Abstract 3362: Diethylaminobenzaldehyde (DEAB) is not a specific inhibitor of ALDH1a1 isozyme

Abstract

Abstract With the cancer stem cell theory, there has been an invigorated new interest in finding more markers for stem cells. Aldehdye dehydrogenase (ALDH) is one such marker that became popular since a flow cytometry based assay, Aldeflour staining, has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been published many years ago as a specific inhibitor for ALDH1 isoform. The Aldeflour assay is promoted as testing the enzymatic activity of ALDH1. Therefore most researchers believe that the high ALDH activity detected in these early progenitors is mainly due to ALDH1. In this study, we explore the specificity of DEAB as an inhibitor of ALDH activity. We used two cancer cell lines known to have very low baseline ALDH activity, K562 leukemia and H1299 lung cancer cell lines. We prepared lentiviral vectors containing the full length of either ALDH2 or ALDH1a2 cDNA and used them to over express the enzymes in the two cell lines. Successful expression was measured by ALDH activity assay as well as by Western blot analysis and RT-PCR. Aldeflour staining assay was also used. The results show that we created 4 cell lines with successful high expression of each of the two enzymes as confirmed by multiple assays including the Aldeflour assay. In order to study the specificity of two known ALDH activity inhibitors, DEAB and tetraethylthiuram disulfide (disulfiram), we incubated each cell line with either inhibitor at 25 μM concentration for 48 hr and then measured the remaining ALDH enzymatic activity. Our results show that both inhibitors reduced ALDH activity of both overexpressed isozymes, ALDH2 and ALDH1a2, by 70-80%. These results suggest that DEAB is not a specific inhibitor for ALDH1a1 and that aldefluor assay is not specific assay for ALDH1a1 either. Therefore, investigators should be careful in their conclusions when using DEAB in studying candidate stem cells and that studies with more specific inhibitors, such as siRNA, are needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3362.