Tolerance to Putrescine Toxicity in Chinese Hamster Ovary Cells Is Associated with Altered Uptake and Export

When Chinese hamster ovary (CHO) cells were cultured with low concentrations of putrescine (< 5 mM) their cell cycle time increased significantly and a fraction of the cells died. A cell line tolerant to the cytotoxic and growth inhibitory effects of millimolar concentrations of putrescine was developed by growing CHO cells over many months in increasing concentrations of the polyamine. A putrescine-tolerant cell line was obtained which was capable of growing in concentrations up to 25 mMputrescine and displayed growth and cell division rates similar to the original untreated/parental CHO cells. The tolerant cells grown in putrescine displayed relatively high intracellular putrescine yet the cell-associated putrescine concentration was estimated to be 10-fold less than the culture medium level. This high concentration of cellular putrescine diminished within 60 min when the cells were changed to non-putrescine-containing media. The putrescine-tolerant phenotype was further characterized in regards to the mechanisms involved in putrescine uptake, efflux, and biosynthesis. The parental and tolerant cell lines had similar or identical levels of cellular spermidine and spermine and no differences in the acetylated polyamine pools or diamine oxidase activity. The activity of ornithine decarboxylase was also similar in the two cell lines in both the presence and the absence of ornithine. The tolerant cells, however, had a decreased uptake rate for putrescine. The tolerant cell line also showed a greatly enhanced ability to export putrescine, especially when treated with ornithine, suggesting that an upregulated polyamine export system may be present in the tolerant cells which could be responsible for the increased cell survival in high putrescine concentrations. The data are discussed in regard to the potential for identifying the transport protein(s) responsible for the maintenance of nontoxic intracellular concentrations of putrescine in a tolerant cell line grown in putrescine.