Insertion mutagenesis of embryonal carcinoma cells by retroviruses.

Mutagenesis was studied in cultured F9 embryonal carcinoma cells infected with a variant of Moloney murine leukemia virus. Proviral insertion induced the inactivation of the hypoxanthine phosphoribosyltransferase locus, and the virus was used to isolate the mutated genes rapidly. Mutagenesis by these methods may be useful for the genetic dissection of the various mammalian cell phenotypes.

Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells.

To study the mode of action of human cytomegalovirus, an important teratogenic agent in human populations, the susceptibility of a pluripotent human embryonal carcinoma cell line to the virus was investigated. Viral antigens were not expressed nor was infectious virus produced by human embryonal carcinoma cells after infection, although the virus was able to penetrate these cells. In contrast, retinoic acid-induced differentiated derivatives of embryonal carcinoma cells were permissive for antigen expression and infectious virus production. Replication of human cytomegalovirus in human teratocarcinoma cells may therefore depend on cellular functions associated with differentiation.

Infected benign cystic teratoma of the mediastinum.

A young man aged 22 years presented with shortness of breath, left sided chest pain, mild dry cough, peripheral cyanosis, fever and generalized weakness for three years. He was diagnosed as having a large infected cystic mediastinal mass with tricuspid regurgitation and severe pulmonary hypertension. On thoracotomy, one litre of pus was aspirated and tumour was excised and sent for histopathology. Biopsy report revealed benign cystic teratoma. This case is reported to highlight the management of a huge infected benign cystic teratoma which is rarely found.

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

We have cloned the polyomavirus mutant fPyF9, which persists in an episomal state in F9 embryonal carcinoma cells (K. Ariizumi and H. Ariga, Mol. Cell. Biol. 6:3920-3927, 1986). fPyF9 carries three copies of exogenous sequences, the prototype of which is a 21-base-pair repeat (box DNA), in the region of the enhancer B domain of wild-type polyomavirus DNA. The consensus sequence, GCATTCCATTGTT, is 13 base pairs long. The box DNA inserted into fPyF9 appeared to come from a cellular sequence and was present in many kinds of DNAs, including F9 chromosomal DNA. The biological function of box DNA was analyzed by chloramphenicol acetyltransferase expression assays, using chimeric plasmids containing box DNA conjugated with simian virus 40 promoter elements. The results showed that box DNA repressed the activities both of the simian virus 40 promoter and enhancer only in transfected undifferentiated F9 cells and not in differentiated LTK- cells. Box DNA functioned independently of orientation and position with respect to the promoter in an enhancerlike manner, although the effect of box DNA was opposite that of the enhancer. The XhoI linker insertion into the consensus sequences of box DNA abolished the repression activity, and the protein(s) recognizing the consensus sequences was identified only in F9 cells, not in L cells. These analyses suggest that box DNA may be a negative regulatory element that functions in undifferentiated cells.

Induction of retrovirus particles in human testicular tumor (Tera-1) cell cultures: an electron microscopic study.

The Tera-1 and Tera-2 cell lines, established from germ-cell tumors of the human testis, were examined by electron microscopy for particles with the morphology of retroviruses. Extracellular and budding particles were observed at low frequencies only in cultures of Tera-1 cells that had been treated with 5-iodo-2'-deoxyuridine and dexamethasone. No particles were detected in untreated cultures of Tera-1 cells or in any preparations of Tera-2 cells.

Cell lines derived from teratocarcinomas.

A variety of cell lines have been isolated in vitro from transplantable teratocarcinomas. Some of them correspond to embryonal carcinoma (EC) cells. They are malignant and represent the stem cells from which all differentiated tissues derive in the tumor or in vitro. EC cells share some biochemical and antigenic properties with multipotential embryonic cells. From one of these EC cell lines, variants have been isolated in vivo and in vitro. Some are of EC type but restricted in their pattern of differentiation; others are altered in their tumorigenicity or in their antigenic characteristics. Another class of such variants corresponds to non-EC types. The most interesting ones correspond to tumoral lines of extra-embryonic tissues. All these cell lines constitute a valuable material for the study of mouse development and differentiation.

A variant F9 embryonal carcinoma cell line which undergoes incomplete differentiation in retinoic acid.

F9 embryonal carcinoma cells treated with 1 microM retinoic acid undergo irreversible differentiation and simultaneously lose their tumorigenicity. Described here are the isolation and characterization of an F9 variant clone (5C), which undergoes partial differentiation in retinoic acid. The behavior of 5C cells indicates that retinoic acid successfully initiates the differentiation pathway but that complete differentiation is not achieved due to a subsequent block in the pathway. The fact that 5C cells do not undergo complete differentiation, or lose their tumorigenicity in response to retinoic acid, indicates that the lesion affects an element involved in the regulation of both these events. Therefore, further genetic and biochemical characterization of this variant should provide information concerning the relationship between the regulation of differentiation and tumorigenicity. Furthermore, the isolation of this variant establishes the feasibility of genetically dissecting the various steps of the differentiation pathway.

Surgical resection of a huge ruptured mature mediastinal teratoma.

Usually slow-growing and benign, mature mediastinal teratomas are rare clinical entities. They may be complicated by rupture into the pleural or pericardial spaces, lungs, or bronchi. Complete surgical resection is the treatment of choice and is usually curative. We report the unusual case of a 24-year-old woman presenting 15 weeks postpartum with a huge ruptured mature mediastinal teratoma superinfected with Mycobacterium avium Catastrophic bleeding from the superior vena cava was encountered on mobilization of adhesions attached to it, requiring extracorporeal membrane oxygenator support for control. Histopathological examination confirmed a 12.0 × 7.8 × 4.5-cm differentiated teratoma without malignant transformation.

Polyomavirus enhancer requirements for expression in embryonal carcinoma cells.

Wild type polyomavirus expression is suppressed in embryonal carcinoma (EC) cell lines. This suppression is alleviated when the EC cells are induced to differentiate. Several characterized host range mutants of polyoma overcome suppression and are able to express and replicate in the undifferentiated EC cells. These previously described isolates were obtained by serial passage of a wild type strain through PCC4 or F9 EC lines. We present a new pyPCC4 isolate (LPT) derived without selection in EC cells. Isolates with host range specificity for a given EC line have been reported to share several common rearrangements and features. These features are also observed in LPT. We report a novel feature shared by these mutants, including LPT, capable of expression in the EC cell line PCC4. In 8 of 10 isolates a novel sequence is created within the enhancer region by rearrangement junctions with near perfect homology to the AP-1 core consensus sequence, 'TGACT(C/A)A'. That the precise location of these junctions varies among these isolates suggest a functional role for this conserved sequence. Our goal is to understand the function of various mutations in host range mutants of polyoma. In order to understand the rearrangements necessary for expression and replication of polyoma in PCC4 cells, we have further characterized the limits of the B enhancer in these cells as compared to those described in permissive cell systems. We have been able to locate the origin proximal limit of the B enhancer for replication close to nt 5189 and distinguish it from the origin proximal limit of the B enhancer for transcription near nt 5215. The two B enhancer cores overlap but do not coincide and are conserved in both cell lines.

Visceral yolk sac-derived tumors.

Externalization of the visceral yolk sac, after fetectomy, induces the development of extra-embryonal fetal tumors in rodents. These tumors are either benign teratomas that appear 3 to 4 weeks after the displacement of the yolk sac or malignant tumors, i.e. yolk sac carcinomas. The latter appear 4 to 8 months after the surgery. If however, Mouse Sarcoma Virus (MSV) is injected in the placentas at the time of fetectomy (day 12 of pregnancy) the malignant tumors develop much earlier (2 to 3 months after surgery) and some display characteristics of embryonal carcinoma. Whether virus induced or not, the yolk sac carcinomas that develop from the displaced visceral yolk sac possess the same morphological and biological characteristics. They are composed of both parietal and visceral yolk sac structures and sometimes trophoblast. The tumors metastasize, grow in ascites form and kill their host. They are readily transplantable in syngeneic rats and grow in tissue culture as an epithelial-like sheet of cells. On the other hand, the benign teratomas are composed of various well differentiated adult tissues. In these tissues, derivatives of all three germ layers are observed. Numerous experiments prove that the stem cells for these various adult tissues are not germ cells. Instead the stem cells are multipotential cells that arise in the displaced yolk sac by a process of dedifferentiation. These poorly differentiated cells originate from the endoderm of the displaced visceral yolk sac. By redifferentiation they give rise to the various adult tissues characteristic for benign teratomas. The multipotential poorly differentiated cells are also likely to be the target cells for malignant transformation. Malignant transformation of these cells, whether induced by a virus or spontaneously occurring in the displaced yolk sac, leads not only to the development of yolk sac carcinomas and eventually embryonal carcinoma but also, although rarely, to choriocarcinoma. The latter tumor is transplantable in allogeneic hosts. It is hormonally active since it secretes lactogen and progesterone. The extra-embryonal fetal tumors and in particular the rat yolk sac carcinomas and choriocarcinoma proved to be a good source for the detection of oncofetal antigens. At least two different oncofetal endodermal antigens were detected with monoclonal antibodies (mab) made after immunization with yolk sac carcinoma. Another mab, made against choriocarcinoma, was found to react specifically with the cytotrophoblast both in the normal placenta and in the tumor. No other placental cells showed a positive reaction.

Organization and state of methylation of endogenous type C retroviral sequences in 129 mouse differentiated and undifferentiated teratocarcinoma cell lines.

Attempts to activate type C endogenous viruses in 129 mouse fibroblasts and in teratocarcinoma-derived cell lines have never been successful, although the genome of these cells contains xenotropic virus-related sequences. We have investigated the arrangement of these sequences and their methylation state by DNA restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization with specific probes. Our results show that the majority of the sequences are organized into two complete provirus families integrated at multiple sites in the cell genome and that they are hypermethylated in embryonal carcinoma cells as compared with differentiated cells. Having previously found a higher expression of viral RNA in 129 derived embryonal carcinoma cells, our data indicate an apparent direct correlation between methylation and type C virogenes expression.

Localization of hepatitis B virus in a primary testicular cancer.

An embryonal carcinoma of the testicle developed in a 29-year-old white man 3 months after recovery from hepatitis B surface antigen-positive hepatitis. Microscopic sections prepared with formalin-fixed tumor tissue were stained with anti-hepatitis B surface antibody and examined by fluorescent microscopy. Appropriate control procedures, including absorption techniques, were also performed. The carcinoma cells contained hepatitis B surface antigen; controls, including sections from normal testicles and three other testicular tumors, were negative. The result indicates that the hepatitis B virus in this case was localized in the tumor cells. The patient was alive and well 4 years after undergoing orchiectomy and chemotherapy.

A teratoma in a duck infected congenitally with duck hepatitis B virus.

A teratoma was diagnosed in an 8-month-old pekin duck based on the presence of tissue derived from embryonic ectoderm, mesoderm, and endoderm in the neoplasm. The neoplasm was examined for the presence of duck hepatitis B virus, because the duck was congenitally infected with the virus, a member of the hepadnavirus family that is associated with hepatic neoplasms in hepadnavirus-infected mammals. Persistent infection occurred in the liver, but no evidence of viral infection was found in the neoplasm.

Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells.

Embryonal carcinoma (EC) mouse cells have been shown to be resistant to infection by retroviruses and small oncogenic DNA viruses, including simian virus 40 and polyoma. When allowed to differentiate, in vitro or in vivo, EC cells become as susceptible to these viruses as differentiated mouse cell lines are. In order to study the relationships between differentiation of EC cells and viral expression, we have isolated and characterized several polyoma mutants that can express early and late functions in undifferentiated EC cells. These mutants, which arose spontaneously during high-multiplicity infection of PCD3 cells (a differentiated fibroblast-like cell line derived from PCC3 EC cells), were selected on PCC4 cells (undifferentiated EC cells) and twice plaque purified. Restriction enzyme analysis of the DNA from several mutants has shown that they all exhibit an additional sequence located in the Pvu II endonuclease fragment 4, close to the junction between Hpa II endonuclease fragments 3 and 5. The size of the insertion varies from 10 to 50 base pairs. The biological properties, including oncogenicity, transforming ability, host range, and burst size of the mutants so far analyzed are similar to those of wild-type virus.

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.

Structural organization of unique retrovirus-like particles budding from human teratocarcinoma cell lines.

Human teratocarcinoma cells cultured in vitro can be induced to produce retrovirus-like particles. The induction procedures are the same as those previously shown to induce the synthesis of animal retroviruses. Electron microscopical evidence is presented that the human teratocarcinoma-derived (HTD) particles are most closely related to the type C retrovirus strains. HTD particles can be banded at 1.16 g/ml in linear sucrose gradients, the characteristic density for retroviruses, and subsequently be used for negative staining and fine structure analysis.

Evidence that HERV-K is the endogenous retrovirus sequence that codes for the human teratocarcinoma-derived retrovirus HTDV.

Human teratocarcinoma-derived viruses (HTDV) are retrovirus-like particles that are regularly observed by electron microscopy at low frequency in cell lines established from human teratocarcinomas. Over the last years, one of our teratocarcinoma cell lines spontaneously began to produce high amounts of HTDV. This cell line is stained in immunofluorescence tests by an antiserum raised against recombinant gag protein of HERV-K, an expressed human endogenous retrovirus sequence. In immunoelectron microscopy of ultrathin frozen sections, this anti HERV-Kgag-specific antiserum reacts specifically with HTDV particles. In Western blots, the antiserum recognizes predominantly a protein with an apparent molecular weight of 30 kDa, presumably the major core protein of HTDV particles. Taken together, these results provide evidence that HERV-K codes for HTDV.