In vivo requirement for asialo GM1 and Thy1 positive leukocytes for antitumor effect of rIL-2 +/- rIFN-gamma.

Localized treatment with recombinant human interleukin-2 (rIL-2) +/- recombinant murine interferon-gamma (rIFN-gamma) results in regression of early B16 melanomas in normal C57BL/6 (B6) mice, but not in syngeneic beige mice, which have defective natural killer (NK) cells. Injection of antibodies to asialo GM1 (a-AGM1) or Thy1 abolishes the tymoricidal effects of rIL-1 +/- rIFN-gamma. Thus, cells activated by these cytokines must be either NK-like cells that are AGM1+ Thy1+, or NK-like cells (AGM1+) cooperating with T lymphocytes (Thy1+), since either antibody (a-AGM1 or a-Thy1) independently abrogates the in vivo antitumor effect.

Eradication of mouse melanoma by combined treatment with recombinant human interleukin 2 and recombinant murine interferon-gamma.

Successful immunotherapy of early s.c. or i.

Successful immunotherapy of mouse melanoma and sarcoma with recombinant interleukin-2 and cyclophosphamide.

Recombinant human interleukin-2 (rIL-2, courtesy of Cetus Corporation, Emeryville, CA, U.S.A.

Ecotropic murine leukemia virus-induced fusion of murine cells.

Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with [3H]thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses.

DNA-mediated transfer of human melanoma cell surface glycoprotein gp130: identification of transfectants by erythrocyte rosetting.

DNA sequences encoding a human melanoma membrane-bound sialoglycoprotein of 130,000 molecular weight (gp130) were introduced into a clonal derivative of mouse B-16 melanoma cells with the selectable neomycin resistance gene (aminoglycoside phosphotransferase). Mouse transfectants were identified by a rapid and precise screening method with mouse monoclonal antibodies and erythrocyte rosetting. The frequency of gp130 transfectants was approximately 1 in 2,000 to 5,000 colonies with neo+ cells.

Efficient DNA-mediated transfer of selectable genes and unselected sequences into differentiated and undifferentiated mouse melanoma clones.

We have found that three phenotypically dissimilar mouse B16 melanoma subclones are competent recipients for DNA-mediated gene transfer. Two of these approach and a third, amelanotic clone B78H1, surpasses mouse LTK cells in frequencies of transferent colony formation after treatment with either of two codominantly selectable plasmid vectors, pSV2gpt or pGCcos3neo. Melanoma transferents incorporate both selectable plasmid-homologous sequences and substantial amounts of unselected donor DNA into their cellular DNAs.

Leukocyte subpopulations elicited by a nontumorigenic variant of B16 melanoma: their role in direct rejection of the melanoma and in prevention of tumorigenesis in Winn assays.

The mechanisms by which various leukocyte subpopulations elicited by an immunogenic, nontumorigenic subclone (C3471) of B16 melanoma caused rejection of the tumorigenic parental melanoma (B559), were investigated. Leukocytes from C3471-immune mice were co-injected with B559 tumor cells in Winn assays into normal syngeneic recipients. Tumor formation by B559 cells was prevented when C3471-immune (a) unfractionated peritoneal leukocytes, or (b) glass-adherent peritoneal cells (90% macrophages), or (c) nylon wool purified nonadherent cells (95% Thy-1.

Relationship between rejection of several syngeneic tumors and retrovirus production by 5-bromodeoxyuridine-grown melanoma cells: lack of protection in natural killer-deficient beige mice.

Bromodeoxyuridine-grown B16 melanoma cells (C3471) immunize mice against not only the parent melanoma but also two other C57BL/6 tumors: a mammary adenocarcinoma and a methylcholanthrene-induced sarcoma. We have shown that the endogenous retrovirus induced in C3471 cells by bromodeoxyuridine can persistently infect feral mouse (SC1) cells and that they then become as efficient as C3471 cells in preventing tumors. Uninfected SC1 cells cannot protect.

Macrophages elicited with heat-killed bacillus Calomette-Guérin protect C57BL/6J mice against a syngeneic melanoma.

We have demonstrated that a murine cytotoxic peritoneal cell can be elicited by intraperitoneal immunization with heat-killed Mycobacterium bovis, strain Bacillus Calmette-Guérin (BCG). When these cells are injected together with cells of clone B(5)59 of B16 melanoma in a Winn-type transfer assay into syngeneic C57BL/6J mice, the tumorigenic potential of the melanoma is completely abrogated. Similarly, mice immunized intraperitoneally with dead BCG are protected against intraperitoneal challenge with a number of B16 melanoma cells sufficient to cause tumors in 100% of control mice.

Recovery of three distinct biologically active type C viruses from cloned C57Bl/6 melanoma cells.

The genome of a single cell derived from the B16 melanoma contains information for the expression of three distinct biologically active viruses: the N- and B-tropic ecotropic viruses and the xenotropic virus. Their release results in reduction of melanin production. A possible relationship of virus replication to differentiation may be involved.

Malignant mouse melanoma cells do not form tumors when mixed with cells of a non-malignant subclone: relationships between plasminogen activator expression by the tumor cells and the host's immune response.

Mouse melanoma clones B559 and B78 are highly tumorigenic when injected into C57BL/6J mice. Tmor formation by these cells is suppressed when they are mixed with nonmalignant bromodeoxyuridine-grown clone C3471 before injection. C3471 cells suppress tumor formation only in immunocompetent hosts; mixtures of B559 and C3471 cells or C3471 cells alone form tumors in antithymocyte serum (ATS)-treated mice.

Co-cultivation of tumorigenic mouse melanoma cells with cells of a non-tumorigenic subclone inhibits plasminogen activator expression by the melanoma cells.

Clone B559 mouse melanoma cells are highly tumorigenic and produce plasminogen activator. Cells of clone C3471, a line obtained by continued growth of B559 cells in medium containing 5-bromodeoxyuridine (1 microgram/ml), have no plasminogen activator and are non-tumorigenic. When B559 cells are co-cultivated with C3471 cells, the ability of B559 cells to activate plasminogen is suppressed.

Reversible suppression of malignancy and differentiation of melanoma cells.

Tumorigenicity is reversibly suppressed in mouse melanoma cells grown with 5-bromodeoxyuridine (BrdU). The nontumorigenic cells are immunogenic, and preinjection of these cells can protect mice against tumors inevitably formed when the parental, untreated melanoma cells are inoculated into inbred strain C57BL/6. A mixture of highly immunogenic clone, C(3)471, with malignant cells is also nontumorigenic.

Suppression of melanoma cell tyrosinase activity and tumorigenicity after incorporation of bromouracil for one or two cell divisions.

We have studied the kinetics of suppression of tyrosinase activity and tumorigenicity in unsynchronized B16 mouse melanoma cells (clone B559) exposed to 5-bromodeoxyuridine (BrdU, 3 mug/ml) for one or two cell divisions, then cultured in BrdU-free medium (RM) for five or six days. Bromouracil replaced about 23% of thymine residues after 24 hours (1 cell division) and almost 40% after 48 hours (2 cell divisions) in the presence of BrdU. Upon subsequent growth in RM the extent of replacement declined in a manner consistent with dilution by new DNA synthesis, reaching 5-10% substitution by day 7 of these experiments.

Correlated suppression by 5-bromodeoxyuridine of tumorigenicity and plasminogen activator in mouse melanoma cells.

The decrease of tumorigenicity by mouse melanoma clone B559 after growth in the presence of 5-bromodeoxyuridine (BrdU) has been correlated with a decrease in detectable cellular plasminogen activator. Reduction of both activities occurs after one to two cell divisions in the presence of this thymidine analog and is virtually complete within three to four cell cycles. These changes are fully reversible; four to five cell divisions in the absence of BrdU are sufficient to allow both tumorigenicity and plasminogen activator levels to return to normal.

Suppression of pigmentation in mouse melanoma cells by 5-bromodeoxyuridine: effects on tyrosinase activity and melanosome formation.

Low concentrations (1-3 microg/ml) of 5-bromodeoxyuridine (BrdU) reversibly suppress pigmentation in a highly pigmented clone (B(5)59) of cultured B16 mouse melanoma cells. We have found that unpigmented cells (clone C(3)471), derived by long-term culture of B(5)59 cells in 1 microg of BrdU/ml, were completely amelanotic with no biochemically or cytochemically detectable tyrosinase activity or ultrastructural evidence of premelanosomes. The process by which pigmentation is suppressed was studied in B(5)59 cells during a 7-day period of growth with BrdU (3 microg/ml).

Tumorigenicity, immunogenicity, and virus production in mouse melanoma cells treated with 5-bromodeoxyuridine.

Bromodeoxyuridine (BrdU), whether administered in a 30-hr pulse of 30 mug/ml or continuously in low concentrations (1-3 mug/ml), significantly increased production of particles with the morphology of murine leukemia virus in a mouse melanoma (B16) cell line. Particles were very rare in control cells, detectable only by electron microscopy. By contrast, in many experiments with BrdU-treated cells the numbers of virus particles counted by electron microscopy increased over 100-fold, and other tests for murine leukemia virus (plaque assay and tests for group-specific antigens 1 and 3 and for Gross cell-surface antigens) became positive.