Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2'-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 micrometer, were gradually increased to 100 micrometer. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2'-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/O). Relative to thymidine uptake by HeLa/O cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzylthioinosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLA/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.