Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

Abstract

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2'deoxyuridine ( FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 x 1 0 -5 M FUdR was very low (0.0001-0.0003) whether the drug was administered during early GI, late GI, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10-7M thymidine or "conditioned" medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.The biological and biochemical activities of several inhibitors of DNA synthesis have been extensively investigated in mammalian cell cultures in order to predict the effectiveness of these compounds as tumor inhibitory agents (Heidelberger, '65; Kim and Eidinoff, '65; Kim et al., '67; Umeda and Heidelberger, '68). Recent studies have demonstrated that some of these inhibitors, such as hydroxyurea and 1-p-D-arabino-furanosylcytosine (ara-C) produce a selective damage upon cells exposed during the period of DNA synthesis (Sinclair, '65, '67, '68), and the minimum time period needed to induce loss of viability would be equal to the duration of the DNA synthetic phase (Kim et al., '68).Whether all inhibitors of DNA synthesis have this selective action upon cells exposed during S is a question of great importance because "in vivo" the cells are not synchronized. Thus, little or no effect would be induced in Gi or G, cells by short periods of exposure to selective compounds. To investigate this question in synchronized Chinese hamster cells the inhibitor 5-fluoro-2-deoxyuridine ( FUdR) was chosen because preliminary results in unsynchronized mouse ELD ascites tumor and cultures of human leukocytes suggested that FUdR affects cells exposed during Gi and G2 as well as during S (Lozzio and Valencia, '65; Lozzio, '67). The results described in the present paper clearly demonstrate that FUdR differs from hydroxyurea and ara-C in that its lethal action is not selectively limited to cells exposed during the period S. MATERIALS AND METHODSCultures of a clone of Chinese hamster cells' with a generation time of 16 hours were employed for these studies. The duration of the cell cycle in this clone was measured by the labeled rnetaphase technique in the logarithmic phase of growth of unsynchronized cultures: S was 8 hours; Gz, 2 hours; and GI plus M, 6 hours.The cells were synchronized by the method of reversal of colcemid inhibition (Stubblefield and Klevecz, '65). A mitotic index of 95-98 per cent was obtained and an almost pure culture of cells in metaphase was plated at time 0. Thes,e cells entered the GI phase of a new cell cycle soon after incubation at 37°C in fresh Eagle's minimal essential medium cont...

Dependence of fluorodeoxyuridine-induced cytotoxicity and megabase DNA fragment formation on S phase progression in HT29 cells

“…Because TS inhibition (and subsequent DNA synthesis inhibition) is such a prominent feature of fluoropyrimidine activity, it is tempting to speculate that cells should be more sensitive to these drugs during S phase. However, whereas some data support this notion [2], other data do not [11]. We have shown previously that the difference in sensitivity to FdUrd of HT29 cells and another human colorectal tumor cell line, SW620, correlates with damage on the parental DNA strand (through uracil misincorporation/misrepair), rather than with inhibition of nascent DNA synthesis [5,6].…”