Tumor-associated antigens in isoantigenic variants of a 3-methylcholanthrene-induced sarcoma.

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.

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.

The bystander effect mediated by the new murine gammaherpesvirus 72--thymidine kinase/5'-fluoro-2'-deoxyuridine (MHV72-TK/5-FUdR) system in vitro.

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.

[Transmission and expression of malignancy in somatic hybrid cell lines (hamster/hamster, mouse/mouse, hamster/mouse)]. / Transmission et expression de la malignité dans les lignées cellulaires d'hybrides somatiques (hamster/hamster, souris/souris/hamster souris)

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.

[Relationship between tumor antigens and histocompatibility antigens at the cell membrane]. / Relation entre antigènes tumoraux et antigènes d'histocompatibilité à la membrane cellulaire

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.

Correlation between resistance to actinomycin D, karyology, agglutination by concanavalin A and tumorigenicity in Chinese hamster hybrid cells.

Subclones isolated from a Chinese hamster hybrid line, derived from fusion of an actinomycin D-resistant and an actinomycin D-sensitive strain, were studied with respect to their resistance to actinomycin D, karyology, transplantability and agglutination by concanavalin A. Statistical analysis of the results allowed the establishment of a classification of the strains based on increasing resistance to actinomycin D. There appeared to be an inverse correlation between actinomycin D-resistance and tumorigenicity and a positive correlation between this resistance and the presence of a marker chromosome.

Functional characteristics of cytotoxic T lymphocyte polyoma virus-transformed fibroblast hybrids.

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.

Identification of a short amino acid sequence essential for efficient nuclear targeting of the Epstein-Barr virus nuclear antigen 3A.

The Epstein-Barr virus nuclear antigen 3A is expressed in the nuclei of cells latently infected by the Epstein-Barr virus. We have previously shown that a fragment of 265 amino acids was essential for the proper subcellular localization of the Epstein-Barr virus nuclear antigen 3A. As described in this paper, we have used deletion analysis to identify a decapeptide, RDRRRNPASR, which is essential for nuclear localization of this protein. Furthermore, this decapeptide is a functional nuclear localization signal as demonstrated by its ability to target expression of beta-galactosidase in the nuclei of transfected cells.

Phenotypic evolution of CTL B-lines in vitro.

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.

Correlation between characteristics of transformation and malignancy of intra and interspecific somatic hybrid cells.

The transformed properties of five hybrid cell lines which had either one or another parent in common were studied and compared with their tumorigenicity. Three hybrid cell lines, derived from the Chinese hamster DC-3F/ADX/Aza line, were resistant to actinomycin-D. This property seemed to be correlated with the presence of a marker chromosome from the common parent. The tumorigenicity was intermediate between those of the parent cell lines. On the other hand, agglutinability by concanavalin A (Con A) was variable. Three hybrid cell lines which had either the A9 or the clone 1D (both derived from mouse fibroblasts) showed very similar transformed characteristics, but two were tumorigenic and one not so. It appears from this study that the properties of the hybrid cell lines can be influenced more by one parent, depending on the genes retained at chromosome segregation. The limits of Con A agglutination as a characteristic of transformation and the validity of the check pouch grafts as tumorigenicity test for malignant human cell lines are discussed.

T-cell-fibroblast hybridoma deformability and concanavalin A-induced agglutination.

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.

Cytolytically active murine T-lymphocyte/polyoma virus-transformed fibroblast hybrids.

It would be of great interest to obtain permanent T-cell lines retaining specific activity without either allogeneic or xenogeneic stimulation. Functionally active hybrids between cytolytic T cells and thymoma were previously reported, but they had to be selected in a TCGF-containing medium. This study contains new results and reports the preparation of a hybrid cell from a cytolytic T cell and a polyoma virus-infected fibroblast, in which the T-cell characteristics dominate over the polyoma-transformed characteristics. A differentiated T-cell function (i.e., cytolysis) persists and the differentiated line does not require TCGF. The loss of cytolytic activity during in vitro evolution may be due to a selection favouring transformed cells, as suggested by concomitant enhancement of the transformed phenotype and chromosome loss.

Tumorigenicity and tumour-graft rejection of polyoma virus-transformed fibroblast-T-lymphocyte hybrids.

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.

Chromosomes involved in production of infectious SV40 particles in mouse/SV40-transformed Chinese hamster cell hybrids.

The clone 6d hybrid, capable of expressing the virus-specific T-antigen but unable to produce infectious virus particles after superinfection, presented a complete mouse (3T3-4E) chromosome complement and a significant loss of Chinese hamster (CHK/SVLP AG) chromosomes. Similar properties were displayed by a BUdR-resistant derivative of the Cl 6d hybrid (Cl 6d.6BU). Three independent superhybrid clones (CL 10B, Cl 10C, Cl 11A) isolated after backcross of the Cl 6d.6BU hybrid with a nontransformed Chinese hamster kidney cell line (CHK/AG) were able to produce infectious SV40 virus. In spite of the loss of mouse chromosomes, there was no significant difference in the average number of chromosomes between the Cl 6d.6BU and the superhybrid clones. Thus, the Chinese hamster chromosomes seemed to compensate for the loss of the mouse chromosomes. Although the effect of Chinese hamster chromosomes cannot be totally disregarded, our data suggested a positive correlation between the inability to produce infectious SV40 and the presence of certain mouse chromosomes.