The aldehyde dehydrogenase (ALDH) superfamily of enzymes consists of a group of NAD(P)-dependent enzymes which can oxidize a structurally diverse group of endogenous and exogenous aldehyde substrates. Seventeen ALDH
genes have been identified in the human genome and have been classified into ten different families based on amino acid sequence identities [1
]. High levels of ALDH1A1 activity can protect human and murine cells from the toxicity of the alkylating agent cyclophosphamide and its oxazaphosphorine analogs. Indeed it was hypothesized that the high expression of ALDH1A1 (formerly known as class 1 ALDH) was responsible for the successful use of cyclophosphamide in tumor cell purging regimens during bone marrow transplantation [3
]. The detection of elevated ALDH1A1 activity in the most primitive human hematopoietic progenitor cells [4
] has provided evidence in support of this hypothesis. However, no physiologic role for this enzyme in the hematopoietic stem cell population has been identified.
Vitamin A (retinol) is the prototype of a class of natural and synthetic chemical compounds named the retinoids. Retinoids are found in a variety of chemical oxidation states, including alcohol, aldehyde, ester, and carboxylic acid functionalities. The acid form, retinoic acid, is a pleiotropic hormone which regulates gene expression in embryonic development, epithelial cell differentiation, hematopoeisis, and tumor cell formation.
Several enzymes, including isoforms of alcohol dehydrogenase, ALDH, and cytochrome P450 (CYP), recently have been shown to be involved in the formation of retinoic acid from retinol and retinal [7
]. The purification, cloning, and characterization of several ALDH enzymes which efficiently catalyze the oxidation of retinal (Figure ) has suggested that regulation of retinoic acid formation could be a key physiologic role for these enzymes. The report of the crystal structure of the homotetrameric ALDH1A1 enzyme from sheep liver revealed a substrate tunnel capable of binding retinal which was absent in ALDH2 and ALDH3A1 enzymes [9
]. Likewise, the crystal structure reported for mouse ALDH1A2 revealed a substrate channel which could provide specificity for retinal and exclude short-chain aliphatic aldehydes [10
Chemical structures for ALDH1A1 substrate and inhibitor. (A) substrate: all-trans retinal. (B) inhibitor: 4-(N,N-dipropylamino)benzaldehyde (DPAB).
The specific role of each enzyme in the regulation of retinoid signalling may depend on the species, cell type, and developmental status of the cell. In vitro,
purified mouse [11
], human [12
], and Xenopus
] ALDH1A1 can efficiently oxidize all-trans
retinal to all-trans
retinoic acid. In vivo
, the introduction of ALDH1A1 mRNA into Xenopus embryos
induces early synthesis of retinoic acid, while ALDH1A1 expression is detected during the tail bud stages of Xenopus
]. ALDH1A2 in the mouse [15
] and rat [16
] has shown even greater specificity and catalytic efficiency for retinal as a substrate. Immuno-histochemical analysis of mouse embryos shows that ALDH1A1 expression occurs primarily in cranial tissues, while ALDH1A2 expression occurs primarily in trunk tissue [17
]. In addition, the targeted knockout of the ALDH1A2
gene in mice results in a significant decrease in retinoic acid biosynthesis and early embryo death [18
The development of inhibitors which can target individual ALDH enzymes provides one approach for addressing the role of a specific ALDH enzyme in the oxidation of retinal to retinoic acid in a given cell type. We have identified 4-(N, N-dipropylamino)benzaldehyde (DPAB) (Figure ) and 4-(N, N-diethylamino) benzaldehyde (DEAB) as potent, selective inhibitors of ALDH1A1, but not ALDH2 or ALDH3, with the dipropyl analog exhibiting 10-fold greater efficacy for inhibition [19
]. The effect of DPAB on ALDH1A2 has not been reported.
DEAB was first used to sensitize the mouse leukemic cell line L1210/CPA, which is resistant to 4-hydroperoxycyclophosphamide (4-HC) by virtue of its overexpression of ALDH1A1. Treatment of L1210/CPA cells in vitro
with 50 μM DEAB abolished the tumor cells' resistance to 4-HC [19
]. DEAB also exhibited in vivo
efficacy as a ALDH1A1 inhibitor, as demonstrated by the toxicity to the intestinal crypt cells in mice receiving co-injections of DEAB and cyclophosphamide [21
]. More recently, DEAB has been used to demonstrate that overexpression of human ALDH1A1 in transfected cell lines is sufficient to cause cellular resistance to the oxazaphosphorines [22
] and 4-HC [23
]. DEAB has also been an effective reagent in fluorescent-activated cell sorting (FACS) techniques to isolate human hematopoietic stem cells based on the expression of ALDH1A1 activity [5
]. In contrast to DEAB and DPAB, daidzin – a natural product isolated from Pueraria lobata
(the Kudzu plant) – shows specificity for inhibiting ALDH2 at concentrations similar to that of DPAB used to inhibit ALDH1A1 [24
]. An inhibitor specific for ALDH1A2 has not yet been reported.
In normal hematopoiesis, terminally differentiated cells are generated daily from a limited number of pluripotent stem cells. The stem cell population must be exquisitely regulated to ensure sufficient self-renewal as well as commitment to progenitor cells which can give rise to mature erythrocytes, platelets, lymphocytes, granulocytes, and macrophages. Retinoic acid appears to elicit a complex response of cell proliferation and/or commitment to a more differentiated cell type, depending on the differentiation state of the cell receiving the signal [26
The HL-60 human promyelocytic leukemia cell line responds in culture to sub-micromolar concentrations of retinoic acid (all-trans
) by undergoing terminal differentiation to a granulocytic cell with the ability to phagocytose and reduce nitroblue tetrazolium [28
]. The response to retinoic acid in HL-60 cells is dependent on the expression of the retinoic acid receptor RAR α [29
]. The use of all-trans
retinoic acid in differentiation therapy for patients with acute promyelocytic leukemia has resulted in prolonged, complete remissions when combined with cytotoxic chemotherapy, and may provide complete, long-term remissions as a single agent [30
However, the enzyme or enzymes responsible for the formation of retinoic acid in the HL60 cells have not been identified. In one report, ALDH1A1 protein was not detected by Western blot, but enzyme activity was detected using flow cytometry and a fluorescent aldehyde substrate [5
]. In a second study, ALDH1A1 protein was detected using an ELISA assay and activity was detected using aldophosphamide as substrate [31
]. In these two studies, designed to look at the relationship of ALDH1A1 activity to antitumor drug resistance, the levels of ALDH1A1 were low in HL-60 compared to other cell types, corresponding to the sensitivity of HL-60 cells to oxazaphosphorines. ALDH activity has been reported in HL-60 cells using 4-hydroxynonenal as substrate [32
]. However, since this molecule is such a poor substrate with ALDH1 compared to ALDH3, the measured activity likely represents only ALDH3 activity. ALDH3 does not oxidize all trans
retinal. Expression of ALDH1A2 activity in HL-60 cells has not been reported. Therefore, we chose the HL-60 cell line to assess the role of ALDH1A1 in the oxidation of retinal.
The collection of results demonstrating that retinal is an excellent substrate for ALDH1A1 in vitro, that ALDH1A1 is responsible for the synthesis of retinoic acid in the cranial tissues of the mouse embryo, and that hematopoietic stem cells are characterized by high expression of ALDH1A1, have led to the hypothesis that the oxidation of retinal to retinoic acid is a key physiologic role for ALDH1A1 in hematopoietic stem cells. Our long-term goal is to use DPAB to investigate the role of ALDH1A1 in retinoic acid biosynthesis at different stages of hematopoiesis. In this paper we show that DPAB is a potent inhibitor of retinal oxidation by murine and human ALDH1A1. However, DPAB only weakly inhibits the retinal-induced differentiation of HL-60 cells, suggesting that ALDH1A1 is not the operative retinal oxidizing enzyme in these cells.