MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

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We use the gpt transgenic Chinese hamster ovary (CHO) cell line, AS52, for molecular studies of mutation. Mutant gpt sequences are amplified using the polymerase chain reaction (PCR) followed by DNA sequence analysis to generate a sequence spectrum of induced mutations. We have studied mutations induced by two general classes of compounds, i) direct acting mutagenic chemotherapeutic agents, Adozelesin (ADZ) and 5-azacytidine (5AC), and ii) known or suspect human carcinogens that require metabolic activation, such as the tobacco specific nitrosamine, NNK. ADZ is a potent antitumor agent and an efficient DNA alkylating agent. ADZ forms bulky DNA adducts predominantly at the N3 position of adenine and shows a sequence specificity of TTA* or AAA*, where (A*) is the modified adenine in the 3' position of either 3-base target. While ADZ induces mostly deletion mutations in AS52 cells, point mutations are induced as well. At least one mutational hotspot which yields AT>TA transversions as well as several insertions, small deletions and rearrangements are observed that appear to be targeted to a sequence containing 3-overlapping consensus ADZ binding sites. 5AC is a potent inducer GC>CG transversion mutations in AS52 cells. This rarely observed type of mutation implicates 5AC:C mispairs during replication. Since C:C mispairs are not extended by prokaryotic or eukaryotic polymerases, these data suggest that structural perturbations of the 5AC:C mispair may allow polymerase extension in vivo. In addition, 5AC appears to induce a class of 6TG resistant colonies that do not carry gpt structural gene mutations. Many of these clones revert to yield a functional gpt gene at frequencies much higher than expected (l0e-4 to l0e-2). In general, such "mutants" do not express gpt mRNA when the cells are grown in 6TG medium and data are accumulating to suggest that these mutants arise as a result of chromatin structural modifications. NNK induces predominantly GC>AT transitions in AS52 cells carrying a functional human CYP2A6 gene. These data suggest that CYP2A6 hydroxylation of NNK mostly generates an intermediate that results in DNA methylation leading to the observed GC>AT transitions.