No abstract

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.

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.