Growth rate regulation and random transition. A study performed on embryonal carcinoma cell lines. I

Analyses of cell-cycle characteristics of the three embryonal carcinoma (EC) cell lines F9, PCC3 N/1 and PCC4 Azal, have been performed. The three lines reflect successive stages in early mouse embryogenesis as regards cell surface antigens and cell-cycle characteristics. In an attempt to understand changes in cell-cycle characteristics occurring during early embryogenesis, the two-random transition probability (TP) model was applied to the EC-cell system--and particularly to the F9 line. By utilizing an intraclonal heterogeneity in intermitotic times found in these EC lines, a growth-regulating point was introduced as a modification of the two-random TP model. The modified model was found to be very useful when demonstrating the cell-cycle growth kinetics of the F9 line. The model is used in an accompanying paper to extend the analysis of cell-cycle characteristics in undifferentiated EC cells.     

Cell growth and cell division: dissociated and random initiated? A study performed on embryonal carcinoma cell lines. II

The 'random transition probability' cell-cycle models have so far failed to convincingly link the transition events with phenomena describable by biochemical methods. The study presented was carried out on the F9 and PCC3 N/1 embryonal carcinoma (EC) cell lines. We now report an extended analysis of the two-random transition probability (TP) model and preliminary results are presented showing that the deterministic L period in that model can be regarded as reflecting the 'cell-growth cycle'. Evidence is presented that suggests that the 'cell-growth cycle' is a supramitotic deterministic phase--i.e. starting in one cell cycle and being completed in the next following G1 period and dissociated from the 'DNA-division cycle'. This phenomenon makes an interesting contribution to the old knowledge of a stepwise G1 prolongation during early embryogenesis in yielding a mechanism by which the cell can alter the ratio of nucleus to cytoplasm prior to the onset of gene expression.     

Cell Growth and Cell Division: Dissociated and Random Initiated?

Abstract. The 'random transition probability' cekycle models have so far failed to convincingly link the transition events with phenomena describable by biochemical methods. the study presented was carried out on the F9 and PCC3 N/1 embryonal carcinoma (EC) cell lines. We now report an extended analysis of the two-random transition probability (TP) model and preliminary results are presented showing that the deterministic L period in that model can be regarded as reflecting the 'cell-growth cycle'. Evidence is presented that suggests that the 'cell-growth cycle' is a supramitotic deterministic phase—i.e. starting in one cell cycle and being completed in the next following G₁ period and dissociated from the 'DNA-division cycle'. This phenomenon makes an interesting contribution to the old knowledge of a stepwise G₁ prolongation during early embryogenesis in yielding a mechanism by which the cell can alter the ratio of nucleus to cytoplasm prior to the onset of gene expression.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

The F9-EC cell line as a model for the analysis of differentiation.

The teratocarcinoma stem cell line F9 has been widely used as a model for the analysis of molecular mechanisms associated with differentiation. This cell line has been considered to be nullipotent and able to differentiate into endodermal-like derivatives upon treatment with retinoic acid. Nevertheless, under definite culture conditions, F9 cells are able to differentiate into derivatives of all three germ layers. The F9 cells express characteristics of early mouse embryonal cells and possess all repression factors known to be present in cells of the early mouse embryogenesis. Induction of differentiation can be achieved not only by adding chemical agents to the culture medium but also by transfection of several oncogenic sequences. In somatic cell genetic experiments, immortalized, differentiated F9-like cells have been shown to express dominantly genes responsible for the appearance of the differentiated phenotype.     

Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9)

Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which allowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC3 and F9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.     

Collagen synthesis in mouse embryonal carcinoma cells: effect of retinoic acid

In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected. In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Collagen Synthesis in Mouse Embryonal Carcinoma Cells: Effect of Retinoic Acid

Abstract. In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, PCC7-S/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected.
In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Determinants of retrovirus gene expression in embryonal carcinoma cells.

The expression of Moloney murine leukemia virus is restricted in embryonal carcinoma (EC) cells. To characterize specific mutations necessary for expression of retroviruses in EC cells, we analyzed the expression of retrovirus mutants and recombinants thereof in EC cell lines F9 and PCC4. DNA sequence comparison and functional studies allowed us to define three point mutations in the enhancer region of the viral mutants at positions -345, -326, and -166 and two point mutations within the 5'-untranslated region of the viral genome at positions +164 and +165 that were essential for retrovirus expression in EC cells. DNA fragments derived from either the wild type or mutant viruses were used to search for sequence-specific DNA-binding factors in nuclear extracts from undifferentiated PCC4 cells. A cellular factor was found to bind strongly to sequences within the enhancer region (-354 to -306) of wild-type viruses but only weakly to sequences derived from mutant viruses. This factor was named ECF-I (for EC cell factor I). Retroviral expression in EC cells correlates with decreased binding affinity for ECF-I.     

Spontaneous differentiation in the colonies of a nullipotent embryonal carcinoma cell line (F9)

Abstract. Experiments were undertaken to reveal the spontaneous differentiation capacity of the nullipotent F9 embryonal-carcinoma (EC) cell line in colonies derived from single cells. Culture conditions which alllowed the development of neuroblasts in colonies of the multipotent EC cell line (PCC3) were worked out, and comparative studies on neuroblast differentiation in PCC 3 and F 9 colonies were conducted. Neural-cell-specific silver impregnation, selective staining of cells having electrically excitable membranes with merocyanine 540 and the observation of nerve processes were considered as differentiation markers. The appearance of neuroblasts in F9 and PCC3 colonies could be detected from the 6th day after seeding. The development of neuroblasts was less prevalent in high-density cultures, especially in the case of F9. By the 8th day in differentiating colonies, PCC3 cells lost much of their colony-forming activity, while F9 cells preserved their original high plating efficiency, in spite of advanced differentiation. The determination of growth parameters during differentiation in colonies led to the conclusion that F9 cells had lost certain growth-control mechanisms which normally restrict the clonal growth of EC cells. It is suggested that the phenomenon of nullipotence may be analysed in terms of the coordinated regulation of proliferation and differentiation of EC cells.     

The glycosidic antigen recognised by a novel monoclonal antibody, 75.12, is developmentally regulated on mouse embryonal carcinoma cells

Monoclonal antibody 75.12 raised against the human ovarian teratocarcinoma cell line PA1 detects a 'Y' or iso-leb glycosidic structure. Using the 75.12 antibody we have established that the Y antigen is expressed on some but not all mouse embryonal carcinoma (EC) lines. The Y or 75.12 antigen-positive EC cell lines F9 and PCC4 cease to express the antigen after differentiation induced with retinoic acid and this decreased expression parallels the morphological differentiation of the EC cells. These results support not only the idea that carbohydrate structures present on embryonic cells undergo marked alteration during differentiation, but also that established mouse EC cells may differ in their differentiation states.     

Minor histocompatibility antigens are developmentally regulated on murine embryonal carcinoma cells and their early differentiated derivatives

Differences in the expression of minor histocompatibility (Hm) alloantigens on two mouse embryonal carcinoma (EC) cell lines and the PYS-2 and T.D.M.-1 differentiated derivatives have been demonstrated by their ability to elicit a cytolytic T lymphocyte response. Experiments involving the use of various responder-target strain combinations and recombinant inbred mice strains have shown that: (1) there are major differences in Hm expression on EC cells compared with differentiated derivatives whose Hm expression appears more like that of adult splenocytes; (2) although both EC cell lines show reduced Hm immunogenicity compared with adult splenocytes, major differences in the expression and possible presentation of Hm between the F9 and PCC3 EC cell lines can be detected by in vivo priming and by in vitro cold competition target experiments. These observations are discussed in relation to the differences in allograft rejection patterns observed with PCC3 and F9 and to possible differences in developmental staging of these cell lines.     

High chemotactic response to platelet-derived growth factor of a teratocarcinoma differentiated mesodermal cell line

Summary Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/ml of PDGF, 2-fold at 2.5 ng/ml of PDGF. In contrast, PDGF induces the same increase in [³H]thymidine incorporation in both cell lines, made quiescent under reduced serum concentration. This high chemotactic response to PDGF seems specific for these mesodermal cells. Among the different teratocarcinoma cells tested, including stem cells (F9, PC 13, PCC4) and endodermal derivatives (PYS, F9 with retinoic acid, PSA 5E), only the visceral endodermlike cells (PSA5E) are slightly attracted by PDGF. This chemotactic response to PDGF is not related to the presence or characteristics of the type B PDGF receptors, which are less numerous in MES-1 cells (10⁵ receptors/cell, KDa 1,2 mM) compared to NIH 3T3 cells (64×10⁴ receptors per cell, KDa 1,8 nM). The MES-1 cell line might be of interest for studying the chemotactic effect of PDGF. These results also suggest a role for this soluble factor in cell migration during early embryogenesis. This investigation was supported by a grant of La Fondation pour la Recherche Médicale.