P53 expression, p53 and Ha-ras mutation and telomerase activation during nitrosamine-mediated hamster pouch carcinogenesis.

Squamous cell carcinomas (SCC) induced in hamster buccal pouch (HBP) by 22 weeks of topical N-methyl-N-benzylnitrosamine (MBN) treatment (twice-weekly, 10 mg MBN/ml propylene glycol) were evaluated for: (i) altered expression of p53 using immunohistochemistry (IHC); (ii) mutations in Ha-ras and p53 using PCR/single strand conformation polymorphism (SSCP); (iii) telomerase activity using the telomerase repeat amplification protocol (TRAP). Precancerous lesions were also evaluated using p53 IHC. Hamsters were killed for lesion analysis at either 3 days (group A, eight hamsters, 89 carcinomas) or 7 weeks (group B, six hamsters, 105 carcinomas) following the final MBN application. Between 3 days and 7 weeks post-treatment the proportion of tumors exhibiting p53 IHC activity (at least 10% of nuclei stained using D07 antibodies for detection of both mutant and wild-type p53) fell from 91 to 50%. However, during this same post-treatment period the frequency of tumors analyzed exhibiting confirmed sequence alterations in the conserved exons (E5-E8) of p53 remained constant (5/15 = 33% in group A versus 14/45 = 31% in group B). Heightened expression of wild-type p53 resulting from DNA damage in the immediate post-treatment period is likely to have contributed to the high proportion of group A tumors exhibiting p53 IHC activity. Nearly 80% of the identified p53 mutations were G-->A and C-->T transitions. The identified p53 point mutations occurred at or near (within three codons) of the corresponding hot-spot codons (175, 245, 248 and 273) of human oral SCC. The proportion of group A and group B tumors analyzed exhibiting Ha-ras mutations was 1/15 (7%) and 7/45 (16%), respectively. Only four of the observed eight Ha-ras mutations occurred in codons known to result in activation of this gene. Telomerase activation was demonstrated in 11 of 13 group A tumors (85%) and in 23 of 24 (96%) group B tumors analyzed. The alterations in p53, Ha-ras and telomerase activity observed in this HBP-MBN model are similar in many respects to those observed in the analogous human lesions of the head and neck. This model may be particularly useful for development of cancer chemoprevention regimens and multimodality cancer therapies.