The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (greater than 30 population doublings) and neonatal prokeratinocytes (greater than 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.

Monolayer cell cultures of macrophages, monocytes, myoblasts, and density-inhibited and transformed fibroblasts form and release cell surface membrane vesicles following exposure to formaldehyde, related low-molecular-weight aldehydes, and disulfide blocking agents. Vesicles have a unique composition of proteins and lipids. They show enrichment of cholesterol and sphingomyelin content and a seven-to tenfold enrichment of 5'-nucleotidase activity. Vesicles also contain intramembranous particles and show a trilamellar unit membrane and no ultrastructural evidence of contamination with other cytoplasmic organelles. The technique is proposed as a novel method for isolating plasma membrane vesicles from cells in culture.

The major glycoprotein of the human erythrocyte membrane has been isolated by treatment with lithium di-iodosalicylate and found to be a single polypeptide chain with a molecular weight of about 50,000. This molecule, which is 60% carbohydrate and 40% protein, carries multiple blood-group antigens, the receptors for influenza viruses, and various plant agglutinins. Four unique carbohydrate-containing peptides (a-i, a-2, a-3, and jB) are produced by tryptic digestion of the isolated glycoprotein; their order in the molecule has been determined by sequential tryptic digestion of intact erythrocyte membranes and partially digested glycoprotein fragments. Cleavage of the native protein with cyanogen bromide produces five fragments; two of these (C-5 and C-i) contain most of the carbohydrate in the molecule and are derived from the N-terminal half of the polypeptide chain. The nonpolar amino acids of this glycoprotein are located predominantly in the C-terminal fragment (C-2).Phytohemagglutinin conjugated to ferritin has been used to map the distribution of glycoprotein receptors over the surfaces of intact erythrocytes by freezeetching and electron microscopy. This label localizes to sites on the membrane that overlie the intramembranous particles. These findings suggest that the glycoprotein is oriented at the cell surface with its oligosaccharide-rich N-terminal end exposed to the exterior, while its C-terminal segment interacts with other components in the interior of the membrane to form intramembranous particles.Glycoproteins comprise about 10% of the total protein of the human erythrocyte membrane (1). Their carbohydrate moieties are antigenic determinants (2, 3) and receptors for viruses and plant agglutinins (4), and their sialic acid residues are responsible for most of the negative charge at the cell surface (5).Glycoproteins have been isolated from erythrocyte membranes with various solvents, such as phenol (6), butanol (7), pyridine (8), sodium dodecyl sulfate (SDS) (9), or formic acid (10 This report summarizes studies on the properties of the major glycoprotein of the human erythrocyte membrane extracted from membranes by a new procedure with lithium di-iodosalicylate as a dissociating agent. A water-soluble glycoprotein has been isolated in high yield that has the chemical properties and biological activities characteristic of the native membrane-bound molecules. This glycoprotein has been partially characterized after tryptic digestion and cyanogen bromide cleavage, and its location in the membrane has been determined by electron microscopy. MATERIALS AND METHODSHuman blood was obtained fresh from donors, and the erythrocytes were washed three times in phosphate-buffered saline (pH 7.4) before preparation of ghost membranes by the procedure of Dodge et al. (11). Glycoprotein was extracted from freeze-dried ghost membranes with 0.3 M lithium diiodosalicylate and purified by phosphocellulose chromatography (12). Samples were electrophoresed in acrylamide gels containing Tris-glycine (pH 8.4), a...

provided helpful comments on an earlier draft. We also appreciate the support of the Kauffman Foundation.

The differentiation of most mammalian cells is preceded by growth arrest in the G1 phase of the cell cycle, but the characteristics of this state have not been established. We now report that the growth arrest that precedes the differentiation of BALB/c 3T3 T mouse proadipocytes must occur at a distinct state in G1 designated GD. GD-arrested cells are characterized by their ability to differentiate in the absence of DNA synthesis and by their unique sensitivity to the mitogenic effect of isobutylmethylxanthine. Proadipocytes induced to become G1 growth arrested at other states by culture in medium deficient in growth factor or nutrients, by contrast, are unable to differentiate in the absence of DNA synthesis and are not stimulated to proliferate by isobutylmethylxanthine even when they are exposed to differentiationpromoting medium prior to arrest. These data support the conclusion that, prior to the expression of a differentiated phenotype, proadipocytes must arrest their growth at a distinct state in the G1 phase of the cell cycle, GD. These data also provide the basis for the hypothesis that carcinogenesis is associated with defects in the coupling of growth arrest and differentiation at the GD state.Regulation of cell proliferation of many cell types is mediated by the, coupling of growth arrest and differentiation. In vivo studies on hematopoietic cells (1) and epithelial cells of the skin (2) and in vitro studies on other cell types (3) have established this fact. Although there is considerable controversy concerning the mechanisms that serve to initiate the differentiation process (4), the available evidence suggest growth arrest in the G1 phase of the cell cycle precedes expression of the differentiated phenotype. It has, however, not been established whether growth arrest occurs at a distinct state in G1 prior to differentiation. This question is of critical importance if the metabolic events that control the coupling of growth arrest and differentiation are to be established and if defects in the coupling process that may be associated with various disease states, such as aging and carcinogenesis, are to be identified. In this paper we report on the first of our studies, which show that, prior to differentiation, proadipocytes arrest their growth at a state in the G1 phase of the cell cycle (GD) that is distinct from the G1 growth arrest states induced by deprivation of growth factor or serum (Gs) or by deprivation of nutrients (GN).MATERIALS AND METHODS Cell Culture, Cell Proliferation, and Cell Differentiation. The proadipocyte cell line, designated 3T3 T, derived from BALB/c 3T3 (A31) mouse embryo cells by L. Diamond (5), was grown at 37°C in a 5% CO2 humidified atmosphere in Dulbecco's modified Eagle's medium (DME medium) containing 10% heat inactivated fetal calf serum unless otherwise stated.All stock cultures were grown in antibiotic-free medium; experimental specimens, however, were cultured in medium supplemented with penicillin (100 units/ml) and streptomycin (100 ,ug/ml). Cells wer...