Animal cells reversibly permeable to small molecules

Abstract

A cell preparation, useful for studying the regulation of metabolism, was developed by making monolayer baby hamster kidney cells permeable. Hypertonically treated cells were permeable to nucleotides, but retained their gross cellular morphology, intact organelles, 100% of their DNA, and 91% of their total protein. The permeable cell synthesized DNA, RNA, and protein rapidly when supplied with the appropriate substrates and cofactors. They either could remain permeable or were able to "reseal" when replaced in complete medium under appropriate conditions. Optimal conditions for DNA synthesis were established for permeable cells, giving rates equal to those of intact cells. Replication rather than repair was shown by the cell-cycle dependence of DNA synthesis and its discontinuous nature. Ribonucleotide reductase was active in permeable cells, permitting equal rates of DNA synthesis when ribonucleotide diphosphates or deoxyribonucleotide triphosphates were provided. Hydroxyurea did not inhibit DNA synthesis in permeable cells supplied with deoxyribonucleotide di- or triphosphates, but drastically inhibited DNA synthesis when ribonucleotide diphosphates were supplied. Hydroxyurea is therefore primarily an inhibitor of ribonucleotide reductase. Permeability was reversed, exposing permeable cells to [(3)H]thymidine triphosphate, which was incorporated, which labeled nuclei of cells that went on to mitosis. The reversible permeability procedure should prove especially useful in studying the functions of poorly penetrating compounds, such as drugs. Intact cells were unaffected by cytosine arabinoside triphosphate, while cells that had been made permeable and resealed were killed.