Correlation between ribosomal RNA production and RNA-directed fluoropyrimidine cytotoxicity

Abstract

The relationship between cytotoxicity and fluoropyrimidine effects on the production of mature cytoplasmic 28S and 18S ribosomal RNA was studied in S-180 cells for the fluoropyrimidines: 5-fluorouracil (FUra), 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd). After a 6-hr drug exposure, the total cytotoxicity in the absence of added thymidine (dThd) was determined by soft-agar cloning and resulted in LC 90 (lethal concentration to 90% of cells) values of 0.6 μM FdUrd, 0.7 μM FUrd, 5.3 μM FUra and 93 μM 5'-dFUrd. The RNA-directed (dThdnonreversible) cytotoxicity was assessed by cloning the cells in the presence of 10 μM dThd. This resulted in an altered order of potency and increased LC 90 values to 5.5 μM FUrd, 20 μM FUra, 265 μM FdUrd and 870 μM 5'-dFUrd. The production of mature cytoplasmic rRNA was determined by measuring the amount of [ 3H]cytidine incorporated into the 28S and 18S rANA species following their separation by agarose gel electrophoresis, compared with the level of [ 3H]cytidine incorporated into the nuclear rRNA. When all four fluoropyrimidines were compared together, the degree of inhibition of cytoplasmic rRNA production was poorly predictive of the total cytotoxicity in the absence of dThd (correlation coefficient, r = 0.77). FdUrd, in particular, had a very minor effect on rRNA production even at very toxic drug concentrations. When toxicity was assessed in the presence of dThd, however, there was a strong and significant correlation between rRNA production and RNA-directed cytotoxicity (r = 0.95, P < 0.001), for all the fluoropyrimidines tested, including FdUrd. Thus, when the inhibition of thymidylate formation was eliminated as a site of drug action and only RNA-directed cytotoxicity was assessed, the impaired production of cytoplasmic rRNA was strongly associated with cytotoxicity. These results demonstrate that the inhibition of mature cytoplasmic rRNA production may be an important common mechanism of RNA-directed cytotoxicity for all the fluoropyrimidines, and not limited to FUrd or FUra.