T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells). Our results imply that replication of wild-type A2 strain Py DNA can occur in EC cells in the absence of a functional T antigen. However, this protein clearly enhances viral DNA replication and is absolutely required in differentiated cells.

Direct inhibiting effects of [D-Trp6]gonadotropin-releasing hormone on the estrogen-sensitive progression of polyoma virus-induced mammary tumors in athymic mice.

The direct antitumoral effects of gonadotropin-releasing hormone (GnRH) analogues on breast tumors have been surmised from clinical observations and in vitro studies. The present study aimed to determine the effects of the GnRH agonist [D-Trp6]GnRH (Decapeptyl) on steps of experimental mammary carcinogenesis, and the mechanisms, other than the chemical castration, involved. We chose a recent model, i.e., mammary tumors induced by wild-type A2 polyoma (Py) virus in BALB/c female nu/nu mice, which displays the following characteristics. Tumors are mammary adenocarcinomas similar to well differentiated breast carcinomas. Tumor promotion period ends 20 days after Py virus inoculation and is estradiol dependent. The first palpable tumors occur 60 days after Py virus inoculation, and tumor growth is ovarian hormone independent. The effects of Decapeptyl treatment on tumor induction and tumor growth were studied in normal or ovariectomized 6-week-old nude mice inoculated with 10(7) plaque-forming units Py virus (day 0 of experiments). Normal mice and ovariectomized mice percutaneously supplemented with 0.6 micrograms 17 beta-estradiol every other day until day 30 (OvE2 mice) were treated with monthly s.c. injections of the sustained release form of Decapeptyl (5 mg/kg) until the end of 180-day experiments. Overall values for latency periods were included within a day 60 to day 130 time interval. Hormone-independent outgrowth was not affected. We focused on tumor progression before the outgrowth. Incidences on tumor appearance kinetics account for effects at this stage. 17 beta-Estradiol repletion strongly antagonized (P less than 0.001) the slowing effect of ovariectomy on the tumor appearance kinetics, indicating that tumor progression is estradiol sensitive in its early stages. [D-Trp6]GnRH treatment antagonized tumor appearance profiles, inducing similar kinetics in both normal and OvE2 mice. In normal mice, the antagonism (P less than 0.01) was concomitant with significant decreases (P less than 0.05) in serum levels of estradiol and prolactin, which are critical hormones for mammary tumor development in mice, suggesting a pituitary-mediated effect. In OvE2 mice, the antagonism (P less than 0.01) occurs independently of estradiol and prolactin, suggesting a direct effect at the mammary cell level. Because of alterations in kinetics, this effect is exerted at the early stages of tumor progression on Py virus-transformed, ovarian hormone-sensitive cells in the mammary tissue. This new animal model of breast cancer is shown to be useful in characterizing direct antitumoral effects of GnRH analogues and studying the basic mechanisms of mammary carcinogenesis.

Specific tissue targeting of polyoma virus oncogenicity in athymic nude mice.

Inbred athymic nu/nu mice (BALB/c and C57BL/6) were injected subcutaneously with polyoma virus A2 strain or with polyoma mutants which are able to infect undifferentiated embryonal carcinoma cells and harbor mutations in their enhancer sequences. Mammary adenocarcinomas were induced exclusively in females in which they represent the majority of the tumors. Both males and females developed sarcomas, mostly osteosarcomas, with a similar low frequency. No other type of neoplasm was observed. Mutations affecting the enhancers do not have any effect on the histotype of the tumors. Multiple copies of intact or defective free viral DNA were detected in all tumors. Such a sex-linked specific tissue targeting suggests a hormonal control of tumor initiation and/or promotion. From a pathological point of view, polyoma-induced adenocarcinomas are very similar to human early breast cancers. Tumor induction in nude mice by polyoma virus therefore represents a unique experimental model which differs from the more extensively used newborn immunocompetent mice.

Estradiol dependence of the specific mammary tissue targeting of polyoma virus oncogenicity in nude mice.

We have previously reported (Berebbi et al., 1988) that in athymic nude mice, Polyoma virus induces mammary adenocarcinomas (MAC) at high frequency and exclusively in females. In the present study we show that in nude mice: (1) Ovariectomy results in a reduced frequency of MAC and a longer latency period of induction. When testosterone is administered to ovariectomized females, tumor induction is drastically reduced. (2) When estradiol is administered continuously to ovariectomized females the incidence and kinetics of MAC induction are the same as in control females. (3) MAC are induced in castrated males administered with estradiol although only osteosarcomas are observed in control males. (4) The tumor cells are found to harbor functional estradiol and progesterone receptors. (5) MAC can be transplanted from females to males, indicating that tumor growth is estradiol independent. (6) Estradiol is required only between day 10 and day 20 following polyoma injection, whereas the first tumors are detected only around day 60. Our results indicate that MAC induction by Polyoma virus in nude mice is estradiol-dependent during a short initiation period and that tumor progression is estradiol-independent in spite of the fact tumor cells carry functional estradiol and progesterone receptors.

Immunophenotypic study of atypical lymphocytes. Generated in peripheral blood and spleen of nude mice After MHV-72 infection.

Inbred athymic nude mice (BALB/c) were injected subcutaneously with the wild-type murine gammaherpesvirus 72 (MHV-72), which has been shown to induce the infectious mononucleosis (IM)-like syndrome in immunocompetent mice. The mice were also injected with UV-irradiated MHV-72. We studied the pattern of acute and chronic infection in the blood cells of the nude mice and detected viral DNA sequences in the infected leukocytes by polymerase chain reaction (PCR) technique up to when the animal died, close to 1 month postinfection. Using the UV-irradiated virus that induces an increase in mouse survival time, the viral sequences were present in the blood up to 3 months postinfection, then disappeared. We detected atypical lymphocytes in the blood of mice infected with both wt and UV-irradiated viruses. These atypical cells were similar in shape to those present in the blood of patients with IM induced by Epstein-Barr virus (EBV). Via Unscheduled DNA Synthesis (UDS), DNA synthesis was demonstrated in the atypical cells whose phenotype is identical to that of B cells, as shown with a panel of monoclonal antibodies. By double immunofluorescence staining, using an hyperimmune anti-MHV-72 serum and an anti-IgG + IgM + IgA monoclonal antibody, we demonstrated that these atypical B cells express some viral antigens. Contrary to the immunocompetent mice, the nude mice did not develop splenomegaly after infection with wt virus, probably due to the lack of T cell subsets. However, we observed an increase of nude mice B cells in the spleen. The nude mice died 1 month postinfection showing a high frequency (40%) of atypical lymphoblast-like B-cells in the blood; the increase in natural killer (NK) cell number was not detected after infection. Such findings suggest that NK cells probably did not play an important role in immune response to the MHV infection in nude mice. Finally, this mouse model could play an important role in antigammaherpesviral therapy of immunocompromised patients.

Transcription of polyoma DNA by Escherichia coli RNA polymerase: influence of ionic strength on promoter selection.

The influence of ionic strength on transcription of polyoma DNA by Escherichia coli RNA polymerase was investigated. At 0.15 M KCl, transcription was highly symmetrical and, due to the lack of reinitiation, a limited extent of RNA synthesis was observed. When the concentration of KCl was raised to 0.45 M, the affinity of the enzyme for its template, as well as its apparent affinity for ribonucleoside triphosphates, was reduced. Under optimal conditions, the rate and extent of RNA synthesis at 0.45 M KCl were greater than at 0.15 M KCl, and transcription was mostly asymmetric. Binding and initiation sites at both ionic strengths were identified; at 0.15 M KCl, transcription was initiated from two major sites, located at 0.99 and 0.06 map unit, whereas at 0.45 M KCl, a unique initiation site, at 0.99 map unit, was selected by RNA polymerase.

DNA-binding proteins in mouse cells infected with polyoma virus.

DNA-binding proteins from uninfected or polyoma virus-infected mouse cells (3T6) were isolated and compared by DNA-cellulose chromatography and SDS-PAGE. During the early phase of infection, few but significant and reproducible differences were observed. In the 0.4 M NaCl eluate, four major new polypeptides were detected in infected cells, with approximate molecular weights of 160 K, 130 K, 86 K and 81 K, the last two being compatible with previous estimates for polyoma T antigen molecular weight.

In vivo induction of tumor-specific immunity by glycolipid extracts of SV40-transformed cells.

Glycolipid extracts were prepared from various Syrian golden hamster cell lines, either SV40-transformed or spontaneously transformed. To detect possible SV40-TSTA activity of the glycolipid preparations, normal hamsters were inoculated with different glycolipid extracts and were subsequently challenged with an SV40 tumor-cell line. Significant immunoprotection against SV40 tumor challenge was induced with glycolipids obtained from SV40-transformed cell lines. This was expressed as complete tumor rejection or as a decrease in tumor growth rate, when compared to controls. No protective effects were induced with glycolipid extracts from spontaneously transformed cells. Results suggest that tumor-specific glycolipids synthesized in cells transformed by SV40 virus could act as tumor transplantation antigens responsible for specific tumor rejection in syngeneic hosts.

In vitro transcription by Xenopus oocytes RNA polymerase III requires a DNA topoisomerase II activity.

Extracts of Xenopus laevis oocytes are able to assemble minichromosomes in vitro when they are supplemented with ATP and Mg2+. We have followed the time course of in vitro DNA supercoiling and transcription of polyoma virus closed circular DNA. The transcriptional activity increased with the assembly time of chromatin in the presence of both ATP and Mg2+, but only residual activity was detected in the absence of either of them. We also found that polyoma DNA as well as 5S RNA gene transcription were carried out mainly by an RNA polymerase III. On the other hand, polyoma RNA and 5S RNA synthesis were inhibited by novobiocin in a way that suggested the requirement of a DNA topoisomerase II or DNA gyrase activity for the initiation of transcription.

Polyoma virus mutants as probes of variety among mouse embryonal carcinoma cell lines.

Embryonal carcinoma (EC) cells are resistant to infection with polyoma virus and become permissive when allowed to differentiate. Polyoma host-range (PyEC) mutants have been selected on two embryonal carcinoma cell lines, PCC4 and F9, which differ in their differentiation potential, both in vitro and in vivo. Since PyEC mutants selected on one line failed to develop, or developed only poorly on the other, we used these two classes of mutants as probes towards several EC lines which differed in their origin and differentiation properties. From their susceptibility to either mutant, and from the effect of temperature upon the efficiency of infection, we inferred a classification of these teratocarcinoma cell lines, which is an agreement with a previous one based upon metabolic coupling. We also discussed results indicating that different EC cell lines might present steps in the process of cell determination at the embryonic level.

Regulation of polyoma virus transcription in murine embryonal carcinoma cells.

Undifferentiated murine embryonal carcinoma (EC) cells are resistant to infection with wild-type polyoma virus. The block appears to be located at the transcriptional level. Polyoma host range mutants capable of expressing early and late functions in EC cells have been isolated. The modifications responsible for the phenotype of these mutants are localized in the noncoding region of polyoma DNA genome, containing regulatory sequences for replication and transcription. We compared the 5' termini of early and late mRNAs of wild-type polyoma and mutant viruses in EC cells and in permissive cells. Our results show that wild-type mRNA is normally spliced in EC cells but present at a very low level. The sequence modifications of the mutant viruses lead to a 100-fold increase in the production of mRNA in these cells, but the major 5' termini of early and late mRNAs are identical to those in wild-type-infected 3T6 cells.

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.

Origin of polyoma virus-associated endonuclease.

Polyoma virus particles purified from infected cells, but not from the culture medium, exhibited an endonuclease activity distinct from the serum contaminant recently described. This endonuclease cleaved form I polyoma DNA once only per molecule, at one of three possible sites corresponding to the known adenosine-ribosylthymine-rich regions of the molecule.

Fine structure of the origin-proximal DNAase I-hypersensitive region in wild-type and EC mutant polyoma.

The chromatin of wild-type polyoma virus displays a unique DNAase I highly sensitive region in situ in the infected nuclei, extending for about 260 nucleotides from the origin of replication to the beginning of the late region. We show that this highly sensitive region is not homogeneous. It displays a well defined pattern of differential sensitivity along its 260 nucleotides, including one protected subregion and two hypersensitive sites, which concern a unique residue or very few nucleotides. All these features were mapped to a precision of +/- 5 bp relative to the DNA nucleotide sequence. In parallel, we studied a PyEC mutant, whose sequence is grossly rear-ranged in this very region. This allows the PyEC to overcome the block in the expression of the early genes in the mouse embryonal carcinoma PCC4 cell line. We show that this mutant, however, displays an identical highly sensitive region with the same fine structure. The mapping of this structure on the mutant nucleotides sequence coincides with that of the wild-type relative to any arbitrary point on the late side of the rearrangement; however, 60% of the mutant molecules display this unique local chromatin structure, instead of 20% for the wild-type ones. Finally, the sequence determinism of this singularity is discussed, as well as its possible role in the control of the early transcription and the establishment of the PyEC phenotype..

Transcriptional activity in 3T3, F9, and PCC4 embryonal carcinoma cells: a systematic deletion and linker-scanning study of the polyomavirus enhancer.

The polyomavirus (PyV) genome is not expressed in undifferentiated embryonal carcinoma (EC) cells such as PCC4 or F9 EC cells. All the viral mutants that have been selected for their expression in these cells harbor mutations or rearrangements within a region which is important for early and late transcription (as transcriptional enhancer) as well as for viral DNA replication. We have studied the role of the different parts of this enhancer on the transcription driven by early PyV promoter. Two series of constructions containing progressive deletions starting from the ends of the region and one series containing linker-scanning mutations were tested in a luciferase assay in three cell lines: 3T3, F9, or PCC4. The results revealed that some regions that have not yet been shown to bind transcription factors are nevertheless important for transcriptional activity. Two subregions between nucleotides 5179 and 5187 and between 5220 and 5227 were found to be inhibitory for the activity of the enhancer in EC cells. The PEA1/AP1 binding site was also unexpectedly shown to be important in F9 EC cells.

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.

Expression of polyoma early functions in mouse embryonal carcinoma cells depends on sequence rearrangements in the beginning of the late region.

Established mouse cell lines, primary cultures of mouse cells, and differentiated cell lines derived from mouse teratocarcinoma are permissive to polyoma virus. No viral early or late functions are expressed upon infection and penetration of multipotential embryonal cell lines. Polyoma mutants capable of growth on these cells were isolated and their DNA was cloned. Both the linear cloned viral DNA and a hybrid composed of mutant Bam HI (0.58) to Bgl I (0.72) 750 bp fragment (containing the origin of replication) ligated to the complementary wild-type 4.5 kb fragment are able to multiply on PCC4 embryonal carcinoma cells. The nucleotide sequence of two mutants indicated a genomic rearrangement on the late side of the origin, in which a deletion starting at nucleotides 46 (Py 204) and 77 (Py97) and terminating for both in nucleotide 107 was replaced by the duplication of a downstream late sequence starting at nucleotide 138 (Py 204) and 157 Py97) and terminating in nucleotide 220. The fact that the sequence rearrangements permit the expression of early and late functions upon infection suggests that this region participates in the control of early transcription. This control is different in embryonal and differentiated mouse cells.

Isolation of polyomavirus mutants multiadapted to murine embryonal carcinoma cells.

Polyomavirus mutants were isolated from PCC4 embryonal carcinoma cells infected with a variant strain of polyomavirus (ev 1001h) and were found to contain a tandem duplication overlapping the enhancers and the origin of replication. These mutants were able to infect several lines of embryonal carcinoma cells, including PCC4, F9, and LT1. The sequence and structure of one of these mutants are presented and compared with those of other PyEC PCC4 mutants previously described.