Faculty Bibliography

Fibronectin isolated from cultures of chicken embryo fibroblasts (CEF) contains phosphorus linked to serine and threonine by monoester bonds. Normal and Rous sarcoma virus (RSV)-transformed cells were incubated with [32P]orthophosphate, and fibronectin was isolated from the cell surfaces and conditioned media. 32P was stably associated with fibronectin during immunoprecipitation, SDS-polyacrylamide gel electrophoresis, phospholipid solvent extraction, and hot acid but not alkaline treatment. After a limited acid hydrolysis of fibronectin, both phosphoserine and phosphothreonine were found. The specific radioactivity of the 32P-labeled fibronectin from the conditioned medium of normal CEF was higher than that from the cultures of transformed CEF

We have examined the effect of the tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), on the actin-containing elements of the cytoskeleton of chick embryo fibroblasts (CEF). TPA at concentrations as low as 7.3 times 10-10M indices a reversible change in the cytoskeleton as visualized by indirect immunofluorescence using anti-actin antibodies. Cells incubated with TPA lose the ordered actin-containing structures found in normal cells and resemble Rous sarcoma virus-transformed cells in that the immunofluorescent actin pattern is diffuse. The TPA effects are both dose-and time-dependent. Analogs of TPA which are inactive as tumor promoters do not induce cytoskeletal changes at the concentrations tested, while a second tumor promoter, PDD, is also able to cause alterations in actin-containing structures. The action of TPA requires de novo synthesis of both RNA and protein. The direct cytoskeletal changes are neither plasmin-dependent nor subject to inhibition by incubating the cells with high levels of protease inhibitors during the exposure to TPA. However, plasminogen does increase the sensitivity of cells to TPA

Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41 degrees C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41 degrees C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(0); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 degrees C resulted in altered kinetics of 35SO4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography

The synthesis of plasminogen activator (PA) by cultured human embryonic lung (HuEL) cells has been examined. The production of PA by these cells was found to be reversibly inhibited by physiological levels of glucocorticoids. The suppression of PA synthesis in HuEL cells was not accompanied by an inhibition of cell growth. Moreover, the glucocorticoid induced deinduction of plasminogen activator synthesis occurred in both growing and non-growing cells. The inhibition of PA production by corticosteroids appeared to have a requirement for DNA-dependent RNA synthesis since the inhibition of DNA-dependent RNA synthesis at the time of exposure of cells to corticosteroids prevented the deinduction of PA

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.

The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells

Cultured normal low-passage embryo fibroblasts, from a number of species, and two untransformed clones of a Balb/3T3 line elaborate increasing amounts of plasminogen activator (PA) as they approach confluence; the low-passage cells then lose this PA activity after reaching confluence, while the 3T3 cells retain it indefinitely. Even at their peaks, however, the PA activities of the low-passage cells remain well below those of the corresponding virally or spontaneously transformed cells. The PA increases in normal cells are probably a result of PA production rather than of adsorption of secreted PA to the cell surface, or of changes in cell-associated protease inhibitors. The elaboration of PA by normal cells is dependent upon their metabolic activity, such that the level of serum supplementation and the growth phase of the culture directly influence the level of cell-associated PA observed. In addition, there may be a component of serum which exerts a negative control on PA production and which is not an acid-labile protease inhibitor