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.     

Differentiation potential of human embryonal carcinoma cell lines

We have established 17 embryonal carcinoma (EC) cell lines from human testicular germ cell tumors by using three different methods of in vitro cultivation. Cultures of only three of these cell lines, and of clones derived from two of them, differentiate extensively when the cells are seeded at low density. A comparison is presented of the results obtained with the three methods used to establish and maintain these cell lines, and some properties of the three pluripotential EC lines are summarized.     

Isolation of cell lines from differentiating embryonal carcinoma cultures

We report the isolation of six cell lines (designated EB cell lines) from cultures of the hypoxanthine guanine phosphoribosyl transferase-deficient (HGPRT-) feeder-dependent embryonal carcinoma cell line PSA4TG12 which have undergone in vitro differentiation, and of clonal derivatives of these lines. Whereas some lines possess quasi-diploid karyotypes similar to that of PSA4TG12, others are markedly aneuploid. Cell line EB26/1 and its clonal derivatives undergo adipogenesis in cultures maintained at confluence; in tumours formed by injection into syngeneic mice they produce muscle-like cells, cartilage and bone in addition to adipose cells. We therefore propose that EB26/1 and its clones are aneuploid derivatives of an uncommitted mesodermal cell. Cell line EB28/5 forms tumours with a histological appearance resembling that of yolk sac carcinoma but does not express biochemical markers characteristic of visceral or parietal endoderm. Cell line EB28/10n has a myoblast-like culture morphology and in tumours is capable of producing muscle-like cells, cartilage and bone. A high specific activity of alkaline phosphatase is present is two of five EB cell lines assayed, and plasminogen activator activity is present in all five. Since the EB cell lines represent populations of cells each expressing a particular subset of the genetic information present in a common ancestral genome, they will be invaluable for studying the developmental regulation of gene expression.     

Two multipotent embryonal carcinoma cell lines irreversibly differentiate in defined media

Two unrelated multipotent embryonal carcinoma cell lines, OC-15S1 and 1003, have been cultured in hormone-supplemented defined media in order to identify the signals that influence their differentiation. Previous studies have shown that F9 embryonal carcinoma cells can be grown for many generations in the defined medium, EM-3, which contains fibronectin, insulin, and transferrin in place of serum. F9 cells, which only differentiate into a few cell types, undergo little or no differentiation in EM-3 unless an inducer is present (A. Rizzino and C. Crowley, 1980, Proc. Natl. Acad. Sci. USA77, 457–461). This report demonstrates that, in contrast to F9, OC-15S1 and 1003 embryonal carcinoma cells do not proliferate in EM-3. Instead, the cells differentiate. However, the differentiated cells do not survive in EM-3 unless it is supplemented with factors such as purified serum lipoproteins. In EM-3 containing high-density lipoprotein, a population of differentiated cells, devoid of embryonal carcinoma cells, is formed. The differentiated cells that appear exhibit an epithelioid morphology throughout the culture. These cells also secrete plasminogen activator and two different criteria argue that it is the type released by parietal endoderm. This suggests that, under the influence of the defined medium, both multipotent embryonal carcinoma cell lines differentiate at high frequency into parietal endoderm. It was also determined that fibronectin promotes the differentiation of OC-15S1 and 1003 in serum-containing media, and this suggests that fibronectin is at least partly responsible for the differentiation observed in EM-3 plus high-density lipoprotein. In light of these findings, it is suggested that fibronectin may directly influence cellular differentiation during early mammalian development.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

Characterization of human embryonal carcinoma cell lines derived from testicular germ-cell tumors

Four human embryonal carcinoma (EC) cell lines (ITO, NEC 8, NEC 14, NEC 15) derived independently from testicular germ-cell tumors were established in vitro. In their xenografted tumor tissues, all of them exhibited histological characteristics consistent with EC. The cell-biological characterization of these human EC cell lines was investigated with reference to well-known murine EC cell lines. This included examination of their morphology, growth, tumorigenic potential, karyotype, cell-aggregate formation, HLA expression, large glycopeptides, AFP and HCG production, plasminogen-activator secretion, and LDH profiles. Three (ITO, NEC 14, NEC 15) of these human EC cell lines shared cell-biological characteristics consistent with typical EC, but one of them (NEC 8) differed from the others with respect to its rapid growth, high tumorigenic potential, formation of solid cell aggregates, and less differentiated, solid histological pattern. Thus, it is suggested that there are several developmentally different types of human EC cells. The relationship between the properties of these human EC cell lines and their differentiation potential is discussed.     

Expression of transcripts of complement components and their receptors during differentiation of embryonal carcinoma cell lines.

Based on our previous finding that TNF-alpha and TNF-beta can be expressed constitutively during early embryonal development [1], we extended our work to identify factors which are generally known to take part in inducing inflammation in adults. They can be regarded as candidate molecules involved in ontogenic inflammation during embryonal development. In this study, we chose the factors which are constituents of either a classical or an alternative pathway of a complement system and found that mRNAs corresponding to those of C2, C3, C4, C5 and to those of receptors CR1 and CR2 were expressed. Among them, mRNA expression of C3, C4, and CR1 was especially constitutive. Contrary to these observations, expression of two kinds of scavenger receptors (SR-I, SR-II) proved to be negative. In this report, the framework of ontogenic inflammation as a regulatory mechanism in embryonal development at the molecular level is discussed.     

Lipid patterns of embryonal carcinoma cell lines and their derivatives: Changes with differentiation

The lipid composition of several teratocarcinoma cell lines has been examined by biochemical and immunological methods in order to identify properties that might be correlated with the state of cell differentiation. The data indicate qualitative and quantitative changes in the phospholipid, cholesterol, and glycolipid composition. In particular, the ratios of cholesterol/phospholipid and of sphingomyelin/phosphatidylcholine are higher in differentiated cells. Gangliosides with short glycosidic chains (GM₃ and GD₃) are characteristic of undifferentiated, multipotent, embryonal carcinoma cell lines. More complex gangliosides (GM₁ and GD_1a) appear early during the course of differentiation. Each differentiated cell line presents a unique ganglioside map. Results are tentatively correlated with a stabilization of the membrane bilayer in differentiated cell lines, whereas a more fluid state of the membrane in embryonal carcinoma cell lines would allow maximal flexibility. Subtle differences in ganglioside composition among embryonal carcinoma cell lines are discussed in relation with their potentialities, and their developmental age.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Evidence of 3 beta-hydroxysteroid dehydrogenase activity in several murine embryonal carcinoma cell lines

This report describes, for the first time to our knowledge, a possible steroidogenic activity in established murine embryonal carcinoma cell lines (PCC3, PCC4, F9), revealed by a 3 beta-hydroxysteroid dehydrogenase activity (revelation of NADH2 by staining, and RIA assessment of delta 4-androstenedione). The remarkable analogy between such totipotent cells and embryonal cells may suggest that this activity could be present before histologic organization of the embryonal testis. Nonmalignant embryonal cells such as fibroblasts (3/A/1/D-3) or myoblasts (T984) were also found to possess a 3 beta-hydroxysteroid dehydrogenase activity, thus suggesting that this enzyme is not specific to hormone-secreting cells, but the sign of a more general phenomenon.     

Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines

Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A)⁺ mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700, 13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells.Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A)⁺ RNA sequences are common to embryonal carcinoma cell polysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A)⁺ RNA fails to hybridize 7–10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.     

Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.     

Cell density and cell cycle effects on retinoic acid-induced embryonal carcinoma cell differentiation.

P19 embryonal carcinoma cells can be induced to differentiate with a pulse of only 4 hr in retinoic acid (RA). The efficiency of RA-induced differentiation is independent of the position of P19 cells in the cell cycle but is critically dependent on the ratio between the number of cells and the number of moles of RA in the culture medium. P19 cultures at lower cell density are more efficiently induced to differentiate than cultures containing cells at higher cell densities. This effect is not mediated by cell-to-cell contact but may be related to the rapid metabolism of RA by the cells. Individual clones of differentiating P19 cells can develop into at least three different cell types suggesting that each cell in the population of embryonal carcinoma cells retains pluripotency.     

Brain cell surface antigens on embryonal carcinoma cells

Allo- and heteroantisera against cerebellar tissue from young postnatal mice detect surface antigens on a nullipotent mouse embryonal teratocarcinoma line, F-9. These antigens are operationally identical to the NS-4 and NS-5 antigens described previously. Antigenic specificities are shared with brain, kidney, and sperm.     

Transglutaminase activity and embryonal carcinoma cell differentiation

Murine embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) modulate transglutaminase (TGase) activity shortly after exposure to the inducer. Compounds that inhibit TGase enzyme activity in vitro can successfully block RA induced EC cell differentiation in culture. These observations suggest that TGase may play a role in mediating RA induced EC cell differentiation.     

Murine lymphocyte and embryonal carcinoma cell surface antigens recognised by rabbit anti-murine embryonal carcinoma serum

Some recent studies have suggested that murine embryonal carcinoma (EC) cells which lack classical H-2 molecules must express some novel class-I-MHC (major histocompatibility complex) type antigens. We have investigated whether rabbit anti-EC sera may recognize such components. Molecules of 40 and 48 kd were immunoprecipitated from EC cells and lymphocytes. The possibility that these molecules may be coded for by genes in the Qa-Tla locus which contains many MHC class-I genes or pseudogenes was investigated. The 40 and 48 kd molecules were not associated with beta 2-microglobulin on EC cells, nor with each other or other molecules through S-S bonds, although they appeared to have intradisulphide structures. Peptide map analysis suggested that the lymphocyte 40 and 48 kd molecules were related to the EC cell 40 and 48 kd molecules. However, these molecules were different from H-2, Ia or Qa-2 molecules from the same mouse strain.     

The response of several murine embryonal carcinoma cell lines to stimulation of differentiation by alpha-difluoromethylornithine

In an attempt to better establish the relationship between polyamine levels and the differentiation of embryonal carcinoma cells, we have examined the ability of alpha-difluoromethylornithine (DFMO), a known inducer of differentiation in one embryonal carcinoma cell line, to stimulate the differentiation of embryonal carcinoma cells from a variety of cell lines. Differentiation was monitored using a variety of criteria including morphological alterations and changes in biochemical and antigenic parameters. Depending on their response to difluoromethylornithine, three classes of cell lines could be identified, those which 1) differentiate extensively, 2) differentiate poorly, and 3) fail to differentiate. Three different classes of embryonal carcinoma cell lines reflect differential changes in polyamine levels resulting from inhibition of ornithine decarboxylase enzyme activity by DFMO. The specific cell lines which exhibit large decreases in both ornithine decarboxylase activity and polyamine levels also show extensive differentiation. The cell lines which show only moderate decreases in enzyme activity and polyamines differentiate poorly while the cell lines which fail to respond to DFMO in that polyamines do not drop below the threshold level necessary to induce differentiation fail to differentiate. These studies suggest that decreases in intracellular polyamines induce EC cell differentiation in vitro.