SV40-induced Sarcoma Research Articles

Differential Extraction of Tumor-Specific Transplantation Antigen and Embryonic Antigen From Simian Virus 40-and Adenovirus 7-Induced Sarcoma Cells of Hamsters With 1-Butanol and 3 M Potassium Chloride2

Simian virus 40 (SV40)-induced sarcomas and adenovirus 7-induced sarcomas (Adv-7) exhibit both specific tumor-specific transplantation antigens (TSTA) and cross-protective embryonic antigens at the cell surface in the LAK:LVG(SYR) strain of Syrian golden hamsters. Specific SV40 TSTA could be released from the surfaces of living hamster sarcoma cells in a 2.5% crude 1-butanol extract (CBE) and served as immunogen to protect syngeneic recipients against subsequent homologous but not heterologous tumor cell challenge. The CBE-extracted SV40-induced TSTA (tumor-specific) was observed to be free of detectable, cross-protective embryonic antigens (EA) by tumor transplantation assays. The induction of cytotoxic lymphocyte-mediated immunity with the CBE-released TSTA was dependent on the administration of a single sensitizing injection of 12-20 micrograms antigen protein. Higher concentrations (50-1,000 micrograms) of the CBE tumor cell extract, given in a single injection, enhanced tumor growth as did two injections of 12.5 micrograms CBE-extracted SV40-induced TSTA at 1-week intervals. A cross-protective antigen(s), not detected in the CBE tumor extracts, was retained in the intact, 1-butanol-extracted SV40 and Adv-7-induced tumor cell lines after completion of the CBE extraction procedure and in similarly extracted 10-day hamster fetal cells. Some alterations in the normal immunogenicity of EA extracted with CBE followed by KCI from SV40-induced sarcoma cells could be detected in the transplantation assays and lymphocyte transformation assays, whereas EA extracted from CBE-KCI-treated Adv-7 cells or 10-day hamster fetal cells retained normal immunogenicity in vivo and in vitro. These procedures provide a means for successful separation of immunogenic SV40- and Adv-7-induced TSTA from detectable, biologically active, cross-protective EA from the surfaces of these sarcoma cells.

Augmentation of specific immune response against a syngeneic SV40-induced sarcoma in mice by depletion of suppressor T cells with cyclophosphamide

When cyclophosphamide was administered to mice before immunization with syngeneic SV40-transformed cells, the specific immune response elicited, as was measured by the tumor cell neutralization assay with a syngeneic SV40-induced sarcoma, was stronger and lasted longer as compared to the response generated in non-cyclophosphamide-treated mice. The augmentation effect of the drug was dependent on cyclophosphamide concentration, being optimal at 100 mg/kg, and on the time of drug administration in relation to antigen immunization, being optimal at 2–4 days before antigen administration. Transfer of T cells from normal syngeneic mice to drug-treated animals abolished the cyclophosphamide-induced augmentation of immune response. These results implied that cyclophosphamide-sensitive T cells suppressed the in vivo generation of specific effector T cells against SV40-induced sarcoma.

Collaboration between distinct subpopulations of T cells in in vitro generation of effector cells capable of growth prevention of syngeneic SV40-induced sarcoma in mice

The concept of T-T cell interaction which was first suggested during cell-mediated immune response to alloantigens was evaluated in a syngeneic tumor system. The combination of lymph node and thymus cells from BALB/c mice immune against syngeneic tumor cells, mKSA, was shown to exhibit collaboration with respect to in vitro generation of effector cells capable of preventing growth of corresponding tumor cells in the tumor cell neutralization assay. While each cell population of either anatomical site did not prevent tumor growth when tested alone, combinations of both did. The antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding lymph node cells. The lymph node and thymus cell populations participating in synergy were found to be thymus dependent. These results suggest that we may be dealing with the same or similar T 1- and T 2-cell populations described before as displaying synergy in response to alloantigens in the graft versus host, mixed lymphocyte, and cell-mediated cytotoxicity reactions.

Augmentation of specific tumor immunity against a syngeneic SV40-induced sarcoma in mice by retinoic acid

The effects of retinoic acid, a vitamin A derivative, which has been shown to have immune adjuvant properties, were studied in vivo in a syngeneic tumor system by the use of the tumor-cell neutralization assay. The effector activity of spleen cells of BALB/c mice immune against a syngeneic SV40-induced sarcoma, mKSA, was specifically augmented by low doses of retinoic acid, whereas high doses had a suppressive effect. In addition, the time required for generation of the effector activity was shortened and the immune activity lasted longer in the retinoic acid-treated mice. With the use of cell-depletion techniques it was demonstrated that thymus-derived lymphocytes were affected by retinoic acid.

T–T cell collaboration in rejection of a syngeneic SV40-induced sarcoma in mice

SYNERGISTIC or collaborative interactions between sub-populations of thymus-dependent lymphocytes were first demonstrated against alloantigens in vivo in graft-versus-host reactions1, and subsequently in vitro in mixed lymphocyte culture responses2 and in generation of cytotoxic lymphocytes3. At least two distinct subpopulations of T cells are involved in the interactions leading to collaborative responses, one acting as an amplifier or helper cell and the other type functioning as the precursor of the effector cell. The interactions between these two T-cell subpopulations yield effects greater than would be expected when each population is examined alone. It is now known that many tumours possess specific antigens which are recognised in vivo and in vitro by syngeneic hosts. There has been extensive study on immune response to SV40-induced syngeneic transplantable mKSA cells of BALB/c mice in our laboratory4–6. This tumour has been shown to possess tumour-associated antigens which can immunise specifically against the growth of mKSA cells. A very useful in vivo assay, the tumour cell neutralisation test (Winn assay), has been used successfully in our laboratory to measure the immune response to SV40 tumour associated antigens and to monitor the activity of various fractions during biochemical antigen purification7–9. The rejection of the tumour was shown to be dependent on the presence of T cells8, however, it has not been established whether different subpopulations of T cells might be involved. There have been no reports on T–T cell collaboration in vivo or in vitro in immune responses to syngeneic tumours. The SV40 tumour system enabled us to investigate this important issue using the in vivo tumour cell neutralisation. Here we describe collaboration between subpopulations of T cells in the rejection of syngeneic SV40-induced tumour in mice.

Syngeneic Antitumor and Antiembryo Immunization Affecting Pregnancy in Mice

Groups of virgin BALB/c female were immunized against methylcholanthrene, SV40-induced or spontaneous syngeneic sarcoma cells or against syngeneic mitomycin-C blocked embryonic cells. Females were then mated to syngeneic males and observed for pregnancy rate and size of litters. To mimic the antiembryo immunization occurring during normal pregnancy, other experimental groups were added in which midgestational embryo fragments were kept in cell-impermeable diffusion chambers placed in the peritoneal cavity of virgin femals for 20 days, and removed before mating these females with syngeneic males. In all cases, antitumor and antiembryo immunization significantly reduced the number of successful pregnancies after the 1st mating while the second pregnancy appeared to be unaffected by the treatment. A significant reduction in the mean litter size was found, in mice immunized with embryonic tissues or with the SV40-induced sarcoma but not in those immunized with methylcholanthrene-induced or spontaneous tumors.

Studies of lymphocyte stimulation by intact tumor-cell and solubilized tumor antigen.

BALB/c mice were rendered immune to syngeneic SV40-induced sarcoma by subcutaneous injection of mKSA-TU5 tissue-culture adapted cells. Spleen cells from immune mice were examined for tumor-cell neutralization in the Winn assay as well as in in vitro lymphocyte stimulation assays. A microculture (200 mul) lymphocyte stimulation (LS) assay utilizing immune spleen cells was employed with mixed lymphocyte/tumor-cell cultures (MLTC) and the papain crude soluble (CS) extracts from mKSA-TU5 cells. Specificity in the LS assay was determined using spleen cells from mice immune to other syngeneic tumors and by soluble antigenic preparation of normal BALB/c spleen cells. The Winn assay studies demonstrated that spleen cells from mKSA-sensitized mice neutralized mKSA tumor cells and this was corroborated by their resistance to direct tumor challenge. Positive lymphocyte transformation responses in MLTC were observed when mKSA-TU5-sensitized spleen cells were mixed with mitocycin-C-treated intact tumor cells or when papain-solubilized antigens of mKSA cells were employed, but not with non-immune spleen cells or with a soluble antigen from normal cells. Papain-solubilized antigen preparations employed in in vitro assays also immunized against challenge with mKSA tumor cells. Specificity of these lymphocyte transformation reactions was demonstrated with non-sensitized lymphoid cells or lymphoid cells from mice sensitized with a syngeneic Kirsten virus-induced respond. Thus, mKSA tumor surface antigens were recognized on intact tumor cells or with microgram quantities of papain-solubilized extracts from these tumor cells. We believe the lymphocyte stimulation assay affords a method for demonstrating the presence of tumor-specific antigen and for monitoring further purification procedures.

Tumour rejection properties of solubilised TSTA from an SV40-induced neoplasm

THE neoplasm mKSA, an SV40-induced sarcoma in BALB/c strain mice has strong, SV40-specific tumour rejection antigens (TSTA). This parental line is also very oncogenic with a tumour dose 50 (TD50) of 102 cells. A subline, passaged in monolayer tissue culture (mKSA-TC), has a much reduced tumour-inducing capacity (TD50 = 2 × 106–2 × 107, cells). Of these mKSA-TC cells 1 × 105 will immunise syngeneic hosts against a challenge of 1 × 106 mKSA cells carried in an ascitic form (mKSA-ASC); immunisation against other SV40-induced BALB/c sarcomas (for example, SVA31C14 and E4) has also been achieved, but not against Adj-PC-5 (ref. 1) nor against Meth A (ref. 2), both neoplasms carrying specific TSTAs and syngeneic with BALB/c but induced by chemical carcinogens.

In vitro neoplastic transformation of newborn hamster salivary-gland tissue by oncogenic DNA viruses.

Explant cultures of newborn hamster salivary-glands were infected with 4 oncogenic DNA viruses: polyoma virus, human adenovirus 12, simian virus 40 (SV40) and the LLE46 strain of adenovirus 7 (the adenovirus 7-SV40 “hybrid”). Only SV40 and LLE46 infected tissues underwent “transformation.” Subcutaneous injection of SV40 transformed cells into irradiated adult hamsters produced fibrosarcomas. Subcutaneous injection of LLE46 transformed cells, however, produced tumors which were bimorphic with 2 distinct histological elements, one composed of small hyperchromatic undifferentiated cells resembling an adenovirus type tumor and the other resembling an SV40-induced sarcoma. No evidence of salivary-gland function as indicated by amylase production could be demonstrated in the transformed cultures or in the tumors which they produced.