Teratocarcinoma stem cells as a model for differentiation in the mouse embryo

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.     

Effect of 5-azacytidine treatment on mouse embryonal carcinoma cells

Several properties of embryonal carcinoma (EC) cell lines, such as multipotent PCC4-aza-1 cells and nullipotent F9 cells originating from murine teratocarcinoma cells, were examined after treatment with 5-azacytidine, which produces undermethylated DNA. Drug-treated PCC4-aza-1 cells exhibited morphological changes and differentiated, whereas azacytidine-treated F9 cells displayed no detectable morphological change. After treatment with 5 azacytidine, PCC4-aza-1 cells, whether or not they differentiated, as well as F9 cells, became permissive for polyoma even though both cell types are usually resistant to polyoma. In contrast, only the differentiated azacytidine-treated PCC4-aza-1 cells became sensitive to SV40 infection, i.e., synthesized T antigen, despite the resistance normally shown by such cells to this viral infection. In some PCC4-aza-1 and F9 cells, drug treatment induced expression of H2 antigen but did not derepress plasminogen activator synthesis. These results suggest that undermethylation of certain cellular genes in PCC4-aza-1 and F9 cells is correlated with the establishment of Py permissivity, SV40 sensitivity, H2 antigen expression, and the triggering of a differentiation process. The relationship between the expression of these characters and differentiation is discussed.     

An adhesion-defective variant of F9 embryonal carcinoma cells fails to differentiate into visceral endoderm

Adhesion-defective EC cells were isolated from a population of mutagenized F9 cells by serial transfer of cells that did not adhere to gelatin-coated dishes. The variant cells grew in suspension as multicellular clusters of loosely aggregated cells. The cells adhered to, but did not flatten on, fibroblast monolayers and extracellular matrix produced by parietal-like endoderm. Two different mutant cell lines exhibited increased sensitivity to the lectin abrin and decreased sensitivity to wheat germ agglutinin, suggesting that changes in cell surface glycosylation are associated with the mutant phenotype. These adhesion-defective mutants were used to study the relationship between cell-cell adhesion and endodermal differentiation. Unlike wild-type cells, when cultured with low concentrations of retinoic acid (RA) in suspension culture, the mutant cells did not form embryoid bodies but remained as loosely adhering strings of cells. Electron microscopic examination revealed that most of the differentiated variant cells resembled parietal endoderm, and this was confirmed by immunofluorescent staining for TROMA-3 marker. The levels of some of the markers that characterize the differentiative pathways were examined by immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). The variant line produced higher levels of laminin and type IV collagen compared to the wild-type cells. alpha-Fetoprotein (AFP) was produced at a significantly lower level by the variant compared to wild-type F9 cells during the differentiative process. The results show that variant cells differentiated toward parietal endoderm but have a very much restricted ability to differentiate to visceral endoderm. We conclude that aggregation and/or compaction provide some essential signals during the differentiation of F9 cells into epithelial layers of visceral endoderm.     

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

Constitutive c-myc expression enhances proliferation of differentiating F9 teratocarcinoma cells

The c-myc protooncogene is expressed in many tumor cells as well as during normal development. In order to study the role of c-myc in differentiation, proliferation and tumorigenicity of F9 mouse teratocarcinoma cells, the pSVmyc1 plasmid constitutively expressing an active c-myc oncogene was introduced into F9 stem cells by cotransfection with the selectable marker RSVneo. Enhanced expression of c-myc did not alter the properties of F9 stem cells. Prolonged proliferation during retinoic acid induced differentiation was observed in cell clones constitutively expressing c-myc. In contrast, as determined by morphology, by immunocytochemistry for markers specific for stem cells and differentiated derivatives, and by Northern hybridization for mRNAs specific for differentiated cells, differentiation was neither inhibited nor delayed by constitutive c-myc expression. Tumorigenicity of stem cells as well as retinoic acid-treated cells--as measured by soft agar cloning efficiency and tumor formation in syngenic mice--was not altered by SVmyc1. We conclude that in F9 teratocarcinoma cells down-regulation of c-myc is related to arrest of proliferation rather than differentiation.     

Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells.

Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.