Selma Silagi

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. In addition they retain the distinctive states of differentiation characteristic of the untreated clones. Frequencies of pGCcosSneo-mediated transfer of neo gene-encoded antibiotic resistance into B78H1 can reach 10−2 in response to treatment with as little as 15 ng plasmid/ml coprecipitate/dish. B78H1 cells readily give rise to “secondary” transferents for the neo gene after treatment with DNA from a “primary” B78H1 neo transferent. This gene transfer system has potential applications for study of regulation of melanoma and neural crest differentiation and malignancy.

Effects of 5-bromodeoxyuridine on tumorigenicity, immunogenicity, virus production, plasminogen activator, and melanogenesis of mouse melanoma cells.

Publisher Summary This chapter discusses the effects of 5-bromodeoxyuridine (BrdU) on tumorigenicity, immunogenicity, virus production, plasminogen activator, and melanogenesis of mouse melanoma cells. Tumorigenicity and expression of PA appeared to be immediately affected by these changes; there was a lag in the time course of reduction and subsequent regaining of tyrosinase activity. The effects of BrdU on total RNA or protein synthesis or on plating efficiency appear insufficient to account for the degree of suppression of function observed. Investigations on the suppression of tumorigenicity by BrdU reveal separate components involved in the tumorigenic potential of melanoma cells. Both reduction in the intrinsic ability of the cells to grow in vivo and changes evoking host defense mechanisms appear to operate in the reduction of tumorigenicity by BrdU treatment. Host response may depend on antigenic changes, some of which may be related to the induction of C-type virus in BrdU-treated cells. The untreated melanoma cells grow in a manner suggestive of transformed cells. The suppression of melanogenesis by BrdU occurs through a coordinated effect on the structural and enzymic proteins required for melanin synthesis. These results are consistent with cessation of synthesis of tyrosinase and melanosomal structural proteins in BrdU-treated cells, and indicate that there may be a “program” of gene activity for melanogenesis, which is regulated as a unit.

Modification of malignancy by 5-bromodeoxyuridine

SummaryTumorigenicity of a clonally derived line of mouse melanoma (B16) cells is completely suppressed after growth for 14 days in 3 μg per ml of 5-bromodeoxyuridine (BrdU) and partially suppressed after growth for shorter periods of time at the same concentration or for long periods at 1 μg per ml of BrdU. The tumorigenicity returns after growth in normal medium. The time course of this return is gradual and similar to the time course of the loss of tumorigenicity, although slower. Several other mouse tumor lines also show a reduction of tumorigenicity after growth in BrdU.Injection of mice with cells (C471) maintained continuously in 1 μg per ml of BrdU produces an inflammatory reaction at the site of injection in C57BL/6J mice and confers immunity to subsequent challenge with untreated tumorigenic melanoma cells. The degree of immunity conferred increases linearly with the number of preinjections, with four preinjections producing 90% immunity. Injections of BrdU-treated cells together with or later than tumorigenic cells confer little or no protection.

Interstrain somatic cell hybrids in the mouse: Chromosome and enzyme analyses☆

Abstract An intraspecific mouse somatic cell hybrid population (C3H X C57BL) and its clonally derived descendants were studied. The modal chromosome number of the cloned hybrid decreased by 12% after 86 tissue culture passages, and by 20% after 2 years of tumor passage. This moderate loss of chromosomes represented a genuine reduction and could not be attributed to the centric fusion of acrocentric chromosomes. Intraspecific isoenzyme genetic variants specific for and different between C3H and C57BL cells were retained in the hybrids. The enzyme variants could be employed to deduce the balance between the input parental genomes in the hybrids.

Immunization of syngeneic mice against malignant melanoma with subcellular membrane fractions of a bromodeoxyuridine-grown variant

SummarySyngeneic C57BL/6 mice immunized with non-tumorigenic B16 melanoma cells and crude membrane fractions are able to reject challenge with the tumorigenic B559 parent clone. The immunogenic C3471 variant was derived from the malignant B559 clone by continuous growth in the presence of 1 μg 5-bromodeoxyuridine (BrdUrd)/ml. Fifty-one percent (35 of 69) of mice immunized by three inoculations of 106 cell equivalents of C3471 crude membranes (CM) isolated by nitrogen cavitation remained tumor-free for at least 50 days after challenge with a tumorigenic dose of B559 cells. This compares favorably with the virtually 100% protection evoked by 106 viable C3471 cells. For those CM-immunized mice failing to reject B559 challenge, the mean latent period for tumor formation was significantly increased (P≥0.001) over controls. In addition, mice immunized with cultured C3471 cells were able to reject, with equal efficiency, challenge with either cultured or tumor-derived B559 cells, indicating that the immunogen(s) present on C3471 cells and CMs was (were) not a tissue culture artifact. Freshly prepared syngeneic fascia cells and membranes as well as CM prepared from cultured malignant B559 cells had no tumor rejection activity. In vivo tumor rejection activity in plasma membrane vesicles prepared from C3471 cells by formaldehyde treatment also demonstrated tumor rejection activity. The host response to CMs, as to C3471 cells, could be transferred by lymphoid cells from mice immunized with C3471 CMs or cells. Co-injection of leucocytes from CM-immunized mice together with a tumorigenic dose of B559 cells into immunocompetent syngeneic mice resulted in abrogation of B559 tumorigenicity. The tumor rejection antigen(s) induced or increased by growth in BrdUrd has not yet been characterized biochemically, but is likely to involve a cell surface component common to both cell types and is retained by crude membrane fractions. The use of subcellular fractions from a spontaneous melanoma grown with BrdUrd, which elicits immunity against the malignant tumor, represents a model with immunoprophylactic potential for human neoplasia.