A somatic mutation in the 5'UTR of BRCA1 gene in sporadic breast cancer causes down-modulation of translation efficiency.

Mutations in the 5' UTR which cause increment/decrement of translation efficiency have been recently described as a novel molecular mechanism of disease. Alterations in the consensus sequence for the translation initiation may promote context-dependent leaky scanning of ribosomes and/or initiation from a downstream AUG codon. Initiation of translation from a downstream in-frame AUG codon in BRCA1 gene was recently identified in normal cells and possibly in breast cancer. Here we present further insight into BRCA1 translational pathophysiology investigating the role of the canonical structure of the initiation consensus sequence of BRCA1. We have analysed the effect of a somatic point mutation (117 G>C) in position -3 with respect to the AUG of the BRCA1 gene, identified in a highly aggressive sporadic breast cancer. We constructed chimeric genes encoding the luciferase reporter sequence downstream of the wild type or the mutated BRCA1 5'UTR. These transcripts were tested for their activity in in vitro and in vivo systems. In in vitro transcription/translation assays the estimated translation efficiency of the construct with the mutated BRCA1 5'UTR was 30-50% lower than that with the wild type BRCA1 5'UTR. The same chimeric genes were analysed for their expression in vivo by transient transfection in human cells. While the two constructs were equally transcribed, the plasmid carrying the mutated sequence produced 70% less luciferase activity compared to the wild type sequence. Finally, to obtain a direct evaluation on translational efficiency in vivo, we analysed mRNA translation on translationally active and non-active ribosomes separated from transfected cells. Mutant mRNA was partially localized in subpolysomal particles analytically confirming a polysome recruitment defect. Thus, characterization of BRCA1 5'UTR and translation efficiency seems to provide new insight into BRCA1 role in breast and ovarian cancer pathogenesis.

Analysis of c-Ha-ras gene mutations in skin tumors induced in carcinogenesis-susceptible and carcinogenesis-resistant mice by different two-stage protocols or tumor promoter alone.

In the present study we describe the molecular analysis of c-Ha-ras gene mutations in 47 papillomas and 17 carcinomas developed in two lines of mice, carcinogenesis-susceptible (Car-S) and carcinogenesis-resistant (Car-R), selectively bred for extreme susceptibility or resistance to chemical skin carcinogenesis initiated and promoted with different doses of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This study also presents the analysis of c-Ha-ras gene mutations in 22 papillomas and 22 carcinomas in Car-S mice initiated with DMBA and promoted with benzoyl peroxide (BzPo) and in seven papillomas and one carcinoma from a group of uniniated Car-S mice that received only BzPo treatment. The data showed that a A(182)-->T transversion in the c-Ha-ras gene was present in 100% and 81% of the skin tumors developed in Car-S and Car-R mice, respectively, after DMBA initiation and TPA promotion, suggesting that differences in genetic susceptibility can influence the frequency of c-Ha-ras mutations in the skin tumors produced. The same A(182)-->T mutation with an incidence of 68% was found in papillomas from DMBA-initiated and BzPo-promoted Car-S mice. The difference in the mutation frequency between DMBA/BzPo and DMBA/TPA papillomas suggested that the promotion step contributes to the final mutation pattern. The tumor induction experiment with BzPo alone showed that this compound can induce tumor development in 26% of Car-S mice, and the molecular analysis of the tumors showed a broad mutation spectrum, including mutations in codons 12, 13, and 61 of the c-Ha-ras gene. Mol. Carcinog. 30:111-118, 2001.