K-RAS CODON-12 AND CODON-61 POINT MUTATIONS IN BROMODEOXYURIDINE-INDUCED AND N-NITROSOMETHYLUREA-INDUCED RAT RENAL MESENCHYMAL TUMORS

The mutagenic thymidine analog bromodeoxyuridine (BrdUrd) may incorrec tly incorporate opposite deoxyguanine in DNA, then pair with deoxyaden osine during subsequent replication. It appears to preferentially targ et the 3'-G of 5'-NGGN-3' sequences in mammalian cells in culture to i nduce G --> A transitions. Ras genes should therefore be vulnerable to activation by mutation at glycine codons 12 (GGT) and/or 13 (GGC) by misincorporation of BrdUrd. There is limited evidence that BrdUrd may be carcinogenic or co-carcinogenic in rats: three renal mesenchymal tu mors, a tumor known to be associated with activating mutations in the c-K-ras-2 oncogene, were reported in 87 rats treated with BrdUrd alone , while N-nitrosomethylurea (NMU) alone or MMU + BrdUrd resulted in in cidences of 12/52 and 26/76, respectively, against a zero incidence in untreated rats. We analyzed renal mesenchymal tumors from rats treate d with BrdUrd for mutations in K-ras exons 1 and 2 and compared the pr evalence and spectrum of mutations with those found in comparable tumo rs induced with NMU. DNAs from 22 paraffin-embedded renal mesenchymal tumors from rats treated 12-15 months earlier with BrdUrd (three speci mens) or NMU (11 specimens) or both agents sequentially (eight specime ns) were amplified by PCR. The base sequence of codons 12-13 and 59-63 of K-ras was determined by the dideoxynucleotide method. Sequencing r esults were confirmed by allele-specific oligonucleotide hybridization . Two of three tumors that appeared in rats given BrdUrd alone contain ed both a codon 12 GGT --> GAT transition and a codon 61 CAA --> CTA t ransversion. One tumor induced by NMU alone also showed a codon 12 GGT --> GAT mutation, while only wild type sequence could be demonstrated in the codon 12-13 region in the remaining ten such tumors. Three NMU -induced tumors also showed codon 61 CAA --> CTA mutations, while the remaining tumors had wild type sequence. While the GGT --> GAT transit ions identified in tumors from BrdUrd-treated rats are consistent with BrdUrd mutagenesis by misincorporation, the co-occurrence of CAA --> CTA transversions, the overall low prevalence of mutations, and the la ck of any difference in mutation spectrum between tumors induced by NM U and those that occurred in BrdUrd-treated rats suggests that in both groups the mutations that did occur did not result from a direct effe ct of either agent.