Androgen regulated expression of the alpha 2u-globulin gene in pancreatic hepatocytes of rat.

Under a copper-deficient regimen, pancreatic cells in the adult rat can be found to undergo differentiation into hepatocytes. Pancreatic hepatocytes induced in male and female rats were examined for the expression of the androgen-inducible hepatic protein, alpha 2u-globulin. Alpha 2u-Globulin protein was demonstrable by immunoperoxidase method in all the pancreatic hepatocytes of male rats. Northern blot analysis confirmed the presence of 1.3 kb alpha 2u-globulin mRNA transcript in the pancreas of male rats with hepatocytes. Orchiectomy resulted in marked decrease of alpha 2u-globulin protein and its mRNA. Administration of dihydrotestosterone to castrated rats resulted in increased levels of alpha 2u-globulin mRNA and the amount of alpha 2u-globulin protein in the pancreatic hepatocytes. Unlike normal males, in intact and ovariectomized females alpha 2u-globulin was not detectable in pancreatic hepatocytes. These results indicate that similar to hepatic parenchymal cells pancreatic hepatocytes synthesize alpha 2u-globulin under androgenic regulation. Furthermore, unlike in liver where it is expressed predominantly in perivenular and midlobular hepatocytes, there is no localized difference in the expression of this gene in the transdifferentiated pancreatic hepatocytes.

Differential expression of neural-cadherin in pulmonary epithelial tumours.

AIMS: Neural (N)-cadherin belongs to a group of transmembrane molecules with a crucial role in tissue morphogenesis and maintenance of an epithelioid phenotype and increased N-cadherin expression is implicated in tumour progression and dedifferentiation. The aim was to determine whether evaluation of N-cadherin in pulmonary tumours might assist in identifying lesions with more aggressive potential. METHODS AND RESULTS: One hundred and fifty-five pulmonary lesions were analysed for N-cadherin expression using immunohistochemistry, including neuroendocrine hyperplasia (n = 3), typical carcinoid (n = 59), atypical carcinoid (n = 12), small cell lung carcinoma (n = 11), large cell neuroendocrine carcinoma (n = 12), adenocarcinoma (n = 35) and squamous cell carcinoma (n = 23). Lymph node status was correlated with immunohistochemical expression. N-cadherin expression was demonstrated in all cases of neuroendocrine hyperplasia, 96% of typical carcinoids, 83% of atypical carcinoids, 63% of the small cell lung carcinomas and 32% of large cell neuroendocrine carcinomas. Over 90% of the adenocarcinomas and 100% of the squamous cell carcinomas were negative. Increased N-cadherin expression in typical carcinoids was associated with negative lymph node status (P < 0.001). DISCUSSION: N-cadherin is differentially expressed in pulmonary tumours and is predominantly observed in neuroendocrine lung lesions with high expression in typical and atypical pulmonary carcinoids. The level of expression of N-cadherin between types of lung tumours does not appear to indicate malignant potential or aggressive behaviour.

Hepatocarcinogenicity of dehydroepiandrosterone in the rat.

Dehydroepiandrosterone, a major secretory steroid hormone of the human adrenal gland, possesses mitoinhibitory and anticarcinogenic properties. It also induces peroxisome proliferation in the livers of rats and mice. Because peroxisome proliferators exhibit hepatocarcinogenic potential, it is necessary to examine the long term hepatic effects of dehydroepiandrosterone since this hormone is contemplated for use as a potential cancer chemopreventive agent in humans. Dehydroepiandrosterone was administered in the diet at a concentration of 0.45% to F-344 rats for up to 84 weeks. At the termination of the experiment, 14 of 16 rats developed hepatocellular carcinomas. Liver tumors induced by dehydroepiandrosterone lacked gamma-glutamyl transpeptidase and glutathione S-transferase (placental form); these phenotypic properties are identical to the features exhibited by liver tumors induced by other peroxisome proliferators. Dehydroepiandrosterone was also shown to markedly inhibit liver cell [3H]thymidine labeling indices, suggesting that cell proliferation is not a critical feature in liver tumor development with this agent. These results show that although dehydroepiandrosterone exerts anticarcinogenic effects in a variety of tissues, the peroxisome-proliferative property makes it a hepatocarcinogen.

An upstream region of the enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase gene directs luciferase expression in liver in response to peroxisome proliferators in transgenic mice.

Peroxisome proliferators, which are structurally diverse nonmutagenic agents, induce hepatocarcinogenesis in rats and mice. Exposure to these xenobiotics leads to a rapid and coordinated transcriptional activation of the genes for the peroxisomal beta-oxidation enzyme system pathway in the liver. We have previously identified a peroxisome proliferator-responsive element in the 5'-flanking region of the rat peroxisomal hydratase/dehydrogenase (PBE) gene, the second enzyme in the beta-oxidation pathway. The peroxisome proliferator-responsive element in the PBE gene was shown to direct the induction of a luciferase reporter gene in vitro. We have now used this 3.2-kilobase 5'-flanking region of the PBE gene fused to the coding region of luciferase to generate transgenic mice. Three independent lines of transgenic mice expressed luciferase in response to ciprofibrate, a peroxisome proliferator. The induction of luciferase is specific to the liver; this agrees with the tissue-specific induction of PBE. Two other hypolipidemic drugs, nafenopin and Wy-14,643, were also capable of inducing luciferase activity in the liver. This study suggests that the PBE upstream element can be used to direct and modulate the expression of cloned genes by changing the levels of peroxisome proliferators. Also, the PBE-luciferase transgenic mouse should be an excellent model system for screening xenobiotics for potential peroxisome proliferator property.

Transformation of epithelial cells stably transfected with H2O2-generating peroxisomal urate oxidase.

Peroxisome proliferators, a group of structurally diverse nongenotoxic agents, induce predictable pleiotropic responses in liver, including the development of liver tumors in rats and mice. These agents transcriptionally activate the three genes of the peroxisomal beta oxidation enzyme system by interacting with the peroxisome proliferator-activated receptor(s). It has been proposed that H2O2 generated by the peroxisomal beta oxidation system leads to DNA damage and neoplastic transformation. Consistent with this hypothesis is that cells stably transfected with H2O2-generating peroxisomal fatty acyl-CoA oxidase cDNA, which encodes the first and rate-limiting enzyme of the beta oxidation system, undergo transformation in the presence of a fatty acid substrate. To test whether H2O2 generated by other peroxisomal oxidases can also lead to transformation, a full-length cDNA encoding rat urate oxidase (UOX), which oxidizes uric acid to allantoin and in the process generates H2O2, was introduced into African green monkey kidney cells (CV-1 cells) under the control of constitutively active human peroxisomal fatty acyl-CoA oxidase gene promoter. Five stably transfected CV-1 cell lines expressing recombinant rat UOX were isolated in which the recombinant protein was targeted to peroxisomes and formed crystalloid structures or cores similar to those present in rat liver peroxisomes. Increased levels of H2O2 were found when cells stably expressing UOX were exposed to the substrate uric acid. These five clones, designated A-U1 to A-U5, exhibited anchorage-independent growth, as demonstrated by the formation of transformed colonies in soft agar in proportion to the duration of exposure to uric acid. These transformants exhibited clonal growth under serum-deprived conditions. One of these transformed cell lines, the A-U3 cell line, was evaluated for tumorigenicity by s.c. injection in nude mice. All five mice injected with transformed A-U3 cells developed adenocarcinomas, but no tumors developed in mice injected with control CV-1 cells or cells stably expressing UOX that were not exposed to uric acid. These results provide further evidence indicating that sustained overexpression of a peroxisomal H2O2-generating oxidase causes cell transformation.

Molecular evolution of the urate oxidase-encoding gene in hominoid primates: nonsense mutations.

Nucleotide sequences of portions of second and fifth exons of urate oxidase encoding gene (UOX) of chimpanzee, gorilla, orangutan, rhesus monkey and squirrel monkey obtained following amplification by polymerase chain reaction have been compared with corresponding sequences of human, baboon and rat UOX. Two or more nonsense mutations are found in the coding regions of this UOX gene thus far analyzed in human, chimpanzee, gorilla and orangutan, but not in the baboon, rhesus monkey and squirrel monkey. Of these nonsense mutations, the stop codon at amino acid position 33 is constant in the human and the three great apes suggesting that this may be the original mutation responsible for the inactivation of the UOX gene during hominoid evolution.

Inhibition of spontaneous testicular Leydig cell tumor development in F-344 rats by dehydroepiandrosterone.

The incidence of spontaneous Leydig cell tumors of testis is very high in old F-344 rats. We have examined the effect of dehydroepiandrosterone (DHEA), a steroid hormone with antimitotic and anticarcinogenic properties, on spontaneous Leydig cell tumorigenesis. Fifteen-week-old male F-344 rats were fed a diet containing DHEA (0.45% w/w) for 84 weeks. At the termination of experiment none of the 13 rats had Leydig cell hyperplasia or Leydig cell tumors. All the eight control rats of comparable age had Leydig cell tumors. These findings suggest that DHEA is a potent inhibitor of spontaneous Leydig cell tumors of testis in aged rats.

Evaluation of liver cell proliferation during ciprofibrate-induced hepatocarcinogenesis.

To determine if the carcinogenic potential of peroxisome proliferators is dependent upon their ability to induce cell proliferation, we have investigated the extent of cell proliferation in the livers of rats fed ciprofibrate, a peroxisome proliferator. Male rats were maintained on a diet containing ciprofibrate (0.025% w/w) and killed at selected intervals following 1 week of continuous [3H]thymidine labeling. Evaluation of labeling indices demonstrated a significant increase in cell proliferation during the first week but not in rats killed at the end of 5 and 20 weeks of treatment. Increases in hepatocyte nuclear labeling were found at 40 and 70 weeks of ciprofibrate administration which coincided with the appearance in livers of putative preneoplastic and neoplastic lesions. In a short-term feeding study, ciprofibrate and ethoxyquin were fed to rats at a dietary concentration of 0.025% and 0.5%, respectively, either alone or in combination for 7 days. Ciprofibrate and ethoxyquin either alone or in combination produced marked hepatomegaly and a significant increase in DNA synthesis as demonstrated by [3H]thymidine incorporation and autoradiographic studies. DNA synthesis in the group receiving ciprofibrate and ethoxyquin simultaneously, was slightly more than in animals that received either compound alone, suggesting a synergistic effect, although chronic feeding of these agents together resulted in inhibition of liver carcinogenesis (Rao, M. S. et al. (1984) Cancer Res., 44, 1072-1076). The results of this study further suggest that cell proliferation induced by peroxisome proliferators may be less important in carcinogenesis than peroxisome proliferation induced by these compounds.

Pathologic features of lung biopsy specimens from influenza pneumonia cases.

Most histopathologic descriptions of influenza pneumonia are based on autopsy findings in fatal cases during epidemics and pandemics of influenza. We describe the histologic findings of influenza pneumonia observed in biopsy material derived from six sporadic cases of influenza (influenza A, four cases; influenza B, one case; and influenza A and B, one case). Four patients recovered completely, one patient died, and one was lost to follow-up. In addition to confirming previously published pathologic descriptions of influenza pneumonia, we also document a spectrum of less severe histologic findings with mild acute lung injury and bronchiolitis obliterans with organizing pneumonia.

Peroxisome proliferator-activated receptor alpha-dependent induction of cell surface antigen Ly-6D gene in the mouse liver.

Spontaneous peroxisome proliferation-related pleiotropic responses occurring in the liver of mice lacking peroxisomal fatty acyl-CoA oxidase (AOX-/-) are attributed to sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by its putative natural ligands that require AOX for their metabolism. In this study, using a gene expression screen, we show that Ly-6 (lymphocyte antigen 6 complex, locus D; mouse ThB), which belongs to a distinctive family of low molecular weight phosphatidyl inositol anchored cell surface glycoproteins, is upregulated in mouse liver with peroxisome proliferation. Increases in Ly-6D mRNA levels are observed in AOX-/- mouse liver with spontaneous peroxisome proliferation and also in the liver of wild-type mice treated with synthetic peroxisome proliferators. Peroxisome proliferators failed to increase hepatic Ly-6D mRNA levels in mice lacking PPARalpha (PPARalpha-/-), suggesting a regulatory role for PPARalpha in the induction of Ly-6D. These observations suggest that changes in certain cell surface proteins also form part of the pleiotropic responses associated with peroxisome proliferation.

Identification of novel peroxisome proliferator-activated receptor alpha (PPARalpha) target genes in mouse liver using cDNA microarray analysis.

Peroxisome proliferators, which function as peroxisome proliferator-activated receptor-alpha (PPARalpha) agonists, are a group of structurally diverse nongenotoxic hepatocarcinogens including the fibrate class of hypolipidemic drugs that induce peroxisome proliferation in liver parenchymal cells. Sustained activation of PPARalpha by these agents leads to the development of liver tumors in rats and mice. To understand the molecular mechanisms responsible for the pleiotropic effects of these agents, we have utilized the cDNA microarray to generate a molecular portrait of gene expression in the liver of mice treated for 2 weeks with Wy-14,643, a potent peroxisome proliferator. PPARalpha activation resulted in the stimulation of expression (fourfold or greater) of 36 genes and decreased the expression (fourfold or more decrease) of 671 genes. Enhanced expression of several genes involved in lipid and glucose metabolism and many other genes associated with peroxisome biogenesis, cell surface function, transcription, cell cycle, and apoptosis has been observed. These include: CYP2B9, CYP2B10, monoglyceride lipase, pyruvate dehydrogenase-kinase-4, cell death-inducing DNA-fragmentation factor-alpha, peroxisomal biogenesis factor 11beta, as well as several cell recognition surface proteins including annexin A2, CD24, CD39, lymphocyte antigen 6, and retinoic acid early transcript-gamma, among others. Northern blotting of total RNA extracted from the livers of PPARalpha-/- mice and from mice lacking both PPARalpha and peroxisomal fatty acyl-CoA oxidase (AOX), that were fed control and Wy-14,643-containing diets for 2 weeks, as well as time course of induction following a single dose of Wy-14,643, revealed that upregulation of genes identified by microarray procedure is dependent upon peroxisome proliferation vis-à-vis PPARalpha. However, cell death-inducing DNA-fragmentation factor-alpha mRNA, which is increased in the livers of wild-type mice treated with peroxisome proliferators, was not enhanced in AOX-/- mice with spontaneous peroxisome proliferation. These observations indicate that the activation of PPARalpha leads to increased and decreased expression of many genes not associated with peroxisomes, and that delayed onset of enhanced expression of some genes may be the result of metabolic events occurring secondary to PPARalpha activation and alterations in lipid metabolism.

Functional expression and peroxisomal targeting of rat urate oxidase in monkey kidney cells.

Humans and hominoid primates lack the enzyme urate oxidase, which catalyzes the oxidation of uric acid to allantoin. In rats and most other mammals, urate oxidase is present as a crystalloid core within the peroxisomes of liver parenchymal cells. To determine whether functionally active recombinantly expressed urate oxidase can be targeted to the peroxisome as well as display the crystalloid core-like structure, we expressed rat urate oxidase cDNA in African green monkey kidney cells (CV-1 cells) under the control of a cytomegalovirus promoter. Cell lines stably expressing urate oxidase were isolated. Northern blot analysis revealed a 1.3-kb transcript and immunoblot analysis confirmed the presence of urate oxidase in the stably transfected cells. The recombinant urate oxidase expressed in CV-1 cells was functionally active. Immunofluorescence microscopy revealed that the expressed protein was visualized as discrete granules in the cytoplasm. Electron microscopy and immunocytochemical localization studies showed that the recombinantly expressed protein formed distinct crystalloid core structures with bundles of tubules within single membrane limited cytoplasmic organelles. On cross section, the recombinant urate oxidase tubular structures are arranged as circles of 10 surrounding a slightly larger circle. This arrangement is reminiscent of urate oxidase-containing cores in rat liver peroxisomes. Immunocytochemical studies confirmed that the recombinantly expressed urate oxidase is correctly targeted to the catalase-containing peroxisomes in these CV-1 cells.

Cloning and expression of the mouse deoxyuridine triphosphate nucleotidohydrolase gene: differs from the rat enzyme in that it lacks nuclear receptor interacting LXXLL motif.

We have previously reported the cloning of rat deoxyuridine triphosphate nucleotidohydrolase (dUTPase) cDNA and demonstrated that the full-length protein as well as the N-terminal 62-amino acid peptide interacts with peroxisome proliferator-activated receptor alpha (PPARalpha). We now report the cloning of mouse dUTPase cDNA and show that it contains a 162-amino acid open reading frame, encoding a protein with a predicted Mr of 17,400 and differs from rat cDNA, which contains additional 43 amino acids at the N-terminal end. Unlike rat dUTPase, mouse dUTPase failed to bind PPARalpha. An evaluation of 205 amino acid containing rat dUTPase cDNA revealed that the N-terminal 43 extra amino acid segment contains an LXXLL signature motif, considered necessary and sufficient for the binding of several cofactors with nuclear receptors, and its absence in murine dUTPase possibly accounts for the differential binding of these enzymes to PPARalpha. In situ hybridization and immunohistochemical studies revealed that, in the adult mouse, dUTPase is expressed at high levels in proliferating cells of colonic mucosa, and of germinal epithelium in testis. At 9.5-day mouse embryonic development, dUTPase expression is predominantly in developing neural epithelium, and hepatic primordium, and in later developmental stages (11.5-, 13.5-, and 15.5-day embryo), the expression began to be localized to the liver, kidney, gut epithelium, thymus, granular layer of the cerebellum, and olfactory epithelium. We also show that the murine dUTPase gene comprises 6 exons and the 5'-flanking region of -1479 to -27, which exhibited high promoter activity, contains a typical TATA box and multiple cis-elements such as Sp-1, AP2, AP3, AP4, Ker1, RREB, and CREB binding sites. These observations suggest the existence of variants of dUTPase, some of which may influence nuclear receptor function during development and differentiation, in addition to catalyzing the hydrolysis of dUTP to dUMP.

Comparative effects of dehydroepiandrosterone and related steroids on peroxisome proliferation in rat liver.

Dehydroepiandrosterone (DHEA) is known to induce peroxisome proliferation and peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (PBE) mRNA in the rat liver. We have compared the effects of 6 intermediate metabolites of DHEA on the induction of peroxisome proliferation and PBE mRNA. Administration of epiandrosterone, etiocholanolone, androstenedione, estrone or estradiol for 2 weeks in the diet at 0.45% concentration to adult male F-344 rats failed to induce significant increases in peroxisome proliferation and PBE mRNA when compared to the parent compound DHEA. Dietary administration of 5-androstene-3 beta,17 beta-diol (ADIOL) for 2 weeks at 0.45% concentration caused an increase in PBE mRNA and peroxisome proliferation but to a lesser extent than DHEA. Following a single intragastric dose of DHEA an increase in PBE mRNA level was observed in the liver at 1 hr and continued to 16 hrs., but not with its metabolites. These results strongly suggest that DHEA or possibly another yet to be identified metabolite might be responsible for peroxisome proliferation.

Hydrogen peroxide generation in peroxisome proliferator-induced oncogenesis.

Peroxisome proliferators are a structurally diverse group of non-genotoxic chemicals that induce predictable pleiotropic responses including the development of liver tumors in rats and mice. These chemicals interact variably with peroxisome proliferator-activated receptors (PPARs), which are members of the nuclear receptor superfamily. Evidence derived from mice with PPARalpha gene disruption indicates that of the three PPAR isoforms (alpha, beta/delta and gamma), the isoform PPARalpha is essential for the pleiotropic responses induced by peroxisome proliferators. Peroxisome proliferator-induced activation of PPARalpha leads to profound transcriptional activation of genes encoding for the classical peroxisomal beta-oxidation system and cytochrome P450 CYP 4A isoforms, CYP4A1 and CYP4A3, among others. Livers with peroxisome proliferation manifest substantial increases in the expression of H(2)O(2)-generating peroxisomal fatty acyl-CoA oxidase, the first enzyme of the classical peroxisomal fatty acid beta-oxidation system, and of microsomal cytochrome P450 4A1 and 4A3 genes. Disproportionate increases in H(2)O(2)-generating enzymes and H(2)O(2)-degrading enzyme catalase and reductions in glutathione peroxidase activity by peroxisome proliferators, lead to increased oxidative stress in liver cells. Sustained oxidative stress resulting from chronic increases in H(2)O(2)-generating enzymes manifests as massive accumulation of lipofuscin in hepatocytes, and increased levels of 8-hydroxydeoxyguanosine adducts in liver DNA; this supports the hypothesis that oxidative stress plays a critical role in the development of liver tumors induced by these non-genotoxic chemical carcinogens. Evidence also indicates that cells stably overexpressing H(2)O(2)-generating fatty acyl-CoA oxidase or urate oxidase, when exposed to appropriate substrate(s), reveal features of neoplastic conversion including growth in soft agar and formation of tumors in nude mice. Mice with disrupted fatty acyl-CoA oxidase gene (AOX(-/-) mice), which encodes the first enzyme of the PPARalpha regulated peroxisomal beta-oxidation system, exhibit profound spontaneous peroxisome proliferation, including development of liver tumors, indicative of sustained activation of PPARalpha by the unmetabolized substrates of acyl-CoA oxidase. With the exception of fatty acyl-CoA oxidase, all PPARalpha responsive genes including CYP4A1 and CYP4A3 are up-regulated in the livers of these AOX(-/-) mice. Thus, the substrates of acyl-CoA oxidase serve as endogenous ligands for this receptor leading to a receptor-enzyme cross-talk, because acyl-CoA oxidase gene is transcriptionally regulated by PPARalpha. Peroxisome proliferators induce only a transient increase in liver cell proliferation and this may serve as an additional contributory factor, rather than play a primary role in liver tumor development. Thus, sustained activation of PPARalpha by either synthetic or natural ligands leads to reproducible pleiotropic responses culminating in the development of liver tumors. This phenomenon of peroxisome proliferation provides fascinating challenges in exploring the molecular mechanisms of cell specific transcription, and in identifying the PPARalpha responsive target genes, as well as events involved in their regulation. Genetically altered animals and cell lines should enable investigations on the role of H(2)O(2)-producing enzymes in neoplastic conversion.