ALDH1 Enzyme Research Articles

Advances in Liver Cancer Stem Cell Isolation and their Characterization.

Over the last decade research on cancer stem cells (CSC) significantly contributed to a better understanding of tumor biology. Given their similarity to normal stem cells, i.e. self-renewal and pluripotency the need arises to develop robust protocols for the isolation and characterization of CSCs. As with other malignancies, hepatic tumors are composed of a heterogeneous population of cells including liver cancer stem cells (LCSC). Yet, a precise understanding of why stem cells become cancerous is still lacking. There is unmet need to develop robust protocols for the successful isolation of LCSCs from human tissue resection material as to assist in the development of molecular targeted therapies. Here we review the research progress made in the isolation and characterization of LCSCs by considering a wide range of cell surface markers and sorting methods, as applied to side populations, microsphere cultures and the gradient centrifugation method. We emphasize the different fluorescence activated cell sorting methods and the possibility to enrich LCSCs by immunomagnetic beads. We review the specificity of functional assays by considering ABCG transporter and ALDH1 enzyme activities and evaluate the in vivo tumorigenicity of LCSCs in highly sensitive bioassays. Finally, we evaluate different LCSC markers in association with viral and non-viral liver disease and explore the potential of novel drug delivery systems targeting CD133, EpCAM, CD13 and CD90 for the development of molecular targeted therapies. Graphical Abstract.

Expression of aldehyde dehydrogenase family 1, member A3 in glycogen trophoblast cells of the murine placenta

IntroductionRetinoic acid (RA) signaling is a well known regulator of trophoblast differentiation and placental development, and maternal decidual cells are recognized as the source of much of this RA. We explored possible trophoblast-derived sources of RA by examining the expression of RA synthesis enzymes in the developing mouse placenta, as well as addressed potential sites of RA action by examining the ontogeny of gene expression for other RA metabolizing and receptor genes. Furthermore, we investigated the effects of endogenous RA production on trophoblast differentiation. MethodsPlacental tissues were examined by in situ hybridization and assayed for RARE-LacZ transgene activity to locate sites of RAR signaling. Trophoblast stem cell cultures were differentiated in the presence of ALDH1 inhibitors (DEAB and citral), and expression of labyrinth (Syna, Ctsq) and junctional zone (Tpbpa, Prl7b1, Prl7a2) marker genes were analyzed by qRT-PCR. ResultsWe show Aldh1a3 is strongly expressed in a subset of ectoplacental cone cells and in glycogen trophoblast cells of the definitive murine placenta. Most trophoblast subtypes of the placenta express RA receptor combinations that would enable them to respond to RA signaling. Furthermore, expression of junctional zone markers decrease in differentiating trophoblast cultures when endogenous ALDH1 enzymes are inhibited. DiscussionAldh1a3 is a novel marker for glycogen trophoblast cells and their precursors and may play a role in the differentiation of junctional zone cell types via production of a local source of RA.

Moderate vitamin A supplementation in obese mice regulates tissue factor and cytokine production in a sex-specific manner

Vitamin A (vitA) regulates obesity, insulin resistance, inflammation, dyslipidemia and hemostasis through its metabolites retinaldehyde (Rald) and retinoic acid (RA) produced in endogenous enzymatic reactions. Combination of at least 3 of these conditions leads to development of metabolic syndrome (Msyn) and, consequently, type 2 diabetes and/or cardiovascular disease. Although many foods are fortified with vitA, it remains unknown what conditions of Msyn are influenced by moderate dietary vitA supplementation. A family of aldehyde dehydrogenase 1 (Aldh1) enzymes is a key contributor to obesity via sex- and fat depot-specific production of RA in adipose tissue. Therefore, we studied effects of moderate vitamin A supplementation of an obesogenic high-fat (HF) diet (4IUvitA/g and 20IUvitA/g HF diet) on multiple conditions and mediators of Msyn in wild-type (WT, C57Bl/6) and Aldh1a1−/− mice. We found that mild vitamin A supplementation did not influence obesity, fat distribution, and glucose tolerance in males and females of the same genotype. In contrast, multiplex analysis of bioactive proteins in blood showed moderately increased concentrations (10–15%) of inflammatory IL-18 and MIP-1γ in vitA supplemented vs. control WT males. Marked decrease (28–31%) in concentrations of lymphotactin and tissue factor, a key protein contributing to thrombogenesis during injury, was achieved by vitA supplementation in WT females compared to control WT females. Aldh1a1 deficiency reduced obesity, insulin resistance, suppressed many pro-inflammatory cytokines, and abolished the effects of vitA supplementation seen in WT mice. Our study revealed specific inflammatory and pro-thrombotic proteins in plasma regulated by dietary vitamin A and the critical role of endogenous vitA metabolism in these processes. The sex-specific decrease of plasma tissue factor concentrations by moderate dietary vitA supplementation could potentially reduce pro-thrombotic states in obese females.

The role of vitamin A metabolites in human and murine B cells

Unraveling mechanisms regulating gammopathies, abnormal immunoglobulin production is necessary to improve treatments of a number of blood cancers, such as multiple myeloma. Retinoic acid and retinaldehyde, a transcriptionally active metabolites of vitamin A, play a key role in differentiation processes. RA is produced by the aldehyde dehydrogenase‐1 family of enzymes (Aldh1a1, a2, a3). Our objective was to identify key genes regulating the cellular levels of these metabolites.Our studies in healthy subjects’ showed that Aldh1a1 is the predominantly expressed Aldh1 enzyme in circulating B cells comprising majority of PBMC fraction. In contrast, two myeloma B cell lines, U266 and RPMI8226 lack Aldh1a1 expression. PBMC from multiple myeloma patients showed an expression profile similar to that of U266. We showed that similar to human B cells, naïve (CD19−) and committed (CD19+) splenic B cells from WT mice predominantly expressed Aldh1a1, whereas in Aldh1a1−/− mice these B cells were complete depleted of Aldh1 expression. In both genotypes B cell differentiation from CD19− to CD19+ phenotype was associated with the loss of Aldh1a1 and a2. The change in the RA production in CD19− and CD19+ cells was accompanied by changed levels of key proteins such as RARa, HoxA10, and PPARg. Our studies suggest that Aldh1 enzymes are important for regulation of Ig production and their deficiency is associated with malignant B cells.Grant Funding Source: Nutrition (ASN)

Potential monovalent cation-binding sites in aldehyde dehydrogenases

Potassium ions are non-essential activators of several aldehyde dehydrogenases (ALDHs), whereas a few others require the cation for activity. Two kinds of cation-binding sites, which we named intra-subunit and inter-subunit, have been observed in crystal structures of ALDHs, and based on reported crystallographic data, we here propose the existence of a third kind located in the central cavity of some tetrameric ALDHs. Given the high structural similarity between these enzymes, cation-binding sites may be present in many other members of this superfamily. To explore the prevalence of these sites, we compared 37 known crystal structures from 13 different ALDH families and evaluated the possible existence of a cation on the basis of the number, distance and geometry of its potential interactions, as well as of B-factor values of modeled cations obtained in new refinements of some reported crystal structures. Also, by performing multiple alignments of 855 non-redundant amino acid sequences, we assessed the degree of conservation in their respective families of the amino acid residues putatively relevant for cation binding. Among the ALDH enzymes studied, and according to our analyses, potential intra-subunit cation-binding sites seem to be present in most members of ALDH2, ALDH1L, ALDH4, ALDH5, ALDH7, ALDH10, and ALDH25 families, as well as in the bacterial and fungal members of the ALDH9 family and in a few ALDH1, ALDH6, ALDH11 and ALDH26 enzymes; potential inter-subunit sites in members of ALDH1L, ALDH3, ALDH4 from bacillales, ALDH5, ALDH7, ALDH9, ALDH10, ALDH11 and ALDH25 families; and potential central-cavity sites only in some bacterial and animal ALDH9s and in most members of the ALDH1L family. Because potassium is the most abundant intracellular cation, we propose that these are potassium-binding sites, but the specific structural and/or functional roles of the cation bound to these different sites remain to be investigated.

Autocrine Function of Aldehyde Dehydrogenase 1 as a Determinant of Diet- and Sex-Specific Differences in Visceral Adiposity

Mechanisms for sex- and depot-specific fat formation are unclear. We investigated the role of retinoic acid (RA) production by aldehyde dehydrogenase 1 (Aldh1a1, -a2, and -a3), the major RA-producing enzymes, on sex-specific fat depot formation. Female Aldh1a1−/− mice, but not males, were resistant to high-fat (HF) diet–induced visceral adipose formation, whereas subcutaneous fat was reduced similarly in both groups. Sexual dimorphism in visceral fat (VF) was attributable to elevated adipose triglyceride lipase (Atgl) protein expression localized in clusters of multilocular uncoupling protein 1 (Ucp1)-positive cells in female Aldh1a1−/− mice compared with males. Estrogen decreased Aldh1a3 expression, limiting conversion of retinaldehyde (Rald) to RA. Rald effectively induced Atgl levels via nongenomic mechanisms, demonstrating indirect regulation by estrogen. Experiments in transgenic mice expressing an RA receptor response element (RARE-lacZ) revealed HF diet–induced RARE activation in VF of females but not males. In humans, stromal cells isolated from VF of obese subjects also expressed higher levels of Aldh1 enzymes compared with lean subjects. Our data suggest that an HF diet mediates VF formation through a sex-specific autocrine Aldh1 switch, in which Rald-mediated lipolysis in Ucp1-positive visceral adipocytes is replaced by RA-mediated lipid accumulation. Our data suggest that Aldh1 is a potential target for sex-specific antiobesity therapy.

Concerted Action of Aldehyde Dehydrogenases in Depot‐specific Fat Formation

Unraveling mechanisms regulating fat distribution in visceral and subcutaneous fat depots is necessary to improve treatments of a number of chronic diseases. Retinoic acid, a transcriptionally active metabolite of vitamin A, plays a key role in differentiation processes. It is produced by the aldehyde dehydrogenase‐1 family of enzymes (aldh1a1,a2,a3). Our objective was to investigate whether differences in RA production by Aldh1, in subcutaneous versus visceral fat, diverge fat formation in these depots.We demonstrated that Aldh1a1 is the major enzyme regulating RA generation in adipocytes in vitro and in vivo. Deficiency in Aldh1a1 inhibits endogenous RA production and suppresses PPARg expression, a key event in 3T3‐L1 adipogenesis, that we rescued by overexpression of Aldh1a1,a3 or both. We identified that visceral fat expressed less Aldh1a2 and a3 than subcutaneous fat in C57/BL6 female mice on regular chow, although Aldh1a1 was predominantly expressed in these depots. Therefore, in Aldh1a1−/− mice, visceral fat essentially lacked all Aldh1 expression, which resulted in 70% decreased expression of PPARg and FABP4 in visceral fat of Aldh1a1−/− vs. WT mice. Consequently, Aldh1a1−/− mice specifically lost visceral fat.Our data suggest that Aldh1 enzymes play a key role in adipogenesis and their concerted action may contribute to differences in fat formation in different depots.

Concerted Action of Aldehyde Dehydrogenases Influences Depot-Specific Fat Formation

Vitamin A metabolite retinoic acid (RA) regulates life-sustaining differentiation processes and metabolic homeostasis. The aldehyde dehydrogenase-1 (Aldh1) family of enzymes (Aldh1a1, a2, and a3) catalyzes RA production from retinaldehyde and thereby controls concentrations of this transcriptionally active metabolite. The hierarchy of Aldh1 functions in adipose tissue has not been elucidated. We hypothesized that Aldh1 enzymes produce endogenous RA and regulate adipogenesis and fat formation in a fat depot-specific manner. We demonstrate that adipogenesis in vitro is accompanied by RA production generated primarily by Aldh1a1. In Aldh1a1-deficient adipocytes, adipogenesis is impaired compared with wild-type adipocytes due to markedly reduced expression of PPARγ regulated through zinc-finger protein 423 (ZFP423)-dependent mechanisms. These effects were recovered to some extent either by RA stimulation or overexpression of any of the Aldh1 enzymes in Aldh1a1(-/-) cells arguing that Aldh1a1 plays a dominant role in autocrine RA production. In vivo studies in C57/BL6 and Aldh1a1(-/-) mice on a regular diet revealed that multiple Aldh1 enzymes regulate differences in the formation of sc and visceral fat. In Aldh1a1(-/-) mice, visceral fat essentially lacked all Aldh1 expression. This loss of RA-producing enzymes was accompanied by 70% decreased expression of ZFP423, PPARγ, and Fabp4 in visceral fat of Aldh1a1(-/-) vs. wild-type mice and by the predominant loss of visceral fat. Subcutaneous fat of Aldh1a1(-/-) mice expressed Aldh1a3 for RA production that was sufficient to maintain expression of ZFP423 and PPARγ and sc fat mass. Our data suggest a paradigm for regulation of fat depots through the concerted action of Aldh1 enzymes that establish RA-dependent tandem regulation of transcription factors ZFP423 and PPARγ in a depot-specific manner.

Targeting tumour-initiating cells to improve the cure rates for triple-negative breast cancer

Tumour recurrence is one of the biggest challenges in breast cancer management because it affects 25-30% of women with breast cancer and the tumours are often incurable. Women with triple-negative breast cancer (TNBC--lacking expression of the oestrogen receptor, progesterone receptor and the receptor HER2/ERBB2) have the highest rates of early recurrence relative to other breast cancer subtypes. Early recurrence might be due to tumour-initiating cells (TICs), which are resistant to conventional therapies, can remain dormant and can subsequently give rise to secondary tumours. In breast cancer, TICs are identified by the cell-surface markers CD44+/CD24-/EpCAM+ and/or possess ALDH1 enzyme activity. This subpopulation has the ability to self-renew, grow as mammospheres and initiate tumour formation. Fuelling the problem of relapse is the fact that chemotherapy and radiation can induce or select for TICs; this was reported in preclinical models and more recently in women being treated for breast cancer. Thus, new therapeutic agents for TNBC are presently being sought to overcome this problem. Here we review the roles of receptor tyrosine kinases, signalling intermediates and transcription factors in sustaining the TIC subpopulation. Particular emphasis is placed on targeting these molecules in order to eliminate and/or prevent the induction of TICs and ultimately reduce the frequency of TNBC recurrence.

Abstract 2300: Overexpression of Snail induces epithelial-mesenchymal transition and cancer stem cell-like phenotypes in CRC cells

Abstract Background: Epithelial-to-mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT has been shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcriptional factor that mediates EMT in a number of tumor types. Its aberrant expression has been found in various cancers including colorectal cancer (CRC). The role of Snail in mediating EMT and CSC function in CRC has not yet been fully elucidated. Methods: The human CRC cell lines HT29 and KM12L4 were transduced with a retrovirus containing the pBabe-puro-Snail expression construct or the empty vector. The Snail expressing cells and controls were selected with the medium containing 2µg/ml puromycin. Changes in cell proliferation and chemo-sensitivity were determined by MTT assay. Migration and invasion were determined using in vitro wound healing assay and modified Boyden Chamber assays, respectively. EMT makers (E-cadherin, vimentin) and the putative CSC marker CD133 was analyzed by Western blotting. ALDH1 enzyme activity was analyzed by the Aldefluor assay. Results: Overexpression of Snail in CRC cells induced an EMT phenotype with elevated expression of vimentin and reduced expression of E-cadherin. Snail-induced EMT did not alter cell proliferation but showed enhanced cell motility, in which Snail-expressing cells were 5- to 6-fold more migratory than the control cells. Snail also promoted the CSC-like phenotype demonstrated by increased expression of the CSC markers CD133. The aldefluor positive cell population in the Snail-expressing cells was increased ∼4 fold. Moreover, the Snail-expressing CRC cells showed increased chemoresistance to 5FU by 50%. Conclusion. High level of Snail expression confers the EMT and CSC phenotype in CRC cells, which enhanced cancer cell invasion and chemoresistance. Our data suggest that Snail may be a potential therapeutic target in metastatic colorectal cancer. 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 2300.

Crystal structure of eta-crystallin: adaptation of a class 1 aldehyde dehydrogenase for a new role in the eye lens.

Eta-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrews, members of an ancient order of mammals. While it retains some activity toward retinal, which is oxidized to retinoic acid, the protein has acquired a number of specific sequence changes that have presumably been selected to enhance the lens role. The crystal structure of eta-crystallin, in common with class 1 and 2 ALDHs, is a dimer of dimers. It has a better-defined NAD binding site than those of related mammalian ALDH1 enzymes with the cofactor bound in the "hydride transfer" position in all four monomers with small differences about the dimer dyads. Although the active site is well conserved, the substrate-binding site is larger in eta-crystallin, and there are some mutations to the substrate access tunnel that might affect binding or release of substrate and product. It is possible that eta-crystallin has lost flexibility to improve its role in the lens. Enhanced binding of cofactor could enable it to act as a UV/blue light filter in the lens, improving visual acuity. The structure not only gives a view of a "natural mutant" of ALDH1 illustrating the adaptive conflict that can arise in multifunctional proteins, but also provides a well-ordered NAD binding site structure for this class of enzymes with important roles in development and health.

Role of the C-terminal tail on the quaternary structure of aldehyde dehydrogenases

To assess the importance of the C-terminal tail in the structure of aldehyde dehydrogenases (ALDH), mutants of tetrameric ALDH1 were generated by adding a tail of 5 amino acids (ALDH1-5aa) or the tail from the class 3 enzyme. A mutant of dimeric ALDH3 was made, where 17 amino acids from the C-terminus were deleted to generate ALDH3ΔTail. The expression and solubility of the ALDH1 mutants was slightly lower than the wild type. Expression of ALDH3ΔTail mutant was similar to wild type, but the solubility was only about 30%. The activity of ALDH1-5aa mutant was 30%, while ALDH1-H3Tail mutant was 60% active, compared to the wild type. The activity of the class 3 mutant was similar to the activity of the parent ALDH3 enzyme. Analysis of stability against temperature demonstrated that ALDH1-5aa was more stable than ALDH1 wild type, while the ALDH1-H3Tail mutant was considerably less stable than ALDH1, showing a stability similar to ALDH3. However, native gel and size exclusion analysis, showed no changes in the oligomerization state of these mutants. ALDH3ΔTail mutant was more stable than wild type; the stability against temperature was similar to ALDH1. The ALDH3ΔTail mutant showed an elution similar to that of ALDH1 from the size exclusion column, indicating that it was possibly a tetramer. These results show that the tail in ALDH3, is involved in the determination of the quaternary structure of ALDH3, but has no effect on the ALDH1 enzyme; the absence of the C-terminal tail is not the only factor participating in holding the dimers together. Thus, the interaction between single residues, or interactions with the N-terminal region might be more important for maintaining stable tetramers.

Coding region-specific destabilization of mRNA transcripts attenuates expression from retroviral vectors containing class 1 aldehyde dehydrogenase cDNAs.

Class 1 aldehyde dehydrogenases (ALDH-1) function as drug resistance gene products by catalyzing the irreversible conversion of aldophosphamide, an active metabolite of cyclophosphamide, to an inert compound. Because the dose-limiting toxicity of cyclophosphamide is myelosuppression, retrovirus-mediated transfer of ALDH-1 to bone marrow cells has been proposed as a protective strategy. Here we show that expression of ALDH-1 vectors was problematic due to low levels of ALDH-1 mRNA accumulation. A number of vectors containing several different ALDH-1 cDNAs were introduced into a variety of different cell lines either by transfection or transduction. Detectable ALDH-1 protein and enzyme activity was only seen in one transfected cell clone. Cells transduced with ALDH-1 retroviral vectors had no detectable protein expression and very low levels of ALDH-1 mRNA. Analogous vectors containing other drug resistance cDNAs led to much higher levels of steady-state mRNA. The mRNA half-life from ALDH-1 vectors was less than 2 hr suggesting that vector-derived mRNAs were destabilized by ALDH-1 coding sequences. These results suggest that methods which increase the stability of ALDH-1 mRNAs will be important for increased drug resistance in retrovirally transduced hematopoietic cells.

Purification and partial characterization of bovine kidney aldehyde dehydrogenase able to oxidize retinal to retinoic acid.

A NAD-dependent enzyme that catalyzes the oxidation of retinal to retinoic acid has been purified to homogeneity from bovine kidney. The procedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chromatography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH1 and ALDH2. The enzymes ALDH1 and ALDH2 were purified about 114- and 65-fold, respectively. Gel filtration chromatography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the purified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate--polyacrylamide gel electrophorsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity oxidizing a wide variety of aliphatic and aromatic aldehydes. The ALDH1 enzyme had a pI of 7.45 and exhibited a low Km (6.37 microM) for retinal, while the ALDH2 enzyme was found to have very low Km for acetaldehyde (0.98 microM). Based on its kinetic properties, it is suggested that the ALDH1 enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.