Effects of VM-26 and lonidamine on a B16 melanoma cell line.

The treatment of exponentially-growing B16 melanoma cells with teniposide causes a dose- and time-dependent decrease of cell survival. By means of the nucleoid technique, the formation of double strand breaks was demonstrated in the nuclei of the treated cells, indicating a possible involvement of topoisomerase II. DNA double strand breaks were rapidly but ineffectively repaired. Morphometric and densitometric analyses showed that teniposide treatment causes a considerable increase of nuclear area, nuclear DNA and cell size, associated with a lowering of the mitotic index to less than one hundredth of that of the controls. The cytocidal effect of VM-26 can be potentiated by the addition of a non-lethal dose of lonidamine, whose synergism is particularly evident at low teniposide concentrations.

Inhibition of tumor growth, invasion and metastasis in papain-immunized mice.

Papain-immunized mice possess serum antibodies which cross-react with cathepsin-B- and cathepsin-H-like endopeptidases isolated from B16 melanoma cells. The growth rate, invasion and metastasis of both the B16 melanoma and the Lewis lung carcinoma were inhibited in mice immunized with papain. These animals presented an increased mean survival time as compared to the tumor-bearing nonimmunized controls. Quantitative microscopy suggested that vasodilation and edema, associated with tumor invasion, are, at least partially, sustained by proteolytic enzymes, being strongly reduced when tumor cells were inoculated in papain-immunized mice.

In vitro and in vivo enhancement of vincristine antitumor activity on B16 melanoma cells by calcium antagonist flunarizine.

Flunarizine, a calcium antagonist commonly employed in therapy for vascular diseases, enhances the in vitro and in vivo antitumor activity of vincristine on B16 melanoma cells. In the presence of flunarizine higher intracellular levels of vincristine were observed in vitro and for a longer time. B16 melanoma bearing mice treated with both the drugs presented a median survival time that was significantly longer than that of the controls. The possible mechanism of the enhancement is herein discussed.

Effects of a calcium-antagonist (flunarizine) on cancer cell movement and phagocytosis.

Flunarizine, a calcium-antagonist drug that binds to calmodulin, was found to inhibit the migration of both B16 melanoma cells and M5076 macrophage-like cancer cells. The migration movement, both under agarose and through a polycarbonate filter membrane, was impaired by the drug. In M5076 macrophage-like cells Fc-dependent as well as Fc-independent phagocytosis were also found significantly reduced by flunarizine. Results are discussed in relationship with the previously observed effects of the drug on the growth rate of B16 melanoma cells in vitro.

Immunosubversive role of PGE2 in tumor bearing mice.

The immunosuppressive activity of tumor cells was studied in vivo and in vitro using C57BL/6 mice and Lewis lung carcinoma (3LL) cells. The SRBC immunization of tumor-bearing mice in vivo gave a lower number of PFC than the control mice. In vitro, employing the Mishell and Dutton technique, the primary immune response of splenocytes from tumor-bearing mice was significantly reduced. The in vitro primary immune response of normal splenocytes was also reduced when the tumor cells or supernatants of tumor cell cultures were present during SRBC immunization. 3LL cells synthesize a large quantity of PGE2 which was also demonstrated in the supernatants of 3LL cell cultures. Nevertheless, as the addition of indomethacin, a potent inhibitor of the prostaglandin synthesis, only partially reduces the tumor cell immunosuppressive action, prostaglandins are conceivably only one of the factors responsible for the immunodepression exerted by the tumor cells.

Amplification by macrophages of prostaglandin-mediated immunosuppression in mice bearing syngeneic tumors.

The role of macrophages in tumor-mediated immunosuppression was examined, using C57B1/6 strain mice bearing four different immunosuppressive transplantable syngeneic tumors (Lewis Lung Carcinoma, B16 Melanoma, and two fibrosarcomas induced by methylcholanthrene in our laboratory). When tested for immunosuppressive activity, in inhibiting the induction of antibody formation by normal spleen cells in response to SRBC in vitro, the splenic and peritoneal macrophages from tumor-bearing mice were all significantly suppressive. The degree of suppression correlated with immunosuppression in tumor-bearing mice challenged in vivo with SRBC. Direct action of tumor cells on normal splenic macrophages in vitro caused them to become suppressive, the extent of suppression dependent on the time of interaction and on the immunosuppressive activity of the tumor cells in vivo. Pretreatment of suppressive splenic macrophages with indomethacin, a potent inhibitor of the synthesis of prostaglandins (PG), reduced significantly their immunosuppressive activity. Also, peritoneal macrophages from tumor-bearing mice produced significantly more PGE in culture than control macrophages. Thus, tumor-activated macrophages, presumably those macrophages that infiltrate the tumor in a host reaction against the tumor, serve to amplify the level of immunosuppression in the host by producing relatively large amounts of PGE that is a key physiological mediator in the activation and function of suppressor T lymphocytes. The stimulation of PGE synthesis in macrophages, as a result of their interaction with syngeneic tumors, is initiated by PGE produced in relatively large amount by the tumor cells.

Effects of a calcium-antagonist (flunarizine) on the in vitro growth of B16 mouse melanoma cells.

Flunarizine, a drug which binds to calmodulin, was found to inhibit both the growth rate and the survival fraction of a B16 mouse melanoma cell line cultured in vitro. Both of these effects, that are dose and time-dependent, occurred at doses which in vitro cause a 50% inhibition of the calmodulin-activated cyclic nucleotide phosphodiesterase prepared from the tumor. Cyclic AMP was increased in treated B16 melanoma cells, while DNA synthesis was inhibited, though the DNA content/cell was increased. The possible mechanisms of the drug's action are discussed herein.

Immunogenicity of Lewis lung carcinoma in C57BL/6 mice. I. Tumor-associated antigen(s) in peripheral blood, thymus and spleen of tumor-bearing mice.

Some aspects of the immunogenicity of the Lewis lung (3LL) carcinoma in C57BL/6 mice are reported. Immediately after i.m. tumor transplant, and for approximately 24 h, tumor antigens were found in the blood stream. They were observed again on the 5th-6th day, when circulating tumor cells could also be noticed. Soluble immune complexes were detected in the blood sera of tumor-bearing mice from the 11th day, indicating that the host had reacted to the neoplastic antigen stimulation with immunoglobulin synthesis. Furthermore, tumor antigens were observed on some circulating lymphocytes, as well as in the thymus and in the spleen. In spleen sections, atypical mitoses also showed the presence of tumor cells. We obtained the in vitro binding of 3LL-antigens, present in 3 M KCl-solubilized extracts, to normal T lymphocytes prepared from healthy C57BL/6 mice. The possibility that circulating T lymphocytes aspecifically bind tumor antigens, carrying them to the thymus, is discussed.

Non-specifically bound immunoglobulins on mouse placental cells.

There seem to be some similarities between mechanisms employed by embryos and cancer cells from the maternal or tumor bearer's immune system. In the present study, we investigated the presence of hemagglutinins on placental cells and their relationship to circulating antibodies in mice immunized against sheep erythrocytes before mating. The results obtained demonstrated that placental cells may fix large quantities of maternal immunoglobulins. The hemagglutinins are probably fixed on placental cells by the Fc, since the combining sites of the antibody were free and able to react with the antigen in the "rosette" test. It was also demonstrated that placental tissue may fix up to 1/4 of the maternal circulating antibodies, while only 1/200 of them were transmitted to the fetuses. Some theoretical implications of the presence in the placenta of antibodies not directed against feto-placental antigens are also discussed.

Tumor-bound immunoglobulins. Hemagglutinins on ascites tumor cells grown in mice immunized with xenologous erythrocytes.

In this study we demonstrated that gamma G2A myeloma cells in syngeneic BALB/c mice and Ehrlich adenocarcinoma cells in outbred Swiss mice, when grown in animals previously immunized against RRBC, have RRBC antibodies bound on their surface. Ascites tumor cells obtained from animals not immunized against RRBC can bind RRBC antibodies from the medium. Tumor cells from RRBC-immunized mice and tumor cells with RRBC antibodies bound in vitro were both able to form 'rosettes' when tested with RRBC, thus demonstrating that the combining site of the antibody molecule was not involved in the binding. Some theoretical implications of the presence of nonspecific antitumor immunoglobulins on cancer cell membrane are discussed.

IgM participation in tumor rejection by immunized mice.

IgG and IgM participation in tumor rejection was studied in DBA/2 mice immunized against L1210 leukemia and in Swiss mice immunized against Ehrlich adenocarcinoma. In both the systems, IgM globulins seem to be implicated and are present on the cell surface of macrophages, lymphocytes and cancer cells, while IgG globulins are present only on some lymphocytes. IgM are also present in the peritoneal washing fluids (obtained 24 h of the control group. In the former group, protein content is about three times higher than that in the control group, while the relative amount of heavy proteins (18 S) and light proteins (7 S) is quite similar. These observations are discussed, as is the possibility that some complement components as C3 may participate in the reaction.

C'3 participation in the rejection of some experimental tumors.

The possibility that C'3 participates in tumor rejection was investigated in DBA/2 mice previously immunized against L1210 leukemia and in Swiss mice previously immunized against Ehrlich's adenocarcinoma. In both the cases, animals treated with an appropriate dose of cobra venom factor to produce a C'3 depletion for some days after the tumor challenge, developed the neoplasia and had a mortality rate analogous to that of non-immunized animals. Studies on the peritoneal washing cells obtained at different times after the challenge revealed that in C'3 depleted immunized mice IgM are present on lymphocytes, macrophages and tumor cells, as in the immunized controls, but no contract between the cells and no macrophage phagocytosis were observed and the number of tumor cells increased progressively. These findings indicate that C'3 is critically involved in the rejection of the experimental tumors considered.