The signal transduction pathways mediated by retinoic acid play a critical role in the regulation of cell growth and differentiation during embryogenesis and hematopoiesis as well as in a variety of tumor cell lines in culture. Following the reports that two members of the superfamily of aldehyde dehydrogenase (ALDH) enzymes, ALDH1A1 and ALDH1A2, were capable of catalyzing the oxidation of
Our results show that DPAB potently inhibits retinal oxidation, with IC50 values of 0.11 and 0.13 μM for purified mouse and human ALDH1A1, respectively. Since the HL-60 human myeloid leukemic cell line has been used extensively to study the retinoic acid induced differentiation of HL-60 cells to granulocytes, and ALDH1A1 activity had previously been reported in HL-60 cells, we investigated the ability of DPAB to block differentiation of HL-60 promyelocytic leukemia cells exposed to retinal in culture. In HL-60 cells coincubated with 1 μM retinal and 50 μM DPAB for 144 hours, cell differentiation was inhibited only 30%. Furthermore, the NAD-dependent oxidation of propanal or retinal was less than 0.05 nmoles NADH formed/min-107 cells in spectrophotometric assays using HL-60 cell extracts.
Although ALDH1A1 may be the major catalytic activity for retinal oxidation in some retinoid-dependent mouse and
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
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 [
Chemical structures for ALDH1A1 substrate and inhibitor.
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.
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
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 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 [
The HL-60 human promyelocytic leukemia cell line responds in culture to sub-micromolar concentrations of retinoic acid (
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 [
The collection of results demonstrating that retinal is an excellent substrate for ALDH1A1
DPAB is a potent inhibitor of retinal oxidation with IC50 values of 0.11 and 0.13 μM for purified mouse and human ALDH1A1, respectively (Table
IC50 values for the inhibition by DPAB of the oxidation of selected aldehyde substrates by mouse and human ALDH1A1
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| 0.11 ± 0.02 | 0.13 ± 0.03 | |
| Propanal | 0.040 ± 0.01 | 0.14 ± 0.04 |
| Phenylacetaldehyde | 0.36 ± 0.04 | 1.2 ± 0.1 |
IC50 values are the mean ± average deviation from at least 3 spectrophotometric assays, each performed in triplicate.
Following continuous exposure to 1 μM retinoic acid for 96 hours, almost 40% of cells have differentiated to granulocytes, as seen by the positive NBT staining. Exposure to 1 μM
Differentiation response of HL-60 cells to retinoids and ALDH inhibitors. Retinoid concentrations were 1 μM for
Although DPAB is a more potent inhibitor of human ALDH1A1 than DEAB, we were concerned that DPAB was not remaining in solution at 50 μM in the culture media. However, the observation that the addition of DEAB, a much more soluble compound, resulted in the same percent inhibition, suggested that solubility was not a factor. Thus, artifacts due to solubility of inhibitor and extracellular oxidation of retinal do not appear to contribute to the observed response of DPAB on retinal oxidation.
In addition, the NAD-dependent oxidation of propanal or retinal was less than 0.05 nmoles NADH formed/min-107 cells in spectrophotometric assays using HL-60 cell extracts, even in the absence of DEAB or DPAB. The NAD-dependent oxidation of these aldehydes was not reproducibly detected above background in spectrofluorometric assays. Therefore, though low levels of ALDH1A1 activity have been reported in HL-60 cells in previous studies [
Although ALDH1A2 appears only to catalyze the oxidation of
Our results suggest an intriguing possibility that regulation of retinoic acid formation in the HL-60 cells, and the subsequent terminal differentiation to granulocytes, could be mediated by one enzymatic acitivy, e.g. a CYP-mediated oxidation of retinal, whereas regulation of retinoic acid formation in the hematopoietic stem cells, and the subsequent maintenance of the self-renewal capacity of the hematopoietic stem cells, is mediated by ALDH1A1 oxidation of retinal (Figure
A schematic depicting the potential roles of different oxidative enzymes in retinoic acid biosynthesis and the differentiation vs. self-renewal response to the retinoic acid ligand at different stages of hematopoiesis. Hematopoietic stem cell (HSC).
We have shown that DPAB can serve as a potent inhibitor of purified human and mouse ALDH1A1 and should be a useful molecule to assess the role of ALDH1A1 in retinal oxidation in a cell system. In addition, our results indicate that in HL60 cells, the prototype cell line for studying retinoic acid induced cell differentiation, ALDH1A1 is not the operative oxidizing enzyme for retinal.
NAD,
The purification of ALDH1A1 from mouse liver and human erythrocytes was performed as in [
Retinal and retinoic acid were dissolved in either DMSO or ethanol and stored as 100 mM stock solutions in foil-wrapped vials at -20°C. For enzyme assays, retinal prepared in DMSO was diluted in a 70% methanol: 30% water (v/v) solution and all assays were run under dim light. DPAB was synthesized as described in [
ALDH1A1 activity was measured on a Shimadzu UV1201 Spectrophotometer at 36.5° (± 0.5°) at 340 nm. Standard assay conditions were as follows: 1 mL final reaction volume; assay buffer (0.1 M sodium pyrophosphate pH 8.4, 1.0 mM EDTA, 5.0 mM dithiothreitol); 1.0 mM NAD; 10 mM pyrazole. The order of addition in the assay was as follows: buffer; pyrazole; NAD; ALDH (10 mU mouse liver ALDH1A1; 5 mU human erythrocyte ALDH1A1; or HL-60 cell extract); inhibitor (if any); and lastly, the aldehyde substrate. One Unit of activity is defined as 1 μmole NADH formed /minute. Background activity was determined for 60–90 seconds after the addition of enzyme and the reaction rate was measured during the 30–60 seconds following addition of retinal substrate Propanal concentration was varied from 500 to 10 μM (20–0.4 times Km), phenylacetaldehyde concentration was varied from 40 to 5 μM (5–0.6 times Km), and retinal concentration was varied from 1–10 μM, all with constant (1 mM) NAD concentration. DPAB concentrations were varied from 20 μM to 0.05 μM. All trials were done in triplicate.
Since there are three molecules with strong absorbances at 340 nm (retinal, retinoic acid, and NADH), we developed a high pressure liquid chromatography (HPLC) assay to assess the ability of DPAB to inhibit retinal oxidation by class 1 ALDH. Assay conditions were: 0.5 mL final reaction volume; assay buffer (0.1 M sodium pyrophosphate, pH 8.4, 1.0 mM EDTA, 5.0 mM dithiothreitol or glutathione); 1.0 mM NAD. The reaction rate was measured at 37°C for 1–10 minutes. The reaction was stopped by the addition of 200 μL butanol. After vortexing for 1 minute, 50 μL of saturated K2HPO4 was added. The sample was vortexed and centrifuged for 2 minutes at 16,000 g. 50 μL of the resulting organic layer was injected onto a reverse phase C18 column (Phenomenex). The mobile phase was 0.5% acetic acid: 20% water: 79.5% acetonitrile (v/v) with a flow rate of 2.0 mL/min.
HPLC was used to analyze the purity of our retinal samples and the stability of retinal under culture conditions. The stability of retinal in tissue culture was determined by incubation of 10 μM retinal in culture media (RPMI 1640 plus 15% fetal bovine serum) for six days at 37°C. At 24 hour intervals, 1 mL of media was removed. 200 mL of 50% butanol:50% acetonitrile (v/v) was added and the mixture was vortexed for 60 seconds, followed by centrifugation for 2 minutes at 16,000 × g. The top organic layer was removed and diluted 1:5 in acetonitrile before injection onto the HPLC column.
HL-60 cells (ATCC, Rockville, MD) were propagated in RPMI-1640 media supplemented with 15% fetal bovine serum and 50 U/mL penicillin and 0.5 mg/mL streptomycin. Cells were subcultured and treated at 2 × 105 cells/mL. Cells were exposed to combinations of 1 μM retinoic acid, 1 μM retinal, and various concentrations of DPAB and DEAB prepared in sterile dimethyl sulfoxide (DMSO) or ethanol. Final concentrations of DMSO or ethanol in the culture medium did not exceed 1% v/v. Cells were exposed for up to 144 hours without replacement of media or washing out of retinoid or inhibitor. Cell differentiation was measured by mixing 100 μL aliquots of cells with 100 μL of a solution containing 1.0% w/v bovine serum albumin, 0.2% w/v nitroblue tetrazolium and 1.0 μg/mL of 12-O-tetradecanoylphorbol 13-acetate (TPA). The mixture was incubated for 45 minutes at 37°C in a 24-well plate. Cells were pipetted onto a Bright Line counting chamber and scored as positive for differentiation by the presence of purple NBT precipitate using bright field microscopy. Two observers each counted two fields of at least 200 cells from each well of treated cells. Cell survival was determined by Trypan blue dye exclusion.
Cytosolic extracts of HL-60 cells were analyzed for ALDH activity using a Perkin Elmer 650 spectrofluorometer with an excitation wavelength of 350 nM and an emission wavelength of 460 nM. Assay conditions were as given for the spectrophotometric enzyme assays above, with propanal or
This work was supported by grants from the M.J. Murdock College Science Research Program, a Stanley R. Rall Research Award, and the Howard Hughes Medical Institute.