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In two tumour sublines (T.wt/BL and T.wt/Bc), established from mammary adenocarcinomas caused by mouse polyoma (Py) infection of nu/nu mice, integration of polyomavirus DNA sequences into the c-myc gene locus was mapped. A complete Py genome was found to be integrated just upstream from the c-myc gene in T.wt/BL cell line, while only a part of the early Py region coding for the early proteins was inserted in the chromosomal DNA of T.wt/Bc cells. An interference of Py sequences with the regulation of c-myc gene expression gives further significance to a Py-derived tumour system that appears to be similar to some human mammary cancers in the modifications of c-myc expression. Both cell lines were found to produce truncated large T antigen and entire middle and small T antigens. In addition, production of VP1 protein was observed in the T.wt/BL cell line. The integration of polyomavirus sequences and/or expression of viral proteins caused an elevation of c-myc expression. The level of the c-myc expression was higher in both tumour cell lines in comparison with control normal murine mammary gland (NMuMG) lines, but substantially lower than in NMuMG cells infected with polyomavirus. Possible co-operation of Py proteins with c-Myc was examined. Through GST fusion protein pull-down experiments, we evidenced, that c-Myc forms a complex with the common part of the Py early antigens in the two tumour cell lines. Co-localisation of the c-myc and LT was observed in cells overexpressing c-Myc and LT.

Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk., (Copyright 2001 Academic Press.)

To investigate the potential of murine gamma-herpesvirus 72 thymidine kinase (MHV-72-TK) to act as a suicide gene, we used a mammalian expression vector on rat fibroblastoid cells deficient in the cellular TK gene. Substrate specificity was assessed in vitro in cells with stable expression of MHV-72-TK. The Herpes simplex virus 1-TK (HSV-1-TK) was used as a reference suicide gene. Unlike HSV-1-TK modified cells, which were sensitive to ganciclovir (GCV) (IC50=9.7 microM), cells modified by MHV-72-TK did not show sensitivity to this drug. The use of 3'-azido-3'-deoxythymidine (AZT) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) did not affect the growth of cells expressing either MHV-72-TK or HSV-1-TK in the range of concentration used for AZT (0-375 microM) and for BVDU (0-50 microM). In contrast, 5'-fluoro-2'-deoxyuridine (5-FUdR) was extremely cytotoxic and effectively killed MHV-72-TK expressing cells (IC50 value 2.1 microM). This value was 16 times lower than that required to kill cells expressing HSV1-TK. To test whether the bystander effect between two heterologous cell types could be mediated by the MHV-72-TK/5-FUdR system in vitro, cells expressing MHV-72-TK were co-cultured with the tumour fibroblastoid cell line NAD for 48 hours before the drug (10.8 microM) was added. The cell mixtures contained various ratios of cells expressing MHV-72-TK (0 to 50% of total cells). Only 1% of MHV-72-TK-expressing cells were needed to enhance mouse tumour cell killing and to decrease the survival rate to 25.6%. The bystander effect was more pronounced when 10% of cells expressing MHV-72-TK were used, decreasing survival to 17.4%. In parallel, the same concentration of 5-FUdR dose only marginally inhibited tumour cell growth in the absence of exogenous TK activity (84% survival). These results demonstrate the efficiency of MHV-72-TK as a suicide gene when 5-FUdR is used as a prodrug. When sequenced, MHV-72-TK proved to be identical to MHV-68 strain TK.

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.

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.

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.

Cell adhesion influences many important immunological functions such as phagocytosis or T-cell-mediated cytotoxicity. Previous work suggested that the ease of inducing intercellular bonds (i.e. binding efficiency) and the difficulty to separate bound cells with mechanical forces (i.e. binding strength) might be parameters of different significances. The present report describes a study made on two T-lymphocyte/polyoma virus transformed fibroblast hybrid subclones (3D1c and 3D1n) with markedly different adhesive properties: indeed, 3D1c cells were at the same time more readily agglutinated with concanavalin A and more easily disagglutinated than 3D1n. In order to understand these differences, a systematic comparison of various properties of 3D1c and 3D1n cells was undertaken. The following parameters were studied: surface density of concanavalin A binding sites, surface electrostatic charge, hydrophobicity, fluorescence polarization measured on individual cells, and ability to spread on a flat substrate in response to volume or surface forces. It is concluded that cell deformability and/or spreading ability might be an important determinant of binding strength, but the factors governing binding efficiency remained incompletely understood. It is suggested that the methods described in the present report might help understanding differences between various tumor cell lines with different malignant potential.

By two different fusions between a cytotoxic T-lymphocyte (CTL) clone and a polyoma virus (Py)-transformed fibroblast line, 40 hybrid clones have been generated. It has been demonstrated that they were all TCGF independent for multiplication. Moreover, some of these hybrids were functional for cytolytic expression, whether or not TCGF was present either at the time of fusion or in the selective media. Two clones generated from the same fusion were markedly cytolytic and were able to remove TCGF from their culture medium, suggesting that they possessed TCGF receptors. These clones also secreted discrete amounts of a TCGF-like factor. The effect of TCGF on hybrid cell proliferation is discussed.

Various characteristics of transformation were studied in subclones isolated from a hybrid cell line obtained by fusion of two Chinese hamster sub-lines having the same origin but presenting different properties, particularly in respect to heterotransplantability. Different subclones were obtained by cloning on semisoft agar. Transplantability, plating efficiency, agglutinability by concanavalin A and actinomycin D resistance were studied in parallel with the evolution of the karyotype to try to find a correlation between these various parameters. A relationship seems to exist between a chromosome marker arising in the hybrid and the percentage of tumours. The second part of this work dealth with the study of intra and interspecies hybrids, one of the parents of which was a normal, fibroblastic cell and the other of which contained the polyoma virus genome. In the hybrid cell this viral genome was expressed at several levels. Firstly, in the formation of specific polyoma virus-induced antigens and secondaryly, in surface properties normally considered related to the expression of tumorigenicity. Nevertheless, tumour development was repressed. Though the presence of characteristic antigens seemed necessary for the expression of malignant transformation, presence alone was not sufficient to induce malignant transformation of the cell. The study of inter-species mouse/hamster hybrids showed that this situation is not general. For this we examined the properties of hybrid cells between, on the one hand, a mouse tumorigenic cell bearing polyoma virus genetic information and, on the other, non-tumorigenic mouse or hamster cell. In this case the complete hamster genome could bot repress malignancy whereas a few mouse chromosomes sufficed to code for the expression of virus-induced tumour antigens and various malignant properties. It may be hoped that these hybrids could be used to pin-point the chromosome localization of the genetic factors of malignancy and could be used in immunoprotection studies or immunotherapy research.

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.

In anticipation of the use of functional T-lymphocyte hybrids in adoptive immunotherapy, the differentiation and tumorigenicity of hybrid clones generated by fusion of a T lymphocyte derived from F1 (DBA/J2 x AKR) mouse spleen, and a polyoma virus-transformed fibroblast initiated from C3H mouse cells, were studied. The hybrid cells grew in suspension and had an appearance (by transmission and scanning electron microscopy) very similar to that of the lymphocytic line. The hybrid and the different clones could induce tumour grafts. Malignancy was dominant in newborn mice where tumours were obtained in all mouse strains (allogeneic or semi-allogeneic) inoculated. In adult mice, the hybrid cells were tumorigenic in C3H and F1 (DBA/J2 x AKR), whereas there was complete tumour rejection in allogeneic (C57/BL6) or semi-allogeneic (DBA/J2 and AKR) mice. The role played by major histocompatibility antigens in the graft rejection is discussed. The histology of the tumour grafts was intermediate between fibrosarcoma and lymphosarcoma.

A sarcoma was induced in an (A.CA X A.BY)F1 mouse. Two isoantigenic variants were selected by loss of one H-2 antigen. The tumor-associated transplantation antigens (TATA) of these variants were compared as to their specificities in (A.CA X A.BY)F1 mice. Both transplantation and indirect membrane immunfluorescence tests revealed that TATA of both variants did not cross-react. Thus selecting angainst different H-2 antigens also selected different TATA. Karyotype studies suggested that both variants originated from a unique clone.

This study demonstrates that cytolytic T-cell lines exhibit progressive in-vitro modifications of their phenotype and of their growth behaviour and may use different pathways for their multiplication. Comparing three established cell lines, we firstly demonstrated that the expression of LFA-I is stable but the Lyt 2, 3 is rapidly lost. In this case, a high lectin-dependent cytotoxicity appears. Secondly, we demonstrated that two of the cell lines used the interleukin 2-interleukin 2 receptors (IL-2-IL-2R) binding pathway. Two different monoclonal antibodies showed that the IL-2 receptors distribution does not correlate with the number of functional sites which determines the IL-2 requirement. In contrast, the third cell line, although bearing high levels of IL-2 receptors, grows without the addition of IL-2; this cell growth is not inhibited by anti-IL-2 receptors monoclonal antibodies. Thirdly, it appears that the new property of IL-2 independence is associated with acquisition of the simultaneous capacity to induce tumour grafts in nude mice. As it has been recently reported that cytolytic T-lymphocytes against tumour cells could be promising immunotherapeutic agents, the spontaneous malignant transformation of such CTL lines should be taken into account before using them for adoptive immunotherapeutic purposes.

The authors outline schematically the major histocompatibility complex as well as the relationship between tumor neo-antigen and pre-existing antigens at the cell surface. They note that these two antigenic systems are not independent and in particular, the major histocompatibility system (H-2) co-segregates with the tumour antigens. Considering the complexity of the H=2 system (a H-2 allele does not correspond to any single transplantation antigen but to a combination of several antigens units simultaneously present), the authors recall Boyse's hypothesis, revised by Haywood and Mc Khann, which propose that tumour antigens are a rearrangement of H-2 substructures. Moreover, it is possible that the relationship between H-2 and resistance to cancer may be attributed directly to the action of genes controlling the immune response (Ir region) which could also intervene in recognition of these tumour antigens. Finally, recent results obtained seem to show that some tumour antigens cross-react with H-2 fractions. This fact prevents the mice bearing these types of alleles from being immunized against the cross-reacting tumours. If this result could be transposed to the human situation, it would explain the frequency of certain types of tumours in defined H-LA groups and could allow the prediction of high-risk groups.