Effect of 12-O-tetradecanoylphorbol-13-acetate on two charateristics of transformation acquired sequentially by ENU-exposed rat brain cells.

Cultures derived from rat brains at different times during the latent period of brain-tumour induction by N-ethyl-N-nitrosourea (ENU) showed increased plasminogen activator (PA) activity before being able to form colonies in agar. Control cultures from buffer-exposed animals showed neither property at comparable passages. More detailed investigations, using a culture derived from foetal brains only 2 days after exposure to ENU and clones from this culture, showed a sequence of low PA activity, then increased activity, followed by the ability to form colonies in agar, suggesting progressive transformation of cells in culture. Continuous culturing in the presence of the mouse skin tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), did not accelerate the rate at which these two properties were acquired, but did cause a much greater increase of PA activity once this started to rise. If included in the assay mixture TPA also increased the PA activity of the cells. It therefore appears that in this system TPA can modulate PA activity under certain circumstances.

Differences in pattern and level of plasminogen activator production between a cloned cell line from an ethylnitrosourea-induced glioma and one from normal adult rat brain.

The production of plasminogen activator (PA) by two cloned cell lines, one from an ethylnitrosourea-induced glioma (A15A5) and the other from normal adult rat brain (ARBO C9), has been investigated. Three assays were used to detect and measure PA in harvest fluids, cells and cell lysates. Similar levels were detected in harvest fluids from both cell lines. However, the cell and lysate assays indicated much higher levels in the tumor line. When actively growing cells were compared A15A5 cells had approximately 16X more fibrinolytic activity than the control cells with a limit of detection in the order of 10(3) cells or 1 microgram protein (cell lysate). In contrast for the control cells PA could only be detected when upwards of 10(4) cells or 5-10 micrograms protein were assayed. Plaminogen activator in as few as 10(3) tumor cells could be detected in the presence of 10(4) non-tumor cells. Plasminogen activator in 26 micrograms protein of A15A5 cell lysate could also be detected in the presence of 44 micrograms protein from ARBO C9 lysates indicating no inhibitory activity in the control cell lysates. Levels of PA in both harvest fluids and cell lysates were determined as cultures progressed through the growth cycle. For cell lysates this showed a build-up of PA in the normal cell line as the cells approached and attained confluence. A much higher level was measured in the tumor cells soon after seeding and maintained to confluence. No differences in growth cycle-associated changes in secreted PA could be determined in harvest fluids: both cell lines showing similar levels at confluence.

Fibrinolytic activity of cultured cells derived during ethylnitrosourea-induced carcinogenesis of rat brain.

Using a fibrin-agarose-overlay technique, high levels of plasminogen-dependent fibrinolytic activity have been demonstrated in cell lines derived from an ethylnitrosourea-induced glioma of the rat brain. Cell lines derived from normal adult rat brain showed only low levels of activity. The degree of lysis produced by a cell line was dependent on the average number of cells per colony, and a different pattern of response was observed for tumour and normal cell lines. A good positive correlation existed between the level of fibrinolytic activity, growth in agar and tumourigenicity of a cell line. Fibrinolytic activity was associated with cell lines derived at various times in the latent period, before the appearance of a visible tumour. Many cell lines derived from rat brains at 57-60 (E7), 90-91 (E8) and 111-112 (E6) days after transplacental exposure to ethylnitrosourea showed fibrinolytic activity, and in the latter group the close association with growth in agar and tumourigenicity was also demonstrated. Results from cell lines derived in the E7 and E8 experiments indicated that the possession of fibrinolytic activity preceded the ability of cells to form colonies in agar.

Physiological modification of alkylating agent induced mutagenesis. II. Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli.

The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment. The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA. After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted. The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell. This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA). Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.

Physiological modification of alkylating-agent induced mutagenesis. I. Effect of growth rate and repair capacity on nitrosomethylurea-induced mutation of Escharichia coli.

The effects of repair capacity and growth rate on the induction of mutations by N-methyl-N-nitosourea (MNUA) was investigated using the trpE reversion system of Escherichia coli WP2 and some repair-deficient derivatives isogenic for this gene. In all these strains reducing the growth rate prior to MNUA-treatment caused a reduction in the mutational response, however major differences were observed between strains. In exrA and recA- bacteria stationary phase cells were 100 times less mutable than cells grown at a mean generation time (m.g.t.) of 30 min, whereas reductions of 12 and 25 times were observed in the uvrA- and wild-type strains respectively. In contrast the mutational response of the polA- mutant varied only slightly with growth rate; the increases at high MNUA concentrations being equal to the increase in the trpE gene number. These results show the increasing importance of the error-prone exrA+/recA+-dependent repair system in mutation-induction by MNUA as the growth rate of the culture is reduced and its relative unimportance for mutation induction in nutrient broth-grown cells (m.g.t. 30 min).