Mutational fingerprint induced by the antineoplastic drug chloroethyl-cyclohexyl-nitrosourea in mammalian cells

Using the pZ189 shuttle vector approach, we determined two chloroethylcyclohexyl-nitrosourea (CCNU)-induced mutation spectra (3 and 6 mM) in African green monkey kidney cells (CV1). One hundred and twenty-one independent clones (101 CCNU induced, 45 at 3 mM and 56 at 6 mM; 20 spontaneous) showing functional inactivation of the supF gene were analyzed. One hundred and five plasmids (91 CCNU induced, 41 at 3 mM and 50 at 6 mM; 14 spontaneous), showing no large deletion/rearrangements, were sequenced. Ninety mutants (81 CCNU induced and 9 spontaneous) showed at least one mutation in the supF region. The analysis of the 122 CCNU-induced mutations (56 and 66 at 3 and 6 mM, respectively) revealed that: (a) the majority of the mutations were GC-targeted base pair substitutions; (b) AT-targeted mutations were significantly more frequent in the CCNU-induced (6 mM) than in the spontaneous mutational spectrum (P <0.0006, Fisher's exact test); (c) mutational spectra obtained at 3 and 6 mM CCNU were significantly different (P <0.008); (d) induced mutations were nonrandomly located in both spectra and generated either a common hot spot (position 123, 5′-GGG-3′) or hot spots exclusive for each CCNU concentration (3 mM: position 159,5′-AGG-3′; 6 mM: position 109,5′-GGG-3′); (e) the occurrence of GC→ AT transitions was significantly different as a function of CCNU concentration (P <0.02, Fisher's exact test), the mutated G being almost exclusively preceded by a purine (5′Pu G) at 6 mM and by either Pu or Py at 3 mM; and (f) by applying Calladine's rules, we found that sequences encompassing the three CCNU hot spots shared identical helix parameters for no more than 2 bp steps 5′ (or 3 bp steps 3′) to the mutated G. Our results are consistent with the hypothesis that O⁶-alkylguanine is responsible, either directly or indirectly, for the majority of GC-targeted mutations, while O⁴-alkylthymine and/or N³-alkyladenine are probably responsible for AT-targeted mutations. The results suggest also that, in CV1 cells, the efficiency of the repair mechanism(s) involved in the removal of O⁶-alkylguanine is influenced by the DNA sequence context All of these factors determine the CCNU mutational fingerprint. CCNU has been implicated in the induction of therapy-related leukemias. The work presented here could be a valuable tool to verify this hypothesis at the molecular level by comparing the CCNU fingerprint with the p53 mutational spectrum obtained in patients formerly treated with CCNU and affected by a therapy-related leukemia.