Abstract B77: Translational effects of alternative NAT1 mRNA isoforms Free

B77

N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of many aromatic amine carcinogens such as 2-aminofluorene and 4-aminobiphenyl. Following N-oxidation by cytochrome P450’s, NAT1 catalyzes the O-acetylation of the N-hydroxy metabolites of many aromatic and heterocyclic amine carcinogens. NAT1 genetic polymorphisms have been associated with differential risks to various cancers related to aromatic and heterocyclic amine carcinogens, but the findings have been inconsistent. Individual susceptibility to these carcinogenic compounds could be modified by genetic polymorphism or by other factors causing differences in NAT1 expression. Our laboratory and others have reported that NAT1 is transcribed from a major promoter, NATb, and an alternative promoter, NATa, resulting in mRNAs with distinct 5’-UTR regions. The 117 nucleotide NATb 5’-UTR contains two noncoding exons, while the 371 nucleotide NATa 5’UTR contains four noncoding exons. The two 5’UTRs share a 79 nucleotide exon immediately upstream of the open reading frame (ORF). We hypothesized that mRNAs including either the NATa or NATb 5’-UTR may be associated with differences in protein expression and that SNPs included in variant NAT1 alleles may modify this effect. We therefore investigated effects on protein expression due to interaction of the different 5’-UTRs with the ORF and region 3’ to the ORF of the reference NAT1*4 allele, and two variant alleles: NAT1*10 and NAT1*11 that are associated with modified risk to arylamine-induced cancers. pcDNA5/FRT plasmid constructs were prepared for transient transfection of full length human mRNAs including either the NATa or NATb 5’-UTR, the ORF, and 885 nucleotides of the region 3’ to the ORF. For each allele of interest, two constructs were made, one including the NATa 5’-UTR and one including the NATb 5’-UTR. Following transient transfection into Chinese hamster ovary cells, NAT1-catalyzed N-acetylation of p-aminobenzoic was measured by HPLC and NAT1 protein expression was measured by Western blot. A difference of approximately 10 fold (p<0.05) in both NAT1 catalytic activity and protein was observed between the NATa and NATb mRNA forms but there was no evidence for allele specific 5’-UTR interactions. These results suggest that differences in NAT1 expression are modified by factors other than polymorphisms in the ORF. Further experiments to examine the possible roles of mRNA production, mRNA stability or differences in mRNA translation efficiency on NAT1 expression are needed. Partially supported by USPHS grants CA034627, ES014559, ES011564, and ES014443.

Citation Information: Cancer Prev Res 2008;1(7 Suppl):B77.