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.     

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.     

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.     

A developmentally regulated activity that unwinds RNA duplexes

We have attempted to use antisense RNA techniques in the developing Xenopus embryo and found that although hybrids form between sense and antisense RNAs, they are transient. Our studies indicate that this is due to the presence of an activity that unwinds RNA:RNA hybrids. The activity is present at low, but detectable, levels in Xenopus oocytes, increases during oocyte maturation, and exists at high levels throughout early embryogenesis. The activity has been characterized in S100 extracts. The denaturation of a specific RNA:RNA hybrid is inhibited by the addition of a second duplexed RNA but not by the addition of single-stranded RNA, single-stranded DNA, or double-stranded DNA.     

An embryonal carcinoma cell line as a model system to study developmentally regulated genes during myogenesis

We have established conditions to efficiently differentiate embryonic carcinoma stem cells of the line P19 into myogenic cells. As inducers for differentiation, a combination of embryoid body formation in conjunction with treatment with dimethyl sulfoxide and retinoic acid proved to be most efficient. Under these conditions we detected an accumulation of myosin- and actin-specific RNA. Also, large amounts of type IV collagen RNA were produced. Type IV collagen is a component of the muscle basement membrane. In analogy to the F-9 system, we found a drastic decrease in stable p53 mRNA under the differentiation conditions used.     

Retroviral vector gene expression in F9 embryonal carcinoma cells.

When F9 embryonal carcinoma (EC) cells are infected with retroviral vectors, the efficiency of expression of selectable genes is considerably lower than that in mouse fibroblasts infected with the same retroviral vectors. In this study, several retroviral vectors with regulatory sequences placed immediately 5' to a selectable gene were constructed, packaged, and used to infect mouse fibroblasts and F9 EC cells. With selection as an assay, there was a hierarchy of relative expression in F9 cells compared with that in mouse fibroblasts. These internally placed regulatory sequences are the source of the mRNAs detected in F9 EC cells, while both retroviral long-terminal-repeat promoters and internal promoters are the source of steady-state mRNAs in mouse fibroblasts. This effect was observable with both the internally placed herpes simplex virus thymidine kinase promoter and the Moloney murine leukemia virus promoter.     

Variant embryonal carcinoma cells lacking SSEA-1 and Forsmann antigens remain developmentally pluripotent

Six embryonal carcinoma (EC) cell lines that are resistant to the cytotoxic, galactose-specific lectin abrin were isolated from mutagenized populations of either PSA-1 or F9 cells. The LD10 for each of the variant lines was at least 150-fold greater than that for parental cells. Indirect cytotoxicity tests demonstrated that all of the variant cell lines lacked both Stage Specific Embryonic Antigen-1 (SSEA-1, less than 1% of wild-type levels) and Forsmann antigen (less than 5% of wild-type levels). When abrin-resistant cells were fused to previously isolated SSEA-1-negative cells (M. J. Rosenstraus (1983), Dev. Biol. 99, 318-323) that express Forsmann antigen, the resulting hybrids expressed SSEA-1. This implies the mutation conferring abrin resistance is in a different gene than that defined by the previously isolated mutation. Thus, we have identified two genes that are required for SSEA-1 expression, one of which also appears to be required for Forsmann antigen expression. The F9-derived variants differentiated into visceral-like or parietal-like endoderm when treated with retinoic acid in the absence or presence of 8-bromo-cAMP, respectively. PSA-1-derived variants formed differentiated teratocarcinomas containing derivatives of all three germ layers. Thus the SSEA-1 and Forsmann haptenic determinants are not required for EC cells to differentiate into a broad spectrum of cell types; nor do they appear to be involved in the cell-cell interactions that are postulated to regulate visceral versus parietal endoderm differentiation.