The enzymatic activity of human 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

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65–90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.

Introduction

Aldehyde dehydrogenases (ALDHs) are a group of NAD(P)⁺-dependent enzymes involved in the metabolism of a wide variety of aliphatic and aromatic aldehydesC According to the latest database, the human genome contains 19 ALDH functional genes and three pseudogenes [3], [4], [5], [6], [7]. The role of some of these ALDHs in endobiotic and xenobiotic metabolism has been reviewed extensively before [1], [2], [8], [9], [10], [11], [12], [13], [14], [15] and the specific metabolic pathways affected have been previously detailed [1]. Many allelic variants within the ALDH gene family have been identified, resulting in pharmacogenetic heterogeneity between individuals which, in most cases, results in distinct phenotypes [1], [15] including intolerance to alcohol and increased risk of ethanol-induced cancers (ALDH2 and ALDH1A1), Sjogren-Larson Syndrome (ALDH3A1), type II hyperprolinemia (ALDH4A1), 4-hydroxybutyric aciduria (ALDH5A1), developmental delay (ALDH6A1), hyperammonemia (ALDH18A1), and late onset of Alzheimer’s disease (ALDH2). Furthermore, knockouts of ALDH1A2 and ALDH1A3 in mouse are embryonic lethal and newborn lethal, respectively [16], [17], [18].

ALDH activity is one of the identifying markers of stem cells, both normal and malignant [19], [20]. Different assays for the measurement of ALDH isozymes have been available including Western blot analysis, RT-PCR, spectrophotometric assay for enzyme activity, and immunohistochemistry. A relatively new flow cytometry-based method, Aldefluor staining, has the advantage of measuring ALDH activity in viable cells. With the introduction and marketing of the Aldefluor assay (StemCell Technologies, Inc.) it has become more feasible to study the significance of ALDH expression in murine and human hematopoietic cells [21], [22], [23], [24], [25], [26], [27]. These studies confirmed that ALDH activity is a good surrogate marker for hematopoietic stem cell activity, both in vitro and in vivo. FACS strategies that employ both surface marker expression and ALDH activity have been used to enrich for hematopoietic stem cells (HSC) and in discerning primitive and progenitor cell populations isolated from umbilical cord blood [27]. With the popularity of the cancer stem cell hypothesis, there have been many publications and reviews about the usefulness of measuring ALDH activity to identify cancer stem cells in the different tissues and its potential as a therapeutic target [19], [20].

It is widely presumed that this Aldefluor assay mostly measures ALDH1A1 isozyme activity due to the report that diethylaminobenzaldehyde (DEAB) used in this assay is a specific inhibitor for ALDH1A1 [28]. Therefore, most researchers believe that the high ALDH activity detected in the early stem cells/progenitors is mainly due to ALDH1A1. On the other hand, ALDH1A1 knockout does not affect hematopoietic stem cells and hematopoiesis [29]. Based on this study and our own observation, we hypothesize that ALDH1A1 is not the only enzyme responsible for the ALDH activity measured by Aldefluor assay and that DEAB is not a specific inhibitor for ALDH1A1. Recently Marcato et al. [30] reported that Aldefluor assay detects ALDH1A3 activity in breast cancer stem cells as the main ALDH isoenzyme. As part of our ongoing interest in ALDH isoenzymes and the search for specific inhibitors [31], we overexpressed two other isoenzymes, ALDH2 and ALDH1A2 in two different cell lines and used them in the present study in order to examine our hypothesis.