Plasma membrane alteration associated with malignant transformation in culture.

Abstract

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 40, 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°). Extremely prominent particle aggregates are present in the plasma membranes ofcells grown at the nonpermissive temperature (410). 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. Malignant transformation of cells in culture has been associated with several changes, such as increased protease production (1), biochemical changes in cell surface components (2-6), increased lectin agglutinability (6, 7), and cell shape changes (8, 9). All of these changes may be directly or indirectly related to modification of the cell surface membrane.Recently, Scott et al. (10,11) reported that differences exist in the internal structure of the membranes of normal and transformed cells. With the freeze-fracture technique, these workers found that at high cell densities the intramembrane particles were aggregated in the membranes of normal cells, while at similar densities, the intramembrane particles were dispersed in transformed cell plasma membranes. Several questions were raised from the data in this earlier study. (i) Were intramembrane particle aggregates a characteristic feature of nontransformed cell membranes, or were they induced by the glycerol treatment that was used for cryoprotection during the preparation of the cells? In other studies on intact cells, it has been demonstrated that glycerAbbreviations: CEF, chick embryo fibroblasts; RSV, Rous sarcoma virus; TS-68, temperature-sensitive Rous sarcoma virus; 3T3, 3T3 mouse cell line; SV40, simian virus 40. ol, as well as dimethyl s...

Cryofixation of monolayer cell cultures for freeze‐fracturing without chemical pre‐treatments

“…In all cases, aldehyde (mainly glutaraldehyde) prefixation and antifreeze (mainly glycerol) impregnation was used. This holds true for cultures of myoblasts (Hourani et al, 1974;Chiquet et al, 1975;Kalderon et al, 1977;Cohen & Pumplin, 1979), fibroblasts (Collins et al, 1975;Furcht & Scott, 1975;Gilula et al, 1975), endothelial cells (Pauli et al, 1977;Ryan et al, 1978), carcinoma cells (Pauli et al, 1977), nerve and glial cells (Pfenninger & Rinderer, 1975;Pfenninger, 1976;Prescott & Brightman, 1978), macrophages (Porvaznik & MacVittie, 1979) and mixed cell cultures (Epstein L ? .…”

“…Different ways were chosen to handle thin cell monolayers for freeze-fracturing; invariably cells were processed by fracturing rather than by cutting. For freezing, cells were in some cases peeled off the substrate (Hourani et al, 1974;Gilula et al, 1975;Furcht & Scott, 1975;Wanson et al, 1977;Porvaznik & MacVittie, 1979) after fixation and cryoprotection; occasionally detachment was facilitated by collodion coating (Prescott & Brightman, 1978). Considering the clear disadvantage of disturbing the original structural arrangement, several recent communications aimed at specifically overcoming this problem.…”

Advances in ultrarapid freezing for the preservation of cellular ultrastructure

“…Such "diaphragms" have been observed both at regions of contact between membranes that do fuse under physiological conditions, and between membranes that do not normally fuse (Chandler and Heuser, 1979). Glycerol can also give rise to Hexagonal Type I1 lipidic particles at sites of membrane fusion (Bearer et al, 1982) and to clustering of intramembrane particles (Gilula et al, 1975;Niedermeyer et al, 1977). All of the penetrating cryoprotectants alter membrane permeability, and so are unsuitable for studies designed to determine the distribution of soluble ions and molecules using microanalytical and cytochemical techniques (see Skaer, 1981).…”

Transmission and Scanning Electron Microscopy and Freeze‐Fracture Replication of Normal Human Melanocytes and Human Melanoma Cells in Tissue Culture

“…The concentration is thought to be related in a general way to metabolic or functional activity, and their density and size can alter according to a change of state of cells as shown for pre-synaptic membranes (Israel et al, 1981) or nerve fibres of diabetic mice and rats (Fukuma et al, 1978;Sharma et al, 1983). An alteration of IMP distribution was also found associated with viral transformation of previously non-malignant cells (Gilula et al, 1975). In this context it is of interest that no significant difference was found in density and size of IMPS between the cultured normal melanocytes and the melanoma cells.…”