Abstract 2563: Identification and functional characterization of a novel UGT2A1 splice variant: Potential importance in tobacco-related cancer susceptibility .

Abstract

Abstract UGT2A1 is a phase II enzyme highly expressed in the respiratory and aerodigestive tracts. In the present study, a novel exon 3 deletion splice variant for UGT2A1 (termed ‘UGT2A1Δexon3’) was characterized. As determined by reverse-transcription polymerase chain reaction, UGT2A1Δexon3 was shown to be co-expressed with wild-type UGT2A1 in various tissues including lung, trachea, larynx, tonsil, and colon. The mRNA expression ratio of UGT2A1Δexon3:wild-type UGT2A1 was highest in colon (0.79±0.08) and lung (0.42±0.12) as determined by real-time PCR; splice variant protein (termed ‘UGT2A1_i2’) to wild-type protein (termed ‘UGT2A1_i1’) ratios were in the range of 0.5-0.9 in these tissues as determined by Western blot analysis using an UGT2A1-specific antibody. In contrast to the UGT2A1 wild-type isoform, homogenates prepared from UGT2A1_i2-over-expressing HEK293 cells exhibited no glucuronidation activity against PAHs, including benzo(a)pyrene-7,8-dihydrodiol (B(a)P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p<0.01) decrease in the UGT2A1_i1 Vmax against 1-hydroxy (OH)-pyrene, 3-OH-B(a)P and B(a)P-7,8-diol; no significant changes in KM were observed for any of the three substrates. Co-immunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; co-expression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and that expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis. Citation Format: Ryan T. Bushey, Gang Chen, Philip Lazarus. Identification and functional characterization of a novel UGT2A1 splice variant: Potential importance in tobacco-related cancer susceptibility . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2013-2563