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.

The percent lipid composition of pooled human sebum analyzed by thin-layer chromatography was: ceramides (13%), fatty acid (47%), cholesterol (7%), cholesterol esters (2%), squalene (11%), triglycerides (3%), and wax esters (17%). Total sebum lipids (2– 4 mg/ml), sonicated into bacterial culture medium, caused 4- to 5-fold log reduction in growth of gram-positive bacteria, Staphylococcus aureus, Streptococcus salivarius and the anaerobe Fusobacterium nucleatum, but was ineffective against most gram-negative bacteria. Fractionation of the sebum lipids showed that both saturated and unsaturated fatty acids contained the bulk of the antimicrobial activity. Lauric acid (C12:0) was the most active saturated fatty acid. The unsaturated fatty acid, palmitoleic acid (C16:1Δ6, cPA) was both the most predominant monoene and the most active antimicrobial fatty acid. Purified cPA (>99%) yielded typical minimal inhibitory concentration (MIC) values of 10–20 µg/ml against gram-positive bacteria. Organically synthesized cPA isomer gave MIC values comparable to the natural material. Both natural and synthetic cPA were found to be the most active sebum lipid fraction in blocking the adherence of a pathogenic strain of Candida albicans to porcine stratum corneum. Ethanol in combination with cPA exerts a synergistic bactericidal activity against gram-negative pathogenic bacteria, including Pseudomonas aeruginosa, Propionibacterium acnes, Escherichia coli, and several methacillin-resistant strains of S. aureus. Palmitoleic acid may be useful in topical formulations for treatment of secondary gram-positive bacterial infections, as a gram-positive bacteria antimicrobial in wound dressings, and as a natural gram-positive antimicrobial preservative in skin and hair care products.

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...

Experimental evidence is presented that supports a cell cycle model showing that there are five distinct biological processes involved in proadipocyte differentiation. These include: (a) growth arrest at a distinct state in the G1 phase of the cell cycle; (b) nonterminal differentiation; (c) terminal differentiation; (d) loss of the differentiated phenotype; and (e) reinitiation of cell proliferation. Each of these events is shown to be regulated by specific human plasma components or other physiological factors. At two states designated GD and GD', coupling of growth arrest and differentiation is shown to occur. We propose that these mechanisms for the coupling of growth arrest and differentiation are physiologically significant and mimic the regulatory processes that control stem cell proliferation in vivo.

Carcinogenesis in humans is a multistage process, and the two major stages have been designated initiation and promotion. Although the biochemical basis for initiation and promotion remains to be established, recent research has provided important insights into potentially significant biologic mechanisms. These data are reviewed, and a new concept of carcinogenesis is presented. This concept suggests that the initiation of carcinogenesis may result from cellular immortalization and the development of defects in the integrated control of stem cell proliferation and differentiation and that the promotion of carcinogenesis may result when such initiated stem cells develop aberrant autoregulatory growth-control properties.