David L. Bronson

Retinoic acid fails to induce differentiation in human teratocarcinoma cell lines that express high levels of a cellular receptor protein

Abstract Human embryonal carcinoma (EC) cells and other human teratocarcinoma-derived cells lines were not observed to differentiate in culture in response to retinoic acid. Nevertheless, they express high levels of a cellular binding protein (cRABP) which, in murine EC cells, appears to be necessary for the induction of differentiation by retinoic acid.

A human embryonal—yolk sac carcinoma model system in athymic mice

Two new human cell lines (1411H and 1411HRQmet) have been established from a patient with metastatic testicular cancer whose primary and metastatic histology included seminoma, teratoma, embryonal carcinoma (EC), and yolk sac tumor (YST). In vitro, the cells have been maintained for more than 70 passages, produce alpha‐fetoprotein (AFP) and human chorionic gonadotropin (hCG), and have a human karyotype. When 2 × 107 cells of either line are inoculated into athymic mice, 87.5% of the animals (21/24) develop tumors. Initially 80% to 90% of the mass is EC, whereas the central portion is YST. After 90 to 390 days in vivo, the tumors achieve a large volume (2.13 ± 0.97 cm3), become cystic, and undergo histologic change. The peripheral rim of the mass remains EC, but the central 80% to 90% becomes YST. The sera of tumor‐bearing mice were positive for hCG and AFP in 11% and 38% of animals, respectively. Tumor cyst fluid was positive for hCG and AFP in 87% and 59% of animals, with mean values of 108 mIU/ml and 2,478 ng/ml, respectively. Tumor cyst fluid also contained placental alkaline phosphatase and human fibronectin. These two cell lines are useful for studies on the interrelationship of EC and YST and the differentiation of human germ cell cancer.

Plasma protein and apolipoprotein synthesis by human yolk sac carcinoma cells in vitro

SummaryThree human yolk sac carcinoma cell lines were characterized for the expression of several markers. Each of the cell lines expressed alpha-fetoprotein, without detectable levels of chorionic gonadotropin, and the level of alpha-fetoprotein expression increased dramatically when the cultures were held without passage for extended periods. The secretion of a number of plasma proteins was documented by metabolic labeling, immunoprecipitation, and gel analysis. The major plasma proteins detected were alpha-1-antitrypsin, alpha-fetoprotein, transthyretin,β-2 microglobulin, and plasminogen, with lower levels of transferrin and complement C4 released. Apolipoproteins B, E, and A1 were secreted in high levels as well and were found in the form of lipoprotein particles. Time course experiments on the synthesis of apolipoproteins E and A1 indicated that, as with alpha-fetoprotein, the level of synthesis increased substantially when the cultures were held without passage. The results indicate that these yolk sac carcinoma cells display a protein expression profile similar to that observed for the human yolk sac, and the possibility that the cells may have the potential to differentiate is discussed.

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.

A comparison of four methods used to concentrate Rous sarcoma virus from tissue culture fluids.

Three methods of pelleting, pelleting followed by Pronase treatment, polyethylene glycol (PEG)-Pronase, and diaflo ultrafiltration (diafiltration) were used to concentrate RSV(RAV-1) from tissue culture fluids. Sucrose-gradient fractions containing virus preparations which had been concentrated by diafiltration or pelleting were heavily contaminated with amorphous debris. This debris was not present in similar, gradient-purified preparations that had been concentrated by the PEG-Pronase or pellet-Pronase methods. Maximum recovery of radiolabelled virus particles and virion-associated RNA-dependent DNA polymerase activity was obtained in gradient fractions containing virus concentrates prepared by the pellet-Pronase and PEG-Pronase methods. Although there were slight differences in recovery by these two methods, the advantages of the PEG-Pronase method make it the preferred method, especially when large volumes of tissue culture fluids are used.

In vitro models of human testicular germ-cell tumors

SummaryMurine teratocarcinoma models have been widely used in the study of several biological processes including early mammalian development and neoplasia. The relatively new human germ-cell tumor models have an important role in the study of human embryogenesis and neoplasia since human embryogenesis differs significantly from species such as mice and the histopathology of human testicular germ-cell tumors differs substantially from tumors obtained in highly inbred strains of mice. In this report we describe the use of human germ-cell lines in the study of cell differentiation; expression of early embryonic antigens; production of the major regulator of erythropoiesis, erythropoietin; and production of retrovirus-like particles.

Interaction of human embryonal carcinoma cells and differentiated derivatives in vitro with simian virus 40, human adenovirus type 7, or PARA

Polyclonal antibodies were used to assay human embryonal carcinoma (EC), differentiating EC, yolk sac carcinoma, and teratoma cells for expression of viral early antigen (T-Ag) after infection with simian virus 40 (SV40). Cells of four EC lines were induced to differentiate by cultivation at low density or by exposure to retinoic acid or dimethyl sulfoxide. After infection with SV40, T-Ag was expressed by 1%, or less, of the cells (presumed to be differentiated derivatives) in only some EC cultures whereas the antigen was synthesized by a significant percentage of the yolk sac carcinoma, teratoma, and differentiating EC cells. Also, viral late proteins were produced by EC cells infected with human adenovirus type 7 (Ad7), and SV40 T-Ag was expressed by EC cells after infection with PARA, which is an Ad7-SV40 hybrid virus containing the SV40 T-Ag sequence controlled by Ad7 late regulatory sequences. Thus, T-Ag is not synthesized by the parental EC cells infected with SV40, but it is expressed in cultures of infected differentiated derivatives. The EC cells produce T-Ag, however, when expression of the viral protein is controlled by the Ad7 regulatory sequences in PARA particles. These results demonstrate that expression of T-Ag after infection with SV40 is an indicator of EC cell differentiation and also raise the possibility that, as in mouse EC cells infected with the virus, the SV40 regulatory sequences controlling T-Ag synthesis are not active in human EC cells.

Human Testicular Germ Cell Tumors In Vitro

Until recently, human testicular germ cell tumors were studied primarily by examination of surgical specimens, and many ideas about their biology are predicated on data from the mouse teratocarcinoma system, which has been developed and exploited widely through in vivo and in vitro research. For example, mouse teratocarcinomas contain a mixture of adult, embryonic, and extraembryonic tissues, in the form of pluripotent embryonal carcinoma (EC) stem cells and their differentiated derivatives, that depict stages in embryogenesis (reviewed by Graham, 1977; Jacob, 1977; Solter and Damjanov, 1979; Martin, 1980). Largely on the basis of these observations, it is believed that human EC cells also differentiate and thus produce the other types of nonseminomatous tumors that comprise embryonic (teratoma) and extraembryonic (choriocarcinoma and yolk sac) tissue. Not all pathologists agree with this thinking, however, and as a result, the classification of human germ cell tumors, on which management of the disease is based, is a controversial subject, as reflected in the number of classification systems proposed (see reviews by Nochomovitz et al., 1977; Mostofi, 1977). The system proposed by the World Health Organization and used in this chapter classifies human testicular germ cell tumors as seminoma, embryonal carcinoma (EC), mature and immature teratoma, teratocarcinoma (EC with teratoma), choriocarcinoma, and yolk sac tumor (Bar and Hedinger, 1976; Nochomovitz et al., 1977).