Quantitative Investigation on the Effect of Puromycin on the Aggregation of Trypsin- and Versene-Dissociated Chick Fibroblast Cells

Abstract

Embryonic chick fibroblast cells were used to test the effects of puromycin on cell adhesion and aggregation. Single cell suspensions were prepared by dissociating the muscle tissues of 5- and 9-day-old chick embryos with either trypsin or versene according to standard cell dissociation procedures. Cell aggregation was quantitatively estimated by a turbidimetric method. Quantitative analyses of the components in cell-free supernatants revealed that both trypsin and versene when used as cell dissociating agents removed substantial amounts of protein from the surfaces of the cells and the intercellular regions. Trypsin removed larger quantities of protein and of N-acetylneuraminic acid than did versene. Nucleic acids were also released during trypsin and versene dissociation, and estimations of these nucleic acids gave a measure of the extent of cell lysis. The proteins and peptides were separated by gel filtration. The solubilized proteins removed by versene treatment were separated into three peaks on Sephadex G. 200. The tryptic digest gave one main peptide peak on Sephadex G.50. Both trypsin- and versene-dissociated cells, on rotation in Hanks's balanced salts solution with or without serum, began to adhere to one another at the start of the test period, indicating that the cell dissociation procedures had not impaired the adhesive properties of the separated cells. When puromycin at a final concentration of 10 µg/ml was introduced at zero time into a cell suspension with or without serum, it did not delay the adhesion of cells to one another, and did not prevent the formation in the first hour of initial aggregates; it succeeded only in preventing progress in aggregation beyond the initial stages. Adding puromycin at the 2nd or 3rd hour of the 4-h test period, when aggregation had already taken place, produced results of a similar pattern, progress in aggregation being brought to a halt an hour later. Puromycin at the above concentration effectively blocked protein synthesis by more than 90% as measured by the incorporation of L-[α-14C]leucine into the proteins of the rotated cells. These results implied that aggregation was not dependent on the synthesis of ‘cell aggregating’ material to replenish the loss of such material from the surfaces of the cells owing to the trypsin treatment. It is significant that 2,4-dinitrophenol, which is known to suppress cellular metabolism by inhibiting the production of ATP, also brought cell aggregation to a halt only after a lag period during which initial aggregates were formed. It is suggested, as evidenced in the results of our investigations, that puromycin may well be exerting its inhibitory effect by ultimately depressing cellular metabolism below the level at which certain basic adhesive mechanisms at the cell surface can operate.