Under-expression of alcohol dehydrogenase in uterine fibroids: Impact on retinoic acid pathway

Abstract

Retinoic acid and precursors play multiple roles in cellular differentiation and development. Alterations in this pathway have been identified in various neoplasms. Furthermore, Boetter-Tong and colleagues (Proc. Soc. Exp. Biol. Med. 1997;215:59) have shown antiproliferative effects of retinoic acid in human tissue, and Tsibris et al. reported a role for retinoic acid in the guinea pig fibroid model system (Cancer Res. 1999;59:5737; BBRC 2003;312:249). Also, Gamage and colleagues found that an RXR ligand induced apoptosis in the Eker rat fiboid model (J. Pharmacol Exp. Ther. 2000;295:677). These findings suggest that components of the retinol pathway are important for fibroid development. The first step in the retinoic acid pathway is the conversion of retinol to retinaldehyde by alcohol dehydrogenase. We have previously identified under-expression of alcohol dehydrogenase in fibroid tissue compared with normal myometrium. We now investigate other genetic alterations within the retinoic acid pathway that help define the molecular fingerprint of fibroids. Screening microarray analysis followed by confirmatory studies. After IRB approval, we collected specific fibroids from patients who were undergoing abdominal hysterectomy. In this study, fibroid / myometrium pairs from five patients were analyzed. RNA isolation was performed using a standard protocol. Samples of 10–15 micrograms of total RNA from fibroid and adjacent myometrium were analyzed by Affymetrix U133 microarray chips. For RT-PCR, primers were designed and added to the reaction mix at 200nM concentration. RT-PCR with Platinum Taq was performed, and primers for GAPDH were included as an internal control. Samples were run on a 2% agarose gel with ethydium bromide. Microarray analysis demonstrated under-expression of alcohol dehydrogenase, class I, beta 1 and 2 in fibroids compared with surrounding normal myometrium (see Table). In addition, retinol and retinoic acid binding proteins were under-expressed in fibroids compared with myometrium. Retinoic acid induced 1, evidence of retinoic acid activity, was also under-expressed, demonstrating decreased retinoic acid activity. Finally, cP450 retinoid metabolizing protein was over-expressed in fibroids compared with myometrium. These results were confirmed by RT-PCR. Molecular alterations in the retinoic acid pathway of fibroids suggest increased retinol activity. Under-expression of alcohol dehydrogenase subunits could result in elevated retinol levels and decreased retinaldehyde and retinoic acid levels. Furthermore, by decreasing binding proteins, levels of free retinol could be further elevated. such alterations could impact retinol pathway signaling, thereby disrupting the normal signals that maintain cell differentiation. By understanding this pathway, it may be possible to generate novel therapies for uterine fibroids.