Excision of 4-Nitroquinoline 1-Oxide Damage and Transformation in Mouse Cells

For elucidation of the role of repair mechanisms for carci nogenesis by chemical agents, cell transformation by 4nitroquinoline 1-oxide (4NQO) and excision repair of 4NQO lesions were studied with mouse A31-714, a subclone de nived from BALB/3T3, with posttneatment incubation in the presence on absence of hydnoxyurea (a known inhibitor of normal semiconservative DNA replication). When hydmoxyu neawas presentinthe medium for12 and 24 hr of incuba tion after 4NQO treatment, the transformation frequency was reduced to 28 and 6%, respectively, of the frequency observed for cells incubated in a growing state without hydmoxyumea. The observed effect of hydnoxyunea resembles the effect of the posttmeatment placement of cells in a den sity-dependent nongrowing state, which also prevented the production of transformed foci by 4NQO. Excision repair of 4NQO damage was measured by autoradiognaphy of un scheduled [3H]thymidine incorporation on by chromato graphic analysis of four kinds of 4NQO-pumine adduct (the major DNA lesions produced by 4NQO) remaining in DNA after posttreatment incubation of cells. The excision repair wasvery rapid in 4NQO-treatedcells kept in the nongmowing state, and it was not affected by the presence of hydmoxyu rea in the incubation medium. Furthermore, the fractions of repaired lesions estimated from the excision and repair synthesis experiments wemeabout 50% and 65 to 90% for the cells incubated for 12 and 24 hr, respectively, paralleling the decrease in the transformation frequency (assuming that the decrease was due to repair of 4NQO lesions). From a combination of these results with others, we sug gest that the four kinds of 4NQO-punine adduct are excisa ble in this mouse cell line by an excision repair system similar to, but possibly not identical with, that which works on UV-induced pymimidine dimers in human cells and that unexcised fractions of these adducts are the major cause of cell transformation by 4NQO. The unique characteristics of the 4NQO-pumine adducts are discussed in relation to exci sion repair, which may have a vital molein chemical carcino genesis. In a previous report Kakunaga (11) demonstrated that A31-714, a subcbone isolated from clone A31 of BALB/3T3, is malignantly transformed by a carcinogen, 4NQO,3 and that the ratio of induced transfonmants to surviving cells increases almost linearly with the concentration of carcino gen oven the dose range studied (0.05 to 1.5 j.@M). The transformed cells show many characteristics considered as indices of malignant cells and produce tumors when in jected into isobogous mice. Kakunaga (12, 13) also has shown that the ability of cells to be transformed is rapidly lost when they are kept, soon after 4NQO or methylcholan threne treatment, in a nongrowing state attained by high cell density. Similar loss of the ability to be transformed has been reported with the morphological transformation by X irradiation of Syrian hamster cells (2) and with the malig nant transformation by X-imnadiationof mouse cells (27). These observations suggest that the loss of the tmansfomma tion may be due to repair of pretnansformational damage by a repair system in these cells. Tada and Tada (24, 25) first showed that tmeatment of mammalian cells with 4NQO produced 4 kinds of 4NQO punine adduct resulting from covalent bonding of enzymati calby activated 4NQO to guanine and adenine moieties. These 4NQO-punine adducts are the major cause of killing and mutation by 4NQO in Escherichia coli (7) and also of killing in mouse A31-714 cells (8). Furthermore, during in cubation after 4NQO treatment, these 4NQO-punine add ucts are removed from the DNA of E. co/i, as well as from that of human cells that have normal excision repair capacity for UV-induced pynimidine dimens, but the adducts are not removed from the DNA of either E. co/i uvrA on XP cells (7, 9), both of which are deficient in excision repair ability for UV-produced pymimidine dimens. From these results one would expect that the 4NQO-punine adducts are the primary DNA lesions that beadto cell transformation by the cancino gen 4NQO and that the adducts can be excised efficiently under conditions that permit a decrease in the transforms tion frequency, as mentioned above. Evidence has been accumulating to indicate that muta tion, cancer, and cell transformation may anise from errors