Faculty Bibliography

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

Mouse teratocarcinoma cells from embryoid bodies were cultured in vitro to permit their differentiation into a number of cell types. Two enzyme activities, creatine phosphokinase (CPK) and the protease plasminogen activator, were studied to follow the developmental sequence of events in these embryoid body-derived cell cultures. CPK activity increased with time in culture, indicating the appearance of new cell types with brain- or muscle-specific enzyme activities. Plasminogen activator was detectable in extracts of embryoid bodies. This protease activity first increased and then decreased to a low level as the embryoid bodies in culture developed into differentiated cell types. These cell cultures also showed a decreased potential for tumor formation in syngeneic mice as a function of time in culture. This decrease in tumorigenic potential was correlated with the appearance of differentiated cells in vitro. Simian virus 40 (SV40) was used to infect and transform cells derived from embryoid bodies in culture. This was done to permit the establishment of cloned teratocarcinoma-derived cell lines. Twenty-nine distinct cloned permanent cell lines (called SVTER) containing the SV40-specific tumor antigen were obtained. None of these cell lines was capable of producing tumors in syngeneic mice. An analysis of the levels of creatine phosphokinase and plasminogen activator in these SVTER cell lines indicated that : (a) some cell lines had high CPK activity and little or no plasminogen activator activity, (b) some cell lines contained high levels of plasminogen activator activity with little or no CPK activity, and (c) some cell lines contained neither of these enzyme activities. No example of a cell line with high levels of both enzyme activities was observed, indicating that these two enzymes may participate in mutually exclusive developmental pathways. The SVTER cell lines may therefore be useful in reconstructing these developmental pathways in vitro.

The intramembrane organization of the plasma membranes of nonmalignant cells in culture has been compared by freeze-fracturing with that of virally-transformed malignant cells. No dramatic differences are present in the distribution of intramembrane particles in the plasma membranes of these cells when the cells are examined without fixation or with mild fixation (glutaraldehyde treatment) prior to freezing. However, a redistribution of intramembrane particles into aggregates occurs in the membranes of nontransformed cells after treatment with glycerol. The aggregation of particles is extensive in normal chick embryo fibroblasts, and less extensive in mouse 3T3 cells. The glycerol-induced particle redistribution is not inhibited at 4 degrees, but it is inhibited by pretreatment with 2.5% glutaraldehyde. A significant number of the cells remain viable after the glycerol treatment, and the process is reversible. Particle aggregation does not appear to be related to either growth rate or cell density. Transformed Rous sarcoma virus/chick embryo fibroblasts and simian virus 40/3T3 cells have few particle aggregates after glycerol treatment. The plasma membranes of chick embryo fibroblasts transformed with a mutant of Rous sarcoma virus (TS-68) that is temperature sensitive for transformation, have few particle aggregates when grown at the permissive temperature (37 degrees). Extremely prominent particle aggregates are present in the plasma membranes of cells grown at the nonpermissive temperature (41 degrees). These observations indicate that there is an alteration in the plasma membrane associated with viral transformation which is related to a glycerol-sensitive mechanism that controls the distribution of intramembrane particles

We have isolated several lines of rat embryo cells transformed by simian virus 40. All these lines are fully transformed with regard to saturation density and serum sensitivity, but they differ greatly in their anchorage dependence, as assayed by efficiency of plating in methyl cellulose suspension. This set of lines reveals a consistent relation of plasminogen activator production to plating efficiency in methyl cellulose. T-antigen-positive transformed lines that synthesize activator grow in methyl cellulose suspension, while T-antigen-positive transformed lines that do not synthesize activator fail to form colonies in suspension. Normal rat embryo cells produce very little plasminogen activator and do not grow in methyl cellulose. Sera that permit high levels of plasmin formation and activity support growth in semi-solid medium better than sera whose plasminogen is activated poorly and/or sera that contain inhibitors to plasmin.