Relationship between hyaluronan production and metastatic potential of mouse mammary carcinoma cells.
(17/1925)
To investigate the roles of hyaluronan produced by cancer cells in cancer metastasis, the metastatic potential of the highly metastatic mouse mammary carcinoma FM3A HA1 cell line was compared with those of hyaluronan-deficient mutant cells. Five different mutant clones showed markedly reduced hyaluronan production and lacked the ability to form hyaluronan-rich pericellular coats. These mutant clones displayed significant decreases in metastatic ability compared with the parental cells after i.v. injection into syngeneic mice. These results suggested that the decreased hyaluronan production caused not only the lack of matrix formation but also decreased metastatic potential of the cancer cells. Expression of mouse hyaluronan synthase 1 (HAS1) by transfection into HAS- cells defective in hyaluronan synthase activity rescued hyaluronan matrix formation as well as hyaluronan production. Lung metastasis after i.v. injection of HAS1 transfectants was also recovered significantly. The results provide direct evidence for the involvement of hyaluronan in cancer metastasis. (+info)
Effect of leptin on cytochrome P-450, conjugation, and antioxidant enzymes in the ob/ob mouse.
(18/1925)
Leptin is a hormone that is secreted by adipocytes and regulates body weight through its effect on satiety and energy metabolism. The ob/ob mouse is deficient in this protein and is characterized by obesity and other metabolic disorders. This study investigated the alterations of several hepatic cytochrome P-450 (CYP), conjugation, and antioxidant enzymes in lean and ob/ob mice and the role leptin plays in the modulation of these enzymes. Lean and ob/ob male mice were injected with leptin (100 microg) or PBS for 15 days. Liver microsomes from ob/ob mice, when compared with lean controls, displayed significantly reduced chlorzoxazone 6-hydroxylation activity (27%); however, 7alpha- and 16alpha- testosterone hydroxylation and pentoxyresorufin O-dealkylation activities were significantly higher (47%, 22%, and 39%, respectively). Leptin administration corrected alterations seen with all P-450 activities. Dealkylation of ethoxyresorufin and omega-hydroxylation of lauric acid activities from ob/ob and lean mice were not statistically different; however, leptin exposure significantly increased ethoxyresorufin activity in lean mice (14%) and decreased the activity in ob/ob mice (36%). UDP-glucuronosyl-transferase and glutathione S-transferase activities were not altered. The antioxidant enzymes, catalase (11%) and glutathione peroxidase (26%), as well as glutathione reductase (17%), were lower in the ob/ob mice and leptin treatment corrected these alterations. The results of this study demonstrate alterations in constitutive expression of CYP2B, CYP2E, CYP2A, catalase, glutathione peroxidase, and glutathione reductase in ob/ob mice that were restored to lean control values following leptin treatment. Additionally, CYP3A activity was increased following leptin treatment in ob/ob mice. The mechanism for the observed alterations may be due to direct leptin effects or via indirect alterations in insulin, corticosterone, and/or growth hormone. (+info)
Glucuronidation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuronosyltransferases: identification of specific UGT1A family isoforms involved.
(19/1925)
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a heterocyclic aromatic amine found in cooked meats and dietary exposure to PhIP has been implicated in the etiology of colon cancer in humans. PhIP, along with other heterocyclic aromatic amines, requires metabolic activation to exhibit genotoxic effects. PhIP is initially oxidized by the activity of cytochrome P4501A2 to produce 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), a reaction occurring primarily in the liver. Whereas subsequent biotransformation of N-OH-PhIP via acetylation or sulfation can produce reactive electrophiles that readily bind to DNA, N-glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), functions as a detoxification mechanism. Although hepatic glucuronidation of N-OH-PhIP has been well characterized, the extrahepatic metabolism of this compound is poorly understood. Studies in our laboratory now indicate that the intestinal tract, and particularly the colon, is a significant site of glucuronidation of N-OH-PhIP. When assays were performed with microsomes prepared from the mucosa of the intestinal tract, it was determined that glucuronidation of N-OH-PhIP occurs throughout the intestinal tract, with activity approximately three times higher in the colon as that found in the upper intestine. Glucuronidation rates from colon microsomes showed considerable interindividual variability and incubation with N-OH-PhIP yielded two glucuronides. HPLC analysis showed that the predominant product formed is the N-OH-PhIP-N2-glucuronide, while the N3-glucuronide accounts for <10% of the total glucuronidation product. These rates approach the rates found in human liver microsomes, demonstrating the significance of extrahepatic metabolism of this food-borne carcinogen. Subsequent assays with human recombinant UGTs demonstrated that at least four human UGT isoforms, all from the UGT1A subfamily, are capable of catalyzing the biotransformation of N-OH-PhIP. Members of the UGT2B family available for this study did not conjugate N-OH-PhIP, although immunoinhibition studies in human liver microsomes strongly suggest the involvement of a UGT2B isoform(s) in this organ. (+info)
Cloning and expression of a novel galactoside beta1, 3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope.
(20/1925)
We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo. (+info)
Cloning and characterization of a novel human olfactory UDP-glucuronosyltransferase.
(21/1925)
Xenobiotic metabolizing enzymes in the olfactory epithelium have been suggested to catalyse inactivation and facilitate elimination of odorants. We report here the molecular cloning and functional characterization of a human olfactory UDP-glucuronosyltransferase (UGT). The cloned protein is composed of 527 amino acids with an identity of 87% with a rat olfactory UGT and of 43-62% with other human UGT isoforms. Based on the sequence homology, it has been designated hUGT2A1. The gene was mapped to chromosome 4q13 by fluorescence in situ hybridization. The expression appeared to be specific for the olfactory tissue. The substrate specificity of this isoform was assessed using Chinese hamster V79 cells stably transfected with the isolated cDNA. The expressed enzyme showed a broad substrate spectrum including a range of phenolic compounds as well as aliphatic and monoterpenoid alcohols, among them many odorants. Furthermore, some steroids, especially androgens, some drugs and carcinogens were conjugated. The results support a role of the enzyme in olfactory perception and in protection of the neural system against airborne hazardous chemicals. (+info)
Human and rat liver UDP-glucuronosyltransferases are targets of ketoprofen acylglucuronide.
(22/1925)
Acylglucuronides formed from carboxylic acids by UDP-glucuronosyltransferases (UGTs) are electrophilic metabolites able to covalently bind proteins. In this study, we demonstrate the reactivity of the acylglucuronide from the nonsteroidal anti-inflammatory drug, ketoprofen, toward human and rat liver UGTs. Ketoprofen acylglucuronide irreversibly inhibited the glucuronidation of 1-naphthol and 2-naphthol catalyzed by human liver microsomes or by the recombinant rat liver isoform, UGT2B1, which is the main isoform involved in the glucuronidation of the drug. A decrease of about 35% in the glucuronidation of 2-naphthol was observed when ketoprofen acylglucuronide was produced in situ in cultured V79 cells expressing UGT2B1. Inhibition was always associated with the formation of microsomal protein-ketoprofen adducts. The presence of these covalent adducts within the endoplasmic reticulum of cells expressing UGT2B1 was demonstrated following addition of ketoprofen to culture medium by immunofluorescence microscopy with antiketoprofen antibodies. Immunoblots of liver microsomes incubated with ketoprofen acylglucuronide and probed with antiketoprofen antibodies revealed the presence of several protein adducts; among those was a major immunoreactive protein at 56 kDa, in the range of the apparent molecular mass of UGTs. The adduct formation partially prevented the photoincorporation of the UDP-glucuronic acid (UDP-GlcUA) analog, [beta-32P]5N3UDP-GlcUA, on the UGTs, suggesting that ketoprofen glucuronide covalently reacted with the UDP-GlcUA binding domain. Finally, UGT purification from rat liver microsomes incubated with ketoprofen glucuronide led to the isolation of UGT adducts recognized by both anti-UGT and antiketoprofen antibodies, providing strong evidence that UGTs are targets of this metabolite. (+info)
Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.
(23/1925)
In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes. (+info)
Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum.
(24/1925)
UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process. (+info)