Proliferation is necessary for both repair and mutation in transgenic mouse cells

Abstract

Proliferating cells are often presumed to be more mutable than quiescent cells because they have less time to repair DNA damage before DNA replication. Direct tests of this hypothesis have been confounded by the need for cell division before a mutation can be detected. We have avoided this problem by showing that the Big Blue mouse cell line permits the dynamic quantification of both lesions and mutations in the complete absence of cell division. These cells carry the bacterial lacI gene in a lambda shuttle vector. Mutant plaques recovered by in vitro packaging of the mouse DNA can arise from mutations sustained either in mouse cells or in the bacteria. The proportion of mutant phage contained within a mutant plaque can distinguish these two types of mutation. Mutations formed in mouse cells yield >90% mutant phage because both DNA strands are mutant. On the other hand, mutations formed in the bacteria from adducted DNA yield </=50% mutant phage, because one of the DNA strands is wild type. Immediately after exposure to a test mutagen, ethylnitrosourea, all induced mutations were formed in the bacteria, but after approximately one cell division, the reverse was true and all mutations arose in the mouse cells. Only one-fifth as many mutations were recovered from quiescent cells and all arose in the bacteria, showing that the mouse cells made no mutations in the absence of proliferation. Surprisingly, the mouse cells did not repair any of the premutagenic damage during 4 days of quiescence. When these quiescent cells were induced to proliferate, however, both repair and mutation fixation ensued.

Figure 1

The generation of mosaic and homogenous mutant plaques. In the first example (Upper), a lesion created within the cell line forms a mutation in the bacterium during phage replication. A lesion fixed into a mutation during the first round of phage replication produces a mosaic plaque containing a 1:1 ratio of mutant to wild-type phage, as indicated after replating the plaque. In the second example (Lower), a mutation sustained within the cell line leads to the formation of a homogenous mutant plaque, which is also made evident on replating.

Figure 2

The effect of serum-free medium on cell division. Control cultures (■) are growing in medium supplemented with 10% (vol/vol) FBS, whereas cells deprived of serum (●) remain quiescent. Cells were counted with a hemocytometer after being removed with trypsin (±SD).

Figure 3

The effect of serum deprivation on DNA synthesis. DNA synthesis was determined after incorporation of [methyl-³H]thymidine into DNA (±SD) during a 1-h incubation (1 μCi/ml). DNA synthesis is expressed relative to that of the exponentially growing control cultures at zero time.

Figure 4

Relative survival of Big Blue mouse cells after serum deprivation as measured by absolute cloning ability (±SE).

Figure 5

The kinetics of repair and mutation in proliferating mouse cells. The frequency (±SD) of bacterial (●) and cellular (■) lacI mutations in proliferating Big Blue mouse cells was measured as a function of time after mutagen (ENU) treatment.

Figure 6

Adduct stability in quiescent cultures. The frequency (±SD) of bacterial (●) and cellular (■) lacI mutations in quiescent Big Blue mouse cells vs. time. Quiescent cultures (12 days after serum removal) were treated with ENU (200 μg/ml) in serum-free medium for 30 min at standard cell culture conditions.

Figure 7

Mutation and repair kinetics in arrested cell cultures induced to proliferate. The frequency (±SD) of bacterial (●) and cellular (■) lacI mutations in quiescent cultures (12 days after serum removal) treated with ENU (400 μg/ml) in serum-free medium for 30 min at standard cell culture conditions. Quiescent cells (red) were induced to proliferate 4 days posttreatment (green). Mutation frequencies less than zero arise when an observed value in the treated group is less than that in the controls.