Cell mutants as a tool to study malignant transformation of fibroblasts.

From 3T3 Balb/c cells, mutants with low adhesiveness to plastic substratum were selected. One of these clones, AD6, was found to be deficient in glucosamine-6-phosphate N-acetylase. A result of this block is a decrease by 60-70% of cell surface carbohydrates, and as a consequence there is a general reduction in the exposure of glycoproteins at the cell surface. This biochemical defect is fully reverted to normal, simply by growing the mutant cells in presence of 10 mM N-acetylglucosamine. This specific and reversible enzymatic block allows us to conclude that the abnormal properties of AD6 cells--low adhesion, round shape, increased agglutinability by lectins, loss of directional locomotion, and absence of microfilament bundles--are the result of the surface carbohydrate defect, since reversion of glycoprotein synthesis to normal results in the general reversion of the altered phenotype. However, in spite of this apparent transformed phenotype, AD6 cells have normal growth control and are not tumorigenic. Using [3H]-2-deoxyglucose suicide, we selected from a spontaneously transformed clone of Chinese hamster lung fibroblasts a mutant (DS7) impaired in glucose metabolism, DS7 has a fourfold to fivefold decreased ability to transport either 2-deoxyglucose or 3-O-methylglucose and produces 14 times less lactic acid than the wild-type when grown on 5 mM glucose. This block in aerobic glycolysis, which is located at the level of phosphoglucose isomerase, makes that cell line dependent exclusively on respiration for its energy requirement. The parenteral line grows at low serum concentration, is anchorage-independent, and is tumorigenic in nude mice. The derived DS7 cells have retained both the in vitro transformed phenotype and the tumor-forming capability. A general conclusion of these studies is that the altered properties and cell behavior of transformed fibroblasts, such as low adhesiveness, round morphology, increased agglutinability by lectins, altered motility, absence of microfilament bundles, and increase in hexose transport and in aerobic glycolysis, are dissociable from malignant transformation. These phenotypic alterations should therefore be considered as events secondary to the mechanism that leads to the loss of growth control.