 mouse teratocarcinoma cells.

Protooncogene c-kit, a transmembrane tyrosine kinase receptor, was recently shown to map to the dominant white spotting locus (W) of the mouse. W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and in adult life. In order to determine the regulation of the c-kit gene in cell differentiation, we investigated its expression during the differentiation of F9 cells. Undifferentiated F9 cells and F9 cells treated with retinoic acid (RA) alone or dbcAMP alone showed little expression of c-kit mRNA if any. The subsequent addition of dbcAMP to F9 cells treated with RA markedly increased the expression of c-kit mRNA. Furthermore, the effect of dbcAMP on c-kit expression is reversible. In differentiated cells treated with RA, c-kit gene expression is induced by agents such as forskolin or theophylline, which are known to elevate cellular cAMP level. These results indicate that the expression of the c-kit gene is regulated by the level of intracellular cAMP in differentiated F9 cells induced by RA.     

14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells

We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells. The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1. Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells. The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells. These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade.