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.

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.

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.

Analysis of transcription factors binding to the duplicated PEA1 and PEA3 sites that are required for polyomavirus mutant expression in PCC4 embryonic carcinoma cells.

Embryonic carcinoma (EC) cell lines, representative of early embryonic undifferentiated cells, are nonpermissive for polyomavirus (PyV) infection as a result of a blockade of viral DNA early transcription and replication. All enhancers of PyV mutants (Py EC-PCC4), selected for the ability to grow on PCC4 EC cells, display a duplication of PEA1 and PEA3 binding sites (sites 1 and 3). However, the Py EC-PCC4 rearrangement is complex and results in variable mutant enhancer activities. We demonstrate here that duplication of sites 1 and 3 is absolutely required for a cooperative cis activation of early Py EC-PCC4 mutant transcription in PCC4 EC cells. In addition, we detect in PCC4 EC cells significant amounts of site 1- and 3-binding proteins, which we characterize as related to the Fos/Jun and Ets protein families, respectively. Wild-type PyV restriction in PCC4 EC cells may be relieved by a cooperation between site 2- and 3-binding proteins that would thereby be activated. Since site 1- or 3-binding factors could be derepressed, we improved the analysis of UV cross-linked DNA-protein complexes and were able to detect a novel factor, called PEA1/2 (for PyV enhancer A site 1- and 2-binding factor). Its DNA binding sequence overlaps sites 1 and 2 (PEA2 binding site) and is not duplicated in the M1 mutant, which exhibits the highest Py EC-PCC4 enhancer activity. he suggest that PEA1/2 is also involved in the regulation of PyV enhancer activity by repressing the site 1-binding activity.

Identification of human endogenous retroviruses with complex mRNA expression and particle formation.

Retroviruses comprise strains with considerable disease potential in animals and humans. In addition to exogenous strains transmitted horizontally, endogenous proviruses are transmitted through the germ line. Some of these endogenous retroviruses can be pathogenic in mice and possibly in other animal species. They may also be considered as mobile genetic elements with the potential to produce mutations. In humans, genomic DNA contains numerous endogenous retroviral sequences detected by their partial relatedness to animal retroviruses. However, all proviruses sequenced so far have been found to be defective. In this communication, we describe the expression of a family of human endogenous retrovirus sequences (HERV-K) in GH cells, a teratocarcinoma cell line producing the human teratocarcinoma-derived retrovirus (HTDV) particles previously described by us. Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic mRNAs are generated by single or double splicing events. This expression pattern is reminiscent of the more complex control of virus gene regulation observed, for example, with lenti- or spumavirus strains, although HERV-K shows no sequence homology to human T-lymphotropic virus or human immunodeficiency virus. Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family.

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.

A general method for the identification of transcribed retrovirus sequences (R-U5 PCR) reveals the expression of the human endogenous retrovirus loci HERV-H and HERV-K in teratocarcinoma cells.

During the past decade, different types of endogenous retroviral sequences have been defined in the human genome usually by low stringency hybridization employing DNA probes of evolutionary conserved animal retrovirus genes. Although all human genomic loci sequenced were found to be defective or interspersed with stop codons, indirect evidence is accumulating that human endogenous retroviral loci are expressed at least in some instances. One example is the synthesis of retroviral particles in human teratocarcinoma cell lines observed by electron microscopy. To establish a link between virus expression and genomic loci we searched for retroviral RNA in human cellular mRNA populations using a generally applicable method. A tRNA-derived primer complementary to a putative retroviral primer binding site was extended by reverse transcription and this product was elongated with a homopolymeric stretch and amplified by PCR (R-U5 PCR). Cloning and sequencing of such products revealed that the endogenous retroviral loci HERV-H and HERV-K are expressed in those human teratocarcinoma cell lines which produce retroviral particles. The size distribution of four HERV-K mRNAs detected in Northern blots is reminiscent of the complex expression pattern seen with a number of exogenous retroviruses.

Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells.

The brain cell specificity of the human papovavirus JC virus was examined by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motif sites, located within the 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, a fact confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites.

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.

Derivatives of Moloney murine sarcoma virus capable of being transcribed in embryonal carcinoma stem cells have gained a functional Sp1 binding site.

The long terminal repeat (LTR) sequences of Moloney murine leukemia virus and its closely related derivative Moloney murine sarcoma virus (Mo-MSV) are incapable of directing transcription in embryonal carcinoma (EC) stem cells. The myeloproliferative sarcoma virus, a derivative of Mo-MSV, has several point mutations in the LTR and is transcribed more efficiently to allow productive infection of F9 EC cells. One of these mutations, at -166 with respect to the transcriptional start, creates a consensus binding site for the well-characterized mammalian transcription factor Sp1. We used gel retardation assays to demonstrate that F9 EC cell extracts form several complexes with the myeloproliferative sarcoma virus sequence around -166. One of these complexes involves a murine Sp1-like protein, which has immunoreactivity, DNA binding specificity, and electrophoretic mobility equivalent to those of purified human Sp1 protein. An equivalent complex forms on the corresponding Mo-MSV sequence but with a fivefold-lower affinity. Consistent with these observations, introduction of the single point mutation at -166 into the Mo-MSV LTR, creating a consensus Sp1 binding site, increases expression in F9 EC cells sixfold.

Nuclear factor 1 activates the feline leukemia virus long terminal repeat but is posttranscriptionally down-regulated in leukemia cell lines.

A recombinant feline leukemia virus (FeLV) proviral clone (T17T-22) with a long terminal repeat (LTR) which differs from prototype FeLV by a point mutation within a conserved nuclear factor 1 (NF1)-binding motif in the LTR enhancer domain was found to be poorly expressed after DNA transfection. The NF1 point mutation reduced in vitro protein binding as assessed by gel shift analysis and reduced promoter activity significantly (2- to 10-fold). However, the degree of promoter impairment due to the NF1 site mutation varied according to cell type and was least severe in a feline leukemia cell line (T3) which had low levels of nuclear NF1 DNA-binding activity. Low NF1 DNA-binding activity was observed in three FeLV-induced leukemia cell lines (T3, T17, and FL74) and in murine F9 embryonal carcinoma cells. While similar levels of NF1 gene mRNA transcripts were detected in all cell lines, Western immunoblot analysis of F9, T17, and FL74 but not T3 nuclear extracts revealed very low levels of nuclear NF1 protein. These results indicate that NF1 activity is down-regulated in FeLV-induced leukemia cells by diverse posttranscriptional mechanisms. We suggest that NF1 down-regulation may be an important characteristic of target cells susceptible to FeLV transformation in vivo and may provide the selective pressure which favors duplication of the LTR core enhancer sequence in T-cell leukemogenic FeLV variants.

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.

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.

Two blocks in Moloney murine leukemia virus expression in undifferentiated F9 embryonal carcinoma cells as determined by transient expression assays.

Transient expression assays were used to investigate the restriction of Moloney murine leukemia virus (MoMuLV) expression in undifferentiated mouse F9 embryonal carcinoma (EC) cells. We previously reported that the MoMuLV long terminal repeat (LTR) is inactive in undifferentiated F9EC cells due to inactivity of the tandemly repeated MoMuLV transcriptional enhancers. Others suggested that the inactivity was due to the presence of negative regulatory elements that interact with the MoMuLV tandem repeats. Two heterologous enhancer sequences that are active in undifferentiated F9 EC cells were inserted into the MoMuLV LTR: the B enhancers from the F101 variant of polyomavirus and a cellular enhancer sequence isolated from EC cells that we previously identified. The chimeric LTRs were then fused to the bacterial chloramphenicol acetyltransferase gene and tested for expression by transfection into F9 EC or NIH 3T3 cells. Insertion of these enhancers either upstream or downstream of the MoMuLV tandem repeats resulted in transcriptionally active LTRs in undifferentiated EC cells, which did not support the existence of negative regulatory elements interacting with the tandem repeats. In our previous MoMuLV enhancer deletion constructs, the GC-rich sequences downstream from the tandem repeats were also deleted, which might have contributed to the inactivity in EC cells. However, restoration of the GC-rich sequences did not yield an active LTR. The experiments also suggested that the EC cellular enhancer was preferentially active in undifferentiated EC cells and inactive in NIH 3T3 cells. The possibility of negative regulatory sequences in the vicinity of the MoMuLV primer-binding site was tested by inserting MoMuLV sequences from +30 to +419 base pairs into the LTR-chloramphenicol acetyltransferase gene constructs downstream of the transcriptional start site. Transient expression assays confirmed that these sequences reduced expression from functional LTRs in undifferentiated F9 EC cells but reduced expression significantly less in NIH 3T3 cells. Moreover, equivalent sequences from myeloproliferative sarcoma virus did not exhibit this effect. These results supported restriction of MoMuLV expression in undifferentiated F9 EC cells at two levels, inactivity of the MoMuLV enhancers and interaction of negative regulatory factors in the vicinity of the primer-binding site.

Differential regulation of viral and cellular genes in F9 mouse embryonal carcinoma cells.

Expression of genes driven by the SV40 promoter/enhancer appears to be under net negative regulatory control in undifferentiated F9 cells, but not in their differentiated derivatives. In cells containing integrated copies of an SV40 promoter-driven marker gene, induction of differentiation by retinoic acid treatment produced a modest increase in transcription from the viral promoter. A much greater increase was observed when differentiated or undifferentiated cells were treated with the protein synthesis inhibitor, cycloheximide. If cycloheximide acts through removal of negative-regulatory molecule(s), then it is apparent that these molecules are present in both differentiated and undifferentiated cells, and that retinoic acid treatment removes only a portion of the total transcriptional repression. RNA levels from a variety of cellular genes activated during F9 cell differentiation were either unaffected or only slightly increased by cycloheximide treatment. This suggests important qualitative or quantitative differences in the regulation mechanism for viral and cellular genes in differentiating F9 cells.

Genome analysis and reverse transcriptase activity of human teratocarcinoma-derived retroviruses.

Five of five human teratocarcinomas cultured in vitro could be induced to synthesize retrovirus-like particles, albeit in extremely low amounts. The accumulation and purification of the human teratocarcinoma-derived retrovirus (HTDV) from one of these cell lines allowed characterization of its genomic material as high mol. wt. (60S) RNA. Partial purification of HTDV-associated RNA-dependent DNA polymerase (reverse transcriptase) has also been achieved. HTDVs are easily distinguishable from the exogenous human T-lymphotropic viruses and human immunodeficiency viruses by morphological, biological and immunological criteria.

A cellular enhancer of retrovirus gene expression in embryonal carcinoma cells.

Murine embryonal carcinoma (EC) cells are refractory to infection by retroviruses because retroviral long terminal repeat (LTR) enhancers have little activity in EC cells. A previous report described the isolation of clonal EC cell lines that express the integrated neomycin-resistance gene (neo) linked to the Moloney murine leukemia virus LTR. The expression of the neo gene was explained by a cis-acting mechanism [Taketo, M., Gilboa, E. & Sherman, M. I. (1985) Proc. Natl. Acad. Sci. USA 82, 2422-2426]. From one such EC cell line, we isolated the flanking cellular sequence 5' to the proviral genome, ligated it to various test constructs, and transfected into the parental EC cells. The cellular sequence increased expression of the LTR-linked neo gene significantly, in a manner independent of its orientation and position. The neo mRNA was initiated at the bona fide promoter of the LTR. By deletion analyses, we defined a region of DNA essential for the enhancer activity and determined its sequence. This region contains distinctly characteristic stretches as well as some similarity to various viral and cellular enhancers. Thus the LTR-linked neo gene is expressed because the provirus is integrated in the vicinity of this enhancer that is active in undifferentiated EC cells.

Interferon inducibility and sensitivity of human teratocarcinoma-derived cell lines.

Three cell lines tera I, tera II, and PA1, derived from human teratocarcinomas were tested for their capacity to produce interferon (IFN) and for their sensitivity to both human IFN-alpha and IFN-beta. When treated with Newcastle disease virus or Sendai virus, or a synthetic polyribonucleotide, poly(rI):poly(rC), tera I cells produced no IFN and the 2',5'-oligoadenylate (2-5A) synthetase enzymatic pathway was not activated, although there was an increase in protein kinase. In contrast, tera II and PA1 cells produced IFN and both enzymatic activities were detected. IFN treatment has no effect on the growth of any of the cell lines. Tera I and PA1 cells did not develop resistance to challenge with vesicular stomatitis virus or encephalomyocarditis virus, but the growth of a type-C baboon retrovirus was inhibited. Tera II cells were protected against all three viruses. It appears that human teratocarcinoma cell lines can thus differ greatly in their ability to produce IFN and to respond to it.