Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice

Transgenic mice harboring the lacZ gene within a plasmid that can be recovered and amplified in Escherichia coli, to establish mutant frequencies and spectra, have provided crucial insights into the relationships between mutations, cancer and aging in vivo. Here, we use embryonic fibroblasts from transgenic lacZ-plasmid reporter mice to determine the relationship between cell proliferation in culture and mutations induced by ultraviolet (UV) light. A single dose of 2.5 J/m² of UVC to actively proliferating cells caused an approximately eight-fold increase in mutant frequency 24 h after irradiation. Identically treated quiescent cells showed a two-fold increase in mutant frequency. Thus, whereas proliferation facilitated the acquisition of mutations, it was not an absolute requirement. Characterization of the UV-induced mutations indicated that the lower mutant frequency in quiescent cells was due mainly to a reduction in point mutations; size-change mutations, indicative of translocations or deletions, were relatively unaffected by the growth state of the cells. To investigate long-term genomic stability after UVC-induced damage, we monitored the lacZ locus in irradiated cells passaged for many generations in culture. The results indicated the emergence of jackpot mutations of rapidly changing frequency, most likely reflecting the successive emergence and decline of dominant cell clones during long-term culture. These findings show that the lacZ-plasmid locus is a valid reporter for studying induced mutations in short-term cultures of both quiescent and proliferating fibroblasts. In long-term cultures, the locus is less suitable for studying induced mutations owing to the instability of the cell population.