Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells

Background  

Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARγ2 receptor, and induction of target genes.     

PSL, a nuclear cell-cycle associated antigen is increased during retinoic acid-induced differentiation of HL-60 cells

PSL(p55) is a nuclear 55kD antigen present in various mammalian cell systems, which has been first identified by use of human autoimmune antibodies (Barque et al. 1983, EMBO J. 2, 743). It has been shown to be associated with interphase chromatine and to be synthesized in during the S phase of the cell cycle. In this work, we have analysed the status of PSL in promyelocytic HL-60 human cells in exponential or stationary growth, or undergoing granulocytic differentiation in presence of Retinoic acid. By use of 2-dimensional electrophoresis, PSL was found to be composed of two acidic proteins designated p55A and p55B. Unexpectedly, estimated 10-20 fold higher amounts of each species were found in cells treated for 5 days with 10(-6)M Retinoic acid, than in asynchronously growing cells or resting cells. Moreover, the p55A protein was phosphorylated during the process. On the basis of these results, PSL appears to be involved in some steps of the granulocytic differentiation process.     

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.     

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells

The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 μM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [³⁵S]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.     

Defective post-translational modification of collagen IV in a mutant F9 teratocarcinoma cell line is associated with delayed differentiation and growth arrest in response to retinoic acid

We have selected a mutant F9 teratocarcinoma stem cell line, RA-5-1, which does not exhibit normal differentiation into parietal endoderm in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). In this report, we demonstrate that the RA-5-1 mutant possesses a prolyl-4-hydroxylase enzyme with a higher Km for a synthetic collagen substrate and that this alteration results in a 6-7-fold reduction in the amount of collagen IV in the medium of RACT-treated mutant cells, as compared to wild type F9 cells. In addition, the collagen IV that is secreted by RACT-treated RA-5-1 cells has an abnormally low molecular weight and contains 6-9-fold less 4-hydroxyproline than the collagen IV secreted by RACT-treated wild type F9 cells. A brief ascorbate treatment can increase the hydroxyproline content of the collagen IV secreted by RACT-treated RA-5-1 cells. A large reduction in the amount of laminin in the medium of RACT-treated RA-5-1 mutant cells is also observed. Concomitant with the reduction in collagen IV and laminin polypeptides in the medium, the expression of several other differentiation-specific mRNAs is delayed in the RACT-treated RA-5-1 cells relative to wild type F9 cells. Moreover, the mutant cells do not exhibit the morphology or the complete growth arrest of wild type terminally differentiated parietal endoderm cells in the presence of RACT. These results suggest that a defect in the post-translational modification of collagen IV in the mutant RA-5-1 prevents the complete expression of the differentiation program in response to RACT. These experiments also demonstrate that the expression of certain differentiation-specific genes is compatible with continued proliferation in the mutant line.     

The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.     

Suppression of ethanolamine-containing glycerophospholipid synthesis in HL-60 cells during retinoic acid-induced differentiation.

Synthesis and degradation of glycerophospholipids in HL-60 cells and retinoic acid (RA)-treated HL-60 cells were examined. The synthesis of each subclass of ethanolamine-containing glycerophospholipids was extremely suppressed in RA-treated HL-60 cells, while that of other glycerophospholipids was not seriously affected. A pulse-chase experiment revealed that about 88% of 1,2-diacyl and 28% of 1-alkenyl-2-acyl glycerophosphoethanolamine were degraded during 4 days in RA-treated HL-60 cells. These characteristics of metabolism observed in RA-treated HL-60 cells might be responsible for the change of subclass composition of ethanolamine-containing glycerophospholipids in HL-60 cells during differentiation to granulocytes.     

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356–5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.