Control of the Initiation of DNA Synthesis in 3T3 Cells: Low-Molecular-Weight Nutrients

Abstract

Sparse 3T3 cells, in excess serum, become quiescent in the early G1 (or Go) phase of the cell cycle if the cells are cultured in low concentrations of amino acids, glucose, or phosphate ion. These quiescent cells then initiate DNA synthesis if the concentration of the limiting nutrient is increased. In normal medium, DNA synthesis in the same 3T3 cell line is controlled by serum factors. The results demonstrate the complexity of the control by external agents of DNA synthesis in these "normal," density-dependent cells.The growth of mouse 3T3 cells is normally controlled by serum factors (1-3). At least four serum factors appear to play a role in the initiation of DNA synthesis in quiescent cells (4). Three of the four serum factors can be replaced by known substances (4), the fibroblast growth factor of Gospodarowicz (5), insulin, and dexamethasone.In the present paper it is shown that the initiation of DNA synthesis in 3T3 cells can also be controlled by concentrations of a number of different low-molecular-weight nutrients in the medium. The results indicate that DNA synthesis and growth in these "normal," density-dependent cells are subject to control by a variety of different external agents. MATERIALS AND METHODSCell Cultures were maintained as described in the previous paper (4). Flow Mllicrofuorometric Analyses. The staining procedure was that of Tobey et al. (6). Trypsin-treated and washed cells were fixed in the cold with a 7.4% formaldehyde solution in saline for 2 hr to 6 days, and then the cells were stained with acriflavine (Aldrich Chemical). The stained cells were analyzed in a Los Alamos design microfluorometer (7) with an argon laser at 488 nm. The photograph obtained from the storage oscilloscope was analyzed graphically, as indicated in Fig. 1 Sephadex G-50-filtered calf serum. Subsequently, daily additions of 1 ml per dish were made of the same medium, containing 10% Sephadex G-50-filtered serum, but only 0.1% of the normal concentrations of amino acids and vitamins. On the third day after the fluid change, 0.5 ml of a 5-fold concentrate of all the amino acids was added to half of the dishes, to raise the concentrations of the amino acids in these dishes to approximately 30% of normal. Controls were fixed at the time of addition of the amino acids. Approximately 17 hr later, the cells to which amino acids were added were fixed and prepared for flow microfluorometric analyses (Fig. 3). Cell counts were made daily throughout the exxperiment (Fig. 4).Arrest and Reinitiation of Growth by Lowering and Raising the Concentration of Phosphate. Growing stock cells were trypsin-treated and plated at 2 X 105 cells per 9-cm dish in medium plus 10% calf serum. One day later the cells were rinsed and fluid changed into medium that contained 1% of the normal Dulbecco-Vogt concentration of sodium phoslhate, plus 10% Sephadex G-50-filtered calf serum. On the third day after the fluid change, 0.1 ml of a 0.1 M sodium phosphate solution (pH 7) was added (to give approximately 1 mM phosphate), or sodium ...

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Selective killing of transformed baby hamster kidney (BHK) cells.

“…Complete inhibition of progress towards S is shown by the curve for cells left with low serum medium during the 9-hr interval; that is, the onset of DNA synthesis is delayed by [9][10] hr after the addition of serum. The drugs caffeine, PAN, and SVA all delayed progress towards S to an extent similar to that of low serum.…”