Expression Of ALDH3 Research Articles

Optimal NaCl Medium Enhances Squalene Accumulation in Thraustochytrium sp. ATCC 26185 and Influences the Expression Levels of Key Metabolic Genes.

Squalene, a natural lipid of the terpenoid family, is well-recognized for its roles in regulating cholesterol metabolism, preventing tumor development, and improving immunity. For large-scale squalene production, the unicellular marine protists—thraustochytrids—have shown great potential. However, the growth of thraustochytrids is known to be affected by salt stress, which can eventually influence the squalene content. Here, we study the effects of an optimal concentration of NaCl on the squalene content and transcriptome of Thraustochytrium sp. ATCC 26185. Under the optimal culture conditions (glucose, 30 g/L; yeast extract, 2.5 g/L; and NaCl, 5 g/L; 28°C), the strain yielded 67.7 mg squalene/g cell dry weight, which was significantly greater than that (5.37 mg/g) under the unoptimized conditions. NaCl was determined as the most significant (R = 135.24) factor for squalene production among glucose, yeast extract, and NaCl. Further comparative transcriptomics between the ATCC 26185 culture with and without NaCl addition revealed that NaCl (5 g/L) influences the expression of certain key metabolic genes, namely, IDI, FAS-a, FAS-b, ALDH3, GS, and NDUFS4. The differential expression of these genes possibly influenced the acetyl-CoA and glutamate metabolism and resulted in an increased squalene production. Through the integration of bioprocess technology and transcriptomics, this report provides the first evidence of the possible mechanisms underscoring increased squalene production by NaCl.

Differential activation of placental nutrient sensor pathways AMPK, insulin/IGF-1, and mTOR in SGA, AGA and LGA newborns from healthy mothers

Intrauterine growth restriction (IUGR) is a complication of pregnancy that has both short- and long-term sequelae for affected mothers and offspring. The pathophysiology of disease stems from poor nutrient and oxygen provision to the fetus, resulting in increased oxidative stress within the placenta. As the milieu within the local microenvironment alters macrophage differentiation, we hypothesized that macrophage plasticity may be altered in placentas associated with IUGR, and that macrophages would show hallmarks of lipid peroxidation including altered aldehyde metabolism.In human placentas taken from normal pregnancies resulting in appropriate-for-gestational-age (AGA) newborns and placentas associated with IUGR, placental macrophages were evaluated by immunohistochemistry and shown in IUGR to resemble pro-inflammatory activated M1-type macrophages. To link oxidative stress to macrophages, the expression of aldehyde dehydrogenase (ALDHs) isozymes ALDH1, ALDH2, and ALDH3 was assessed.All three isozymes displayed preferential staining for distinct cellular populations within the term human placenta. ALDH1 and ALDH2 were strongly expressed in placental Hofbauer and decidual stromal cells. ALDH3, in contrast, was present in extravillous trophoblasts. Comparing AGA and IUGR-associated placentas, ALDH1 and ALDH2 trended to have greater expression in macrophage populations but lower expression in decidual cell populations in IUGR-associated placentas. ALDH3 had higher expression in IUGR-associated placentas but localized specifically to extravillous trophoblast populations.Therefore, we speculate that specific ALDH isozymes have cell-specific functions related to differentiation, inflammation, or oxidative stress responses that are altered in IUGR-associated term human placentas. This family of isozymes may be a novel method to identify human placentas affected by placental insufficiency/IUGR.

Aldehyde dehydrogenase isoforms and inflammatory cell populations are differentially expressed in term human placentas affected by intrauterine growth restriction

ObjectiveIntrauterine growth restriction (IUGR) is a complication of pregnancy that has both short- and long-term sequelae for affected mothers and offspring. The pathophysiology of disease stems from poor nutrient and oxygen provision to the fetus, resulting in increased oxidative stress within the placenta. As the milieu within the local microenvironment alters macrophage differentiation, we hypothesized that macrophage plasticity may be altered in placentas associated with IUGR, and that macrophages would show hallmarks of lipid peroxidation including altered aldehyde metabolism. MethodsIn human placentas taken from normal pregnancies resulting in appropriate-for-gestational-age (AGA) newborns and placentas associated with IUGR, placental macrophages were evaluated by immunohistochemistry and shown in IUGR to resemble pro-inflammatory activated M1-type macrophages. To link oxidative stress to macrophages, the expression of aldehyde dehydrogenase (ALDHs) isozymes ALDH1, ALDH2, and ALDH3 was assessed. ResultsAll three isozymes displayed preferential staining for distinct cellular populations within the term human placenta. ALDH1 and ALDH2 were strongly expressed in placental Hofbauer and decidual stromal cells. ALDH3, in contrast, was present in extravillous trophoblasts. Comparing AGA and IUGR-associated placentas, ALDH1 and ALDH2 trended to have greater expression in macrophage populations but lower expression in decidual cell populations in IUGR-associated placentas. ALDH3 had higher expression in IUGR-associated placentas but localized specifically to extravillous trophoblast populations. ConclusionTherefore, we speculate that specific ALDH isozymes have cell-specific functions related to differentiation, inflammation, or oxidative stress responses that are altered in IUGR-associated term human placentas. This family of isozymes may be a novel method to identify human placentas affected by placental insufficiency/IUGR.

PO-034 Inhibition of lung carcinoma growth and pulmonary metastasis with DIMATE as single agent and in combination with cisplatin

IntroductionAberrant cell proliferation in NSCLC causes an aberrant redox state that leads to the production of toxic reactive species and aldehydes. To keep oxidative stress until a threshold, above which oxidative damage can be detrimental to cell viability, cancer cells must actively upregulate multiple antioxidant systems. The aldehyde dehydrogenase (ALDH) gene superfamily encodes enzymes that are critical for certain life processes and detoxification of numerous endogenous and exogenous aldehyde substrates, including pharmaceuticals and environmental pollutants.Material and methodsIn this study, a meta-analysis was conducted based on multiple microarray data from The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) repositories, spanning lung adenocarcinoma and lung squamous cell carcinoma datasets. Twenty-six NSCLC cell lines with different oncogenic driver alterations were used to analyse the molecular and cellular consequences of ALDHs inhibition. Preclinical studies with orthotopic xenografts of NSCLC and lung metastatic breast cancer were performed to evaluate the effect of the inhibition of ALDH class 1 and class3 activity on tumour growth.Results and discussionsHere, we found that increased expression of aldehyde dehydrogenase isoenzymes ALDH1A1, ALDH1A3 and ALDH3A1 in human NSCLC tumours has a strong impact on chemotherapy resistance and patient overall survival, correlating with poor prognosis. We showed that inhibition of class 1 and class 3 ALDH activity with the novel irreversible ALDH1/3 inhibitor DIMATE suppresses tumour growth in orthotopic human lung cancer xenograft model and inhibits lung metastasis of human breast cancer in athymic nude mice. Accumulation of HNE-protein adducts and depletion of intracellular GSH are main responsible of DIMATE-induced cell death. Consistently, we found that alteration of tumour redox balance further enhances sensitivity of NSCLC cells to DIMATE. Finally, we demonstrate that the combination of DIMATE with Cisplatin-induced ROS generation in an orthotopic lung cancer model can significantly enhance the cytotoxicity of these two drugs in NSCLC cells with a synergistic promotion of cell death and marginal systemic toxicity in animals.ConclusionTargeting the detoxification machinery of ALDHs constitutes a novel therapeutic avenue for NSCLC. Patients with increased expression of ALDH1 or ALDH3 might greatly benefit from a combination therapy that include drugs interfering with the activity of these enzymes to overcome patient-specific drug resistance.

Template Curvature Influences Cell Alignment to Create Improved Human Corneal Tissue Equivalents.

To accurately create corneal stromal equivalents with native-like structure and composition, a new biofunctionalized, curved template is developed that allows the precise orientation of cells and of their extracellular matrix. This template is the first demonstration that curvature alone is sufficient to induce the alignment of human corneal stromal cells, which in turn are able to biofabricate stromal tissue equivalents with cornea-like shape and composition. Specifically, tissues self-released from curved templates show a highly organized nanostructure, comprised of aligned collagen fibrils, significantly higher expression of corneal stroma-characteristic markers keratocan, lumican, decorin, ALDH3, and CHST6 (p = 0.012, 0.033, 0.029, 0.003, and 0.02, respectively), as well as significantly higher elastic modulus (p = 0.0001) compared with their planar counterparts. Moreover, curved tissues are shown to support the growth, stratification, and differentiation of human corneal epithelial cells in vitro, while maintaining their structural integrity and shape without any supporting carriers, scaffolds, or crosslinking agents. Together, these results demonstrate that corneal stromal cells can align and create highly organized, purposeful tissues by the influence of substrate curvature alone, and without the need of additional topographical cues. These findings can be important to further understand the mechanisms of corneal biosynthesis both in vitro and in vivo.

FBXL12-Mediated Degradation of ALDH3 is Essential for Trophoblast Differentiation During Placental Development.

How stem cells maintain their stemness or initiate exit from the stem cell state for differentiation remains largely unknown. Aldehyde dehydrogenase (ALDH) activity is a hallmark of stem cells-including embryonic, adult tissue, and cancer stem cells-and is essential for their maintenance. The mechanisms by which ALDH activity is regulated in stem cells have remained poorly understood, however. We now show that the ubiquitin-dependent degradation of ALDH3 mediated by FBXL12 (F box and leucine-rich repeat protein 12) is essential for execution of the differentiation program of trophoblast stem cells (TSCs). FBXL12 is present only in eutherian mammals, and its expression is largely restricted to the placenta during mouse embryogenesis. FBXL12 was found to interact specifically with members of the ALDH3 family and to mediate their polyubiquitylation. Most mice deficient in FBXL12 died during the embryonic or perinatal period probably as a result of abnormal development of the placenta, characterized by impaired formation of the junctional zone. ALDH3 accumulated in the FBXL12-deficient placenta, and forced expression of ALDH3 in wild-type TSCs phenocopied the differentiation defect of FBXL12-deficient TSCs. Conversely, inhibition of ALDH3 activity by gossypol rescued the phenotype of FBXL12 deficiency. Our results suggest that FBXL12 plays a key role in the downregulation of ALDH3 activity in TSCs and thereby initiates trophoblast differentiation during placental development.

A Novel Aldehyde Dehydrogenase-3 Activator Leads to Adult Salivary Stem Cell EnrichmentIn Vivo

To assess aldehyde dehydrogenase (ALDH) expression in adult human and murine submandibular gland (SMG) stem cells and to determine the effect of ALDH3 activation in SMG stem cell enrichment. Adult human and murine SMG stem cells were selected by cell surface markers (CD34 for human and c-Kit for mouse) and characterized for various other stem cell surface markers by flow cytometry and ALDH isozymes expression by quantitative reverse transcriptase PCR. Sphere formation and bromodeoxyuridine (BrdUrd) incorporation assays were used on selected cells to confirm their renewal capacity and three-dimensional (3D) collagen matrix culture was applied to observe differentiation. To determine whether ALDH3 activation would increase stem cell yield, adult mice were infused with a novel ALDH3 activator (Alda-89) or with vehicle followed by quantification of c-Kit(+)/CD90(+) SMG stem cells and BrdUrd(+) salispheres. More than 99% of CD34(+) huSMG stem cells stained positive for c-Kit, CD90 and 70% colocalized with CD44, Nestin. Similarly, 73.8% c-Kit(+) mSMG stem cells colocalized with Sca-1, whereas 80.7% with CD90. Functionally, these cells formed BrdUrd(+) salispheres, which differentiated into acinar- and ductal-like structures when cultured in 3D collagen. Both adult human and murine SMG stem cells showed higher expression of ALDH3 than in their non-stem cells and 84% of these cells have measurable ALDH1 activity. Alda-89 infusion in adult mice significantly increased c-Kit(+)/CD90(+) SMG population and BrdUrd(+) sphere formation compared with control. This is the first study to characterize expression of different ALDH isozymes in SMG stem cells. In vivo activation of ALDH3 can increase SMG stem cell yield, thus providing a novel means for SMG stem cell enrichment for future stem cell therapy.

Aldehyde dehydrogenase 3 gene regulation: studies on constitutive and hypoxia-modulated expression

We have previously shown that expression of the Class 3 aldehyde dehydrogenase gene (ALDH3) is abrogated by hypoxia. This phenomenon occurs in rat hepatoma systems in which ALDH3 expression is xenobiotic-inducible as well as in rat primary corneal epithelial cells that exhibit high constitutive ALDH3 expression. We have begun to test various segments of the ALDH3 5′ flanking region for elements that may mediate this effect using CAT reporter gene constructs. In addition, although the involvement of the Ah receptor nuclear translocator (ARNT) in xenobiotic induction of ALDH3 is well established, the role of ARNT in constitutive ALDH3 expression is not clear. Moreover, ARNT is also a component of the hypoxia inducible factor-1 (HIF-1) bipartite transcription factor complex that mediates hypoxic induction of a variety of genes. Concomitant activation of the xenobiotic and hypoxia pathways results in cross-talk and functional interference. It has been hypothesized that this interference is due to limiting levels of ARNT. To examine if ARNT levels are limiting during hypoxic and xenobiotic induction in the context of ALDH3 expression and to examine possible roles of ARNT in constitutive expression of ALDH3 in corneal epithelial cells we co-transfected rat corneal epithelial cells and H4-II-EC3 rat hepatoma cells with ALDH3 5′ UTR-CAT reporter genes and expression vectors containing either wild type or dominant negative forms of ARNT. Our results indicate that during hypoxia and xenobiotic induction of ALDH3 in H4-II-EC3 cells ARNT is not the limiting transcription factor. Further, neither wild type nor dominant negative ARNT had effects on constitutive ALDH3 expression in corneal epithelial cells.

Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells

Aldehyde dehydrogenases (ALDHs) are a superfamily of several isoenzymes widely expressed in bacteria, yeast, plant and animals. Three major classes of ALDHs have been traditionally identified, classes 1, 2 and 3. Both exogenous and endogenous aldehydes, including aldehydes derived from lipid peroxidation, are oxidized by the ALDH superfamily. Several changes in ALDH isoenzyme expression take place in hepatoma cells, in particular cytosolic class 3 ALDH (ALDH3), not expressed in normal hepatocytes, appears and increases with the degree of deviation. It has been demonstrated that cytosolic ALDH3 is important in determining the resistance of tumor cells to antitumor drugs, such as cyclophosphamide. Moreover, hepatoma-associated ALDH3 seems to be important in metabolizing aldehydes derived from lipid peroxidation, and in particular the cytostatic aldehyde 4-hydroxynonenal (4-HNE). We demonstrated previously that restoring endogenous lipid peroxidation in hepatoma cells by enriching them with arachidonic acid causes a decrease of mRNA, protein and enzyme activity of ALDH3 and that this decrease reduces cell growth and/or causes cell death, depending on basal class 3 ALDH activity. To confirm the correlation between inhibition of class 3 ALDH and reduction of cell proliferation, we exposed hepatoma cells to antisense oligonucleotides (ODNs) against ALDH3. In JM2 hepatoma cell line, with high ALDH3 activity, the exposure to antisense ODNs significantly decreases mRNA and enzyme activity (90%). At the same time, cell growth was reduced by about 70%. The results confirm that in hepatoma cells ALDH3 expression is closely related with cell growth, and that its inhibition is important in reducing the proliferation of hepatoma cells overexpressing ALDH3.

Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids.

The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene.

Expression of human aldehyde dehydrogenase-3 associated with hepatocellular carcinoma: promoter regions and nuclear protein factors related to the expression.

The human ALDH3 gene is constitutively expressed in stomach, lung, esophagus, and cornea, but hardly detectable in the normal liver. However, it is highly activated in the hepatocellular carcinoma tissues from approximately 50% of patients. The nuclear DNA binding factors exist in both ALDH3-positive cancerous liver and ALDH3-positive HepG2 cells, but not in ALDH3-negative Hep3B cells and normal liver tissues. South-western blot hybridization showed the existence of two nuclear-binding protein components, 35 and 14 kDa, in ALDH3-positive cancerous liver tissues. These two DNA binding proteins were not found in normal stomach tissues and stomach carcinoma KATO III cells. DNaseI footprint analysis identified two protective regions within the ALDH3 promoter. The first protected region has one putative CCAAT-box and one putative Sp1-site. The second protected region contains a putative HiNF-A binding sequence. These findings suggest that a high level of expression of ALDH3 in cancerous liver tissues resulted from the expression or activation of at least two nuclear proteins reacting to the ALDH3 promoter region.

CAMP-dependent Negative Regulation of Rat Aldehyde Dehydrogenase Class 3 Gene Expression

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.

Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter.

The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and gel mobility shift assays indicate that Sp1-like proteins interact with two proximal DNase I footprinted sites to confer strong promoter activity. Two distal DNase I footprinted sites are found within a region that inhibits rat ALDH-3 promoter activity. This negative region is bound by NF1-like proteins and/or unique proteins. This 1 kb 5' flanking region of rat ALDH-3 may act constitutively in many cell types. In contrast with other Ah receptor regulated genes, no DNA elements or transcription factors acting within this region appear to be involved in regulating xenobiotic-inducible expression of rat ALDH-3.

Hepatocyte expression of tumor associated aldehyde dehydrogenase (ALDH-3) and p21 Ras following diethylnitrosamine (DEN) initiation and chronic exposure to di(2-ethylhexyl)phthalate (DHEP).

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP) either promote or inhibit rat liver tumorigenesis depending on the carcinogenesis protocol. In this study, we examined the expression of two histochemical markers, the tumor associated isozyme of aldehyde dehydrogenase (ALDH-3) and the oncoprotein p21 Ras, in the livers of male F344 rats. The rats were initiated with DEN and further treated with either DEHP (a known inhibitor of hepatocarcinogenesis), phenobarbital (PB, a known promoter of hepatocarcinogenesis), or a combination of DEHP and PB. The studies were designed to examine the expression of these markers in both normal appearing liver and hepatic hyperplastic and neoplastic lesions and to correlate the early expression of the markers at 26 weeks in the normal appearing liver to later tumor incidence at 52 weeks. The expression of each marker was detected by immunohistochemical methods on formalin-fixed paraffin embedded sections of normal appearing liver or liver lesions. We found that ALDH-3 and p21 expression were significantly enhanced in rats receiving PB after DEN initiation at 26 weeks and that the incidence of hepatocellular carcinomas was likewise increased compared to control or DEN only treated animals. DEN initiation followed by a combination of PB and either 0.1 or 0.5% DEHP significantly reduced ALDH-3 but not p21 Ras expression at 26 weeks compared to DEN plus PB only. These treatment regimens also reduced the incidence of hepatocellular carcinomas at 52 weeks. DEN followed by any of the three doses of DEHP without PB resulted in ALDH-3 expression similar to DEN alone. However, p21 Ras expression was significantly increased after these treatments. For all treatment groups, both the early (26 weeks) expression of p21 Ras and ALDH-3 correlated with hepatocellular carcinoma incidence at 52 weeks. However, the correlation between hepatocellular carcinoma and ALDH-3 expression was better than p21 Ras or the other markers we have studied. We concluded that ALDH-3 expression is significantly downregulated after DEHP treatment, and that expression of the isozyme correlated with later hepatocarcinoma incidence and may indicate a significant relationship between ALDH-3 expression and hepatocarcinogenesis during DEHP treatment.

Comparison of expression of aldehyde dehydrogenase 3 and CYP1A1 in dominant and recessive aryl hydrocarbon hydroxylase-deficient mutant mouse hepatoma cells

The mouse hepatoma cell line Hepa-1 is inducible by 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) for both CYP1A1 (aryl hydrocarbon hydroxylase, AHH) and class 3 aldehyde dehydrogenase (ALDH3) enzymes. To test the hypothesis of a common regulatory mechanism, several AHH deficient mutants of Hepa-1 were studied for their ALDH3 activities and specific mRNA levels before and after TCDD treatment. The recessive (with respect to the wild-type Hepa-1) mutants have defects in Cypla-1 structural gene (mutant c1) or in the Ah (aryl hydrocarbon) receptor (mutants c2 and c6 with decreased levels of Ah receptor; mutant c4 defective in the DNA binding of the Ah receptor). The results with these mutants suggested that Ah receptor nuclear translocator protein, ARNT, is needed for ALDH3 expression. Two dominant mutants, one of which is characterized by preventing the binding of the Ah receptor complex to DNA, were also studied. Surprisingly, these mutants possessed elevated levels of ALDH3 mRNA and enzyme activities which were also inducible by TCDD. The binding of Ah receptor-ligand complex to DNA was thus not needed for the expression of ALDH3. A dominant repressor for Cypla-1 gene transcription did not prevent the derepression or induction of ALDH3. The results thus suggest that Aldh-3 gene is regulated by a mechanism independent of the Ah receptor.

Structure of the 5′ Flanking Region of Class 3 Aldehyde Dehydrogenase in the Rat

Class 3 aldehyde dehydrogenase (ALDH-3) is induced by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and during chemical carcinogenesis. These inductions as well as the basal expression of ALDH-3 vary significantly in different organs. In order to identify DNA elements controlling ALDH-3 expression, we have cloned and analyzed approximately 5.5 kb of the 5′ flanking region of the ALDH-3 gene. Deletion analysis showed that the 5′ flanking region contains at least three functional domains: a strong promoter proximal to the transcription start site, inhibitory regions upstream of the promoter, and TCDD-responsive enhancers. The TCDD-responsive enhancers in the ALDH-3 gene were functionally similar to xenobiotic responsive elements in the cytochrome P450IA1 gene. These results indicate that transcription of the ALDH-3 gene is controlled by cooperation of at least three functional domains.

Biochemical genetic analysis of human and rodent aldehyde dehydrogenase (ALDH)

ALDH isozymes have been characterized in terms of substrate and coenzyme specificity, heat stability, tissue distribution and electrophoretic properties. The activity of the isozymes has also been examined in rodent-human somatic cell hybrids in order to map the structural genes to specific chromosomes and to study the control of gene expression. One isozyme, designated ALDH3, which is very active against benzaldehyde, was found to show variable expression in hybrids made between rat hepatoma cells and human fibroblasts or fetal liver. Segregation analysis of these hybrids indicates that the structural locus for human ALDH3 may be on chromosome 17. The expression of rodent ALDH3 in these hybrids was extremely variable and not correlated with the appearance of the human enzyme. In hybrids expressing human and rodent ALDH3 no heteromeric isozymes were observed. The human “cytosolic” ALDH1 and “mitochondrial” ALDH2 isozymes did not appear to be expressed in any of the somatic cell hybrids examined.