Showing papers by "Paul B. Fisher published in 1979"

Effect of Phorbol Ester Tumor Promoters on the Expression of Melanogenesis in B-16 Melanoma Cells

Abstract: Cells of the C 3 clone of B-16 melanoma synthesize melanin only at confluence after which they senesce and can no longer be passaged. Addition to the cultures of 10 −8 −10 −7 m 12- O -tetradecanoylphorbol-13-acetate (TPA) shortly after plating delayed by about 2 days the onset of melanogenesis. TPA did not, however, affect the growth of the cells or the time at which they reached confluence. The ability of a series of phorbol esters to delay melanogenesis correlated with their tumor-promoting activity on mouse skin. The optimum time for addition of TPA was within the first 24 hr after plating; the inhibitory effect decreased when TPA was added at later points. α-melanocyte-stimulating hormone (5 × 10 −7 m) added to B-16 cultures 24 hr after plating slowed the growth of the cells and caused them to differentiate when still subconfluent. TPA also inhibited this α-melanocyte-stimulating hormone-induced melanogenesis. These results suggest that TPA inhibits a very early stage in a stepwise process that leads to the differentiation of these cultures. For reasons that are not apparent, the cells eventually escape from this inhibition. The B-16 melanoma cell culture system may be useful for studying the mechanism by which TPA and related tumor promoters affect cellular differentiation.

Phenotypic Properties and Tumor Promoter-induced Alterations in Rat Embryo Cells Transformed by Adenovirus

Abstract: Seven clones of secondary rat embryo cells transformed by a temperature-sensitive mutant of human adenovirus type 5 were analyzed to determine whether transformants obtained from cultures treated with chemical carcinogen prior to virus infection had a different phenotype than those obtained from cultures treated with virus alone. In addition, the effects of prolonged serial passage and the effects of exposure of the clones to 12- O -tetradecanoylphorbol-13-acetate on the expression of several markers of transformation were also determined. When compared to normal secondary rat embryo cells, most of the transformants had a reduced populationdoubling time, increased saturation density, reduced serum requirement, increased plasminogen activator production, reduced large external transformation-sensitive protein, increased lectin agglutinability, and decreased anchorage dependence ( i.e. , growth in agarose and agar). Prolonged serial passage of the transformants led to a spontaneous increase in cloning efficiency in agar. There was no consistent difference between the phenotypes of transformants obtained from cultures treated with carcinogen plus virus or those of transformants obtained with virus alone, although clones obtained from the carcinogen-plus-virus treatment appeared to express anchorage independence at earlier subpassages. The most striking finding was that 12- O -tetradecanoylphorbol-13-acetate caused an appreciable enhancement of the growth in agar of all of the transformants. Two early-passage-transformed clones grew in agar only in the presence of 12- O -tetradecanoylphorbol-13-acetate. The tumor promoter also increased the saturation densities and enhanced the cloning efficiencies in liquid medium of most of the transformants. These effects were not as striking with normal secondary rat embryo cells. These results suggest that following adenovirus transformation there is a spontaneous progression in the expression of markers of transformation and that the phorbol ester tumor promoters can accelerate this process.

Selecting interspecific human-mouse and Chinese hamster-mouse hybrids using a new half-selection technique with a polyene antibiotic.

Abstract: Interspecific human-mouse and Chinese hamster-mouse hybrids were isolated from polyethylene glycol fused cells by a new half-selection technique employing a structurally modified polyene macrolide antibiotic, amphotericin B methyl ester (AME), and HAT media. Unfused parental cells were killed as a result of innate sensitivity to AME or their genetic deficiency, absence of thymidine kinase (TK-) or hypoxanthine guanine-phosphoribosyl transferase (HGPRT-). In contrast, hybrid colonies were isolated after two to three weeks growth in three or four changes of HAT-AME media and subsequent growth in HAT media alone. The ability of hybrid cells to proliferate using this selective protocol indicates that genetic complementation resulted, and polyene antibiotic resistance was expressed as a dominant phenotypic property in the hybrids. Hybrid selection was dependent on: (1) the number of cells of each parental cell type co-cultivated; (2) the level of polyene antibiotic administered; and (3) the time interval before selection was initiated. The half-selection technique described in this report is simple to use, very effective in eliminating unfused parental cells and increases the potential types of hybrids which can be formed. Only one parental cell type need contain a biochemical defect, whereas the second parental type can be genetically normal.