Molecular radiobiology of human cell lines: II. Effects of thymidine replacement by halogenated analogues on cell inactivation by decay of incorporated radiophosphorus

Abstract

Techniques were developed to study the inactivation of human cells by radioactive decay of DNA-incorporated 32P. When compared with bacteria or phages, human cells were found to be highly resistant to this type of inactivation, with cell death ensuing after approximately 4000 32P to 32S transmutation events within the DNA complement of the cell (α = 2·4 × 10−4). Incorporation of thymidine analogues, 5-chloro-, 5-bromo-, and 5-iododeoxyuridine (CUdR, BUdR, and IUdR), into the cell DNA increases the sensitivity to 32P-decay inactivation by a factor of 2·1 (CUdR and BUdR) to 3·3 (IUdR) under the experimental conditions employed (60% thymidine replaced by the analogues). Studies on the mechanism of this phenomenon revealed that enhancement of the cell's sensitivity to the lethal effects of 32P decay requires that the halogen atoms be incorporated into the strand opposite to that bearing the 32P atoms. In other words, uninlar halogen labeling of the same DNA strand as that bearing the 32P atoms, while the other strand remains unlabeled (achieved by placing the cells in 32P- and BUdR-containing medium for only one round of DNA replication), does not alter the rate of cell inactivation observed with unifilar 32P labeling alone. These data indicate that scissions through both strands of the DNA molecule constitute the lethal events, and that the halogen, by its electrostatic interaction with the proximate phosphorus atom (Szybalski, 1961), labilizes the phosphate-ester bond, and thus enhances the probability of the double chain scission event. The low killing efficiency of DNA-incorporated 32P for mammalian (human) cells indicates either that the double scission occurs only rarely in the vulnerable regions of the DNA complement, i.e. those regions indispensable for cell growth under tissue culture conditions, or that some very effective mechanism exists for repair of a constant fraction of the molecular lesions or for annulment of their lethal consequences by other means.