Abstract 4361: The enzymatic activity of aldehyde dehydrogenases 1A2 and 2 (ALDH1A2 and ALDH2) is detected by aldefluor, inhibited by diethylaminobenzaldehyde and has significant effects on cell proliferation and drug resistance

Abstract

Abstract With the cancer stem cell theory, there has been an invigorated new interest in using ALDH activity as one marker for stem cells and such use 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. In this study, we explore the effects of specific ALDH isozymes, ALDH1A2 and ALDH2, and the specificity of DEAB as an inhibitor of their activity. We prepared lentiviral vectors containing the full length of either ALDH2 or ALDH1A2 cDNA and used them to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by spectrophotometric ALDH activity assay, Western blot analysis, RT-PCR, as well as aldeflour staining assay. In addition, gene expression profiles were assessed by quantitative real-time RT-PCR using micro fluidic cards (Taqman Low Density Arrays, TLDA, Applied Biosystem), covering 19 ALDH and three housekeeping genes. The expression levels were compared between sorted K562 with high versus low ALDH activity. The results show that we created 4 cell lines with successful high expression of each of the two enzymes and high ALDH activity which was detected by aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-HC and doxorubicin. 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 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%. TLDA analysis revealed that K562 cells express high levels of isozymes in the ALDH1, ALDH3, ALDH7 and ALDH8 families. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that aldefluor assay is not specific assay for ALDH1A1 either. In addition, other ALDH isozymes seem to play a major role in the biology and drug resistance of various malignant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4361. doi:10.1158/1538-7445.AM2011-4361