Several clones of transformed chick embryo fibroblasts infected with wild-type B77-C or Prague-C strain of Rous sarcoma virus have been isolated from sof agar suspension. These clones were screened for plasminogen activator activity by overlaying monolayer cultures with medium containing agar, casein, and chicken plasminogen. Twenty-three percent of all of the isolated clones showed little caseinolytic activity, 42% had intermediate activity, and 35% had high activity. Although the clones with low plasminogen activator activity had no more than twice the activity shown by uninfected fibroblasts, they did not differ significantly from clones possessing high levels of plasminogen activator in their morphology, 2-deoxyglucose transport, or efficiency of colony formation in soft agar.
We have previously isolated, from agar suspension culture, clones of chicken embryo fibroblasts transformed by B77 and Prague strains of Rous sarcoma virus (RSV) that varied in the expression of plasminogen activator activity [Wolf, B. A. & Goldberg, A. (1976)
Proc. Natl. Acad. Sci. USA
73, 3613-3617]. All of the clones exhibited an altered cellular morphology, an increased rate of sugar transport, and a high efficiency of colony formation in agar suspension regardless of the level of plasminogen activator. Because B77 and Prague strains of RSV replicate as well as cause sarcomas in chickens, the tumorigenicity of the transformed cells could not be evaluated with clones of these cells. In order to determine the oncogenicity of clones with various levels of plasminogen activator, it was necessary to isolate cells transformed by the replication-defective Bryan strain of RSV, which release noninfectious virus. All of the agar suspension clones of transformed cells, derived by infection of chicken embryo cells with replication-defective Bryan RSV, fell within the continuum observed for B77- and Prague-transformed clones with respect to altered morphology, increased rate of sugar transport, efficiency of colony formation in agar suspension, and variations in plasminogen activator activity. All of the clones, regardless of the level of plasminogen activator, produced tumors when as few as 5 × 10
2
cells were injected into the wing web of 1-day-old chicks. The latency period for tumor formation after injection of cells was similar regardless of the level of plasminogen activator of the injected cell. Primary explants of tumors resulting from inoculation of clones having low, intermediate, or high activator activity displayed a spectrum of activator activity.
Sakiyama et al. ('72) reported the isolation of a line of hamster cells (NIL l c l ) which contains only three glycolipids, hematoside, ceramide monohexoside and ceramide dihexoside. The incorporation of radiolabeled palmitate into hematoside during 24 hours was three fold higher in normal confluent, non growing cells than sparse, growing ones, Polyoma transformed cells did not exhibit this effect.We have continued studies with the untransformed cell line and have found that the higher incorporation of radiolabeled palmitate into hematoside by normal confluent cells is not due to a higher rate of turnover of hematoside at confluence but represents a true chemical increase. We have also found that this increase is not a gradual process during cell growth but instead occurs only when the cells become confluent and stop growing. The increase of hematoside at confluence is not due to a higher rate of synthesis of hematoside during G I ,
Three oxidative dyes were screened for mutagenic and teratogenic potential. A range of mutagenic response was observed using Salmonella typhimurium tester strains, TA 1538 and TA 98 in the presence or absence of metabolic activation. M-phenylenediamine exhibited activity with both strains while 4-chlororesorcinol showed no response in either strain. Pyrogallol exhibited a weak response that showed no linear correlation only in TA 98 in the absence of activation. In teratogenicity studies, the 3 oxidative dyes were administered by gavage to Sprague-Dawley rats on days 6 through 15 of gestation at the following dose levels: 4-chlororesorcinol at 50, 100, and 200 mg/kg; m-phenylenediamine at 45,90, and 180 mg/kg; and pyrogallol at 100,200, and 300 mg/kg. A significant reduction in mean maternal weight gain was noted at the high dose level of each dye. The high dose levels of 4-chlororesorcinol and pyrogallol were embryolethal as indicated by an increase in resorptions. The high dose level of pyrogallol was fetotoxic as evidenced by a decrease in fetal body weights. Evaluation of the number of gross, visceral, or skeletal anomalies/variations revealed no statistically significant differences between dye treated and control groups. Exposure to the positive control agents, Vitamin A and aspirin, resulted in a statistically significant increase in abnormal fetuses with a broad spectrum of gross visceral and skeletal anomalies ranging from a frequency of 18-60%. These studies indicated that the predictability of teratogenicity from bacterial mutagenicity screening is low with the chemical categories under investigation.
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