Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR–RFLP method

Abstract

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells

“…Based on studies of B[a]P-induced mouse skin papilloma it has been suggested that the H-ras mutations are generated by misrepair, rather than misreplication, of the apurininc sites (Chakravarti et al, 1998). The B[a]P-induced apurinic sites in cellular DNA are 15-to 120-fold higher than those formed spontaneously, suggesting that this large increase in apurinic sites has the potential of overwhelming the cell repair capacity before replication occurs (Chakravarti et al, 1995;Cavlieri and Rogan, 1998).…”

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene

“…An area of untreated belly skin was excised from each mouse as control. Other methods for the determination of mutation spectra in the H-ras gene have been described previously (Chakravarti et al, 1998b(Chakravarti et al, , 2000.…”

“…Carcinogen-induced DNA damage causes cells to enter into a period of replication block (Slaga et al, 1973(Slaga et al, , 1974 and repair induction (Sawyer et al, 1988;Gill et al, 1991) before continuing with the cell cycle (Kaufmann, 1995;Maher and McCormick, 1987). Oncogenic mutations may arise from errors committed during pre-replication repair (Chakravarti et al, 1998b(Chakravarti et al, , 2000 and, also, during subsequent replication in cells harboring unrepaired DNA lesions (Maher and McCormick, 1987;Kaufmann, 1995;Moriya et al, 1996).…”

“…When SENCAR mouse skin is treated with DB[a,l]P, 99% depurinating adducts are formed in skin DNA . AP sites generated by these depurinating adducts induce pre-replication repair that is error-prone and forms mismatched heteroduplexes leading to transforming mutations in the H-ras gene at codon 61 (CAA to CTA) (Chakravarti et al, 1998b(Chakravarti et al, , 2000. In contrast, a metabolite of DB[a,l]P, (+)-anti-DB[a,l]P-11,12-diol-13,14-epoxide (anti-DB[a,l]PDE), which forms 97% stable adducts in mouse skin , does not appear to induce mismatched heteroduplexes in these early preneoplastic stages (Chakravarti et al, 2000).…”

Tumour necrosis factor-α mediates tumour promotion via a PKCα- and AP-1-dependent pathway

“…To investigate this further, papillomas resulting from DMBA/TPA-treatment of wt and TNF-a 7/7 mouse skin were analysed for the presence of the characteristic c-Ha-ras gene mutation (A?T transversion in codon 61), thought to be the key event in tumour initiation (Balmain et al, 1984). Mutational analysis of the c-Ha-ras gene by PCR is possible since an XbaI restriction site is introduced into the c-Ha-ras PCR product if the codon 61 (A?T) mutation is present (Chakravarti et al, 1998). All papillomas tested from both wt and TNF-a 7/7 mice contained the codon 61 mutation in c-Ha-ras ( Figure 1b), suggesting that TNF-a does not affect the frequency of codon 61 c-Ha-ras mutations by DMBA.…”