Frequent codon 12 Ki-ras mutations in mouse skin tumors initiated byN-methyl-N?-nitro-N-nitrosoguanidine and promoted by mezerein

Abstract

The skin tumor initiators N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA) differ in effectiveness when tumor formation is promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Even at high doses, MNNG is less effective, producing fewer benign and malignant tumors with a longer latent period. In DMBA-initiated skin, 10 wk of TPA promotion produced a maximal tumor response. With MNNG, 20 wk of TPA promotion was required, producing nearly four times as many papillomas as 10 wk of promotion. Promotion of MNNG-initiated skin with mezerein induced the appearance of very rapidly-growing papillomas within 5 wk, 3 wk earlier than the first TPA-promoted papillomas. Thus, MNNG may induce a novel mutation resulting in a population of initiated cells that respond especially well to mezerein. Since ras mutations are common in experimental tumors in many tissues, we determined the frequency of activating mutations in the Ha-ras, Ki-ras, and N-ras oncogenes. Activating Ha-ras mutations were present in essentially all DMBA-initiated tumors and about 70% of MNNG-initiated tumors. No N-ras mutations were found in tumors lacking other ras mutations. Surprisingly, 41% of the papillomas arising in the first 11 wk in MNNG-initiated, mezerein-promoted mice bore mutations in codon 12 of the Ki-ras oncogene. Activating Ki-ras mutations were also found in more than 60% of squamous cell carcinomas and 40% of keratoacanthomas. Although mutations in Ha-ras are frequently detected in mouse skin tumors, mutations in Ki-ras are rare. This is the first report of mutated Ki-ras in skin tumors from mice initiated by MNNG.

Methylation of the O6‐methylguanine‐DNA methyltransferase promoter suppresses expression in mouse skin tumors and varies with the tumor induction protocol

“…The same tumor samples were assayed earlier for ras mutations. 19 Tumor type (papilloma, squamous cell carcinoma or keratoacanthoma) was verified by examination of hematoxylin and eosinstained slides.…”

“…19 For comparison, DNA was extracted from frozen samples of shaved back skin and 1 cm of mouse tail.…”

“…19 To examine whether methylation of the MGMT promoter region is associated with tumor development and progression in mouse skin, we analyzed DNA from 184 primary skin tumors using the MSP assay. Methylation of the MGMT promoter was found in 81 tumors (44.0%) (Table I), but was not found in 26 normal mouse skin samples (Fig.…”

“…17 In earlier studies, we showed that the phenotype of induced skin tumors (indicated by the optimal duration of promotion, time of appearance and growth rate, and frequency of conversion to malignancy) varied with different initiators and promoters. 18,19 Compared to the most commonly used protocol of DMBA initiation followed by TPA promotion, promotion with mezerein resulted in fewer papillomas that developed more slowly, but the rate of conversion of papillomas to squamous cell carcinomas was significantly higher. 18 The increased conversion rate with mezerein promotion was not found with MNNG as initiator, but a population of rapidly-growing papillomas developed in the MNNGmezerein group.…”

“…18 The increased conversion rate with mezerein promotion was not found with MNNG as initiator, but a population of rapidly-growing papillomas developed in the MNNGmezerein group. 19 The optimal duration of promotion also differed for TPA and mezerein, 19 suggesting that these promoters may select for different populations of initiated cells. An activating mutation in Ha-ras was found in essentially all DMBA-initiated tumors (A-T mutation in codon 61) and 70% of MNNG-initiated tumors (G-A mutation in codon 12).…”