Profiling gene expression of whole cytochrome P450 superfamily in human bronchial and peripheral lung tissues: Differential expression in non-small cell lung cancers

Susceptibility to lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is largely influenced by the metabolic capacity of lung tissues. This capacity is partly determined by the expression profile of the cytochromes P450 (CYPs), a superfamily of enzymes that have relevant catalytic properties toward exogenous and endogenous compounds. Using quantitative real-time RT-PCR, we conducted a comprehensive analysis of the expression profile of the 57 human CYP genes in non-tumoral (bronchial mucosa and pulmonary parenchyma) and tumoral lung tissues of 18 patients with non-small cell lung cancer. This study highlights (i) inter-individual variations in lung expression for some CYPs, (ii) different CYP expression patterns between bronchial mucosa and pulmonary parenchyma, that indicate distinctive susceptibility of these tissues toward the deleterious effects of inhaled chemical toxicants and carcinogens, (iii) high intertumoral variability, that could have major implications on lung tumor response to anti-cancer drugs.     

Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2′-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.     

Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism

Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult mouse (C57BL/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic as well as biochemical techniques and by the use of various peroxisomal marker proteins (Pex13p, Pex14p, ABCD3, beta-oxidation enzymes and catalase). In contrast to previous reports, we have found that peroxisomes are present in all cell types in human and mouse lungs. However, they differ significantly and in a cell-type-specific manner in their structure, numerical abundance and enzyme composition. Whereas catalase showed significant differences between distinct cell types, Pex14p proved to be the marker of choice for labeling all lung peroxisomes. In alveolar type II cells and alveolar macrophages peroxisomes contained significant amounts of the lipid transporter ABCD3 and beta-oxidation enzymes, suggesting their involvement in the modification and recycling of surfactant lipids and in the control of lipid mediators and ligands for nuclear receptors of the PPAR family. Possible connections between ROS and lipid metabolism of lung peroxisomes are discussed.     

Carcinogen mmetabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control study on lung cancer patients

Individual variations in activity of pulmonary enzymes that metabolize tobacco-derived carcinogens may affect an individual's cancer risk from cigarette smoking. To investigate whether some of these enzymes (e.g., cytochrome P450IA-related) can serve as markers for carcinogen-induced DNA damage accumulating in the lungs of smokers, non-tumorous lung tissue specimens were taken during surgery from middle-aged men with either lung cancer (n = 54) or non-neoplastic lung disease (n = 20). Phase I (AHH, ECDE) and phase II (EH, UDPGT, GST) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were analyzed for DNA adducts, using ³²P-postlabeling.

Data analysis of subsets or the whole group of patients yielded the following results. (1) Phase I and II drug-metabolizing enzyme (AHH, EH, UDPGT, GST) activities in histologically normal surgical specimens of lung parenchyma were correlated with the respective enzyme activities in bronchial tissues of the same subject. (2) In lung parenchyma, enzyme (AHH, ECDE, EH, UDPGT) activities were significantly and positively related to each other, implying a similar regulatory control of their expression. (3) Mean activities of pulmonary enzymes (AHH, ECDE) were significantly (2- and 7-fold, respectively) higher in lung cancer patients who had smoked within 30 days before surgery (except GST, which was depressed) than in cancer-free subjects with a similar smoking history. (4) In the cancer patients, the time required for AHH, EH and UDPGT activities to return to the level found in non-smoking subjects was several weeks. (5) Bronchial tree and peripheral lung parenchyma preparations exhibited a poor efficiency in activating promutagens to bacterial mutagens in Salmonella. However, they decreased the mutagenicity of several direct-acting mutagens, an effect which was more pronounced in tissue from recent smokers. GSH concentration and GST activity were positively correlated with mutagen inactivation in the same sample. (6) In recent smokers, AHH activity in lung parenchyma was positively correlated with the level of tobacco smoke-derived DNA adducts. (7) Pulmonary AHH and EH activity had prognostic value in tobacco-related lung cancer patients. (8) An enhanced level of pro-oxidant state in the lungs was associated with recent cigarette smoking. Malondialdehyde level in lung parenchyma was associated with the degree of small airway obstruction, suggesting a common free radical-mediated pathway for both lung cancer induction and small airway obstruction.

These results demonstrate the pronounced effect of recent cigarette smoke exposure on pulmonary xenobiotic metabolism and lipid peroxidation and lend further support to the hypothesis that the inducibility of pulmonary AHH activity (cytochrome P450IA1 levels) in tobacco smokers is associated with lung cancer risk. Results on DNA adducts in smokers' lung tissue may help to explain why a certain metabolic phenotype accumulates more DNA damage in lung cells.     

TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia

Gram-negative bacteria release lipopolysaccharide (LPS) into the bloodstream. Here, it engages Toll-like receptor (TLR) 4 expressed in human lung microvascular endothelia (HMVEC-Ls) to open the paracellular pathway through Src family kinase (SFK) activation. The signaling molecules that couple TLR4 to the SFK-driven barrier disruption are unknown. In HMVEC-Ls, siRNA-induced silencing of TIRAP/Mal and overexpression of dominant-negative TIRAP/Mal each blocked LPS-induced SFK activation and increases in transendothelial [(14)C]albumin flux, implicating the MyD88-dependent pathway. LPS increased TRAF6 autoubiquitination and binding to IRAK1. Silencing of TRAF6, TRAF6-dominant-negative overexpression, or preincubation of HMVEC-Ls with a cell-permeable TRAF6 decoy peptide decreased both LPS-induced SFK activation and barrier disruption. LPS increased binding of both c-Src and Fyn to GST-TRAF6 but not to a GST-TRAF6 mutant in which the three prolines in the putative Src homology 3 domain-binding motif (amino acids 461-469) were substituted with alanines. A cell-permeable decoy peptide corresponding to the same proline-rich motif reduced SFK binding to WT GST-TRAF6 compared with the Pro → Ala-substituted peptide. Finally, LPS increased binding of activated Tyr(P)(416)-SFK to GST-TRAF6, and preincubation of HMVEC-Ls with SFK-selective tyrosine kinase inhibitors, PP2 and SU6656, diminished TRAF6 binding to c-Src and Fyn. During the TRAF6-SFK association, TRAF6 catalyzed Lys(63)-linked ubiquitination of c-Src and Fyn, whereas SFK activation increased tyrosine phosphorylation of TRAF6. The TRAF6 decoy peptide blocked both LPS-induced SFK ubiquitination and TRAF6 phosphorylation. Together, these data indicate that the proline-rich Src homology 3 domain-binding motif in TRAF6 interacts directly with activated SFKs to couple LPS engagement of TLR4 to SFK activation and loss of barrier integrity in HMVEC-Ls.     

Expression of multi-drug resistance 1 mRNA in human and rodent tissues: reduced levels in Parkinson patients

The membrane transporter multi-drug resistance 1 (MDR1, P-gp) regulates the bioavailability of endogenous and exogenous compounds and has been implicated in disorders such as Parkinson's disease, cancer, epilepsy, human immunodeficiency virus disease, and inflammatory bowel disease. To promote further understanding of the role of MDR1 in disease, we have characterized cellular MDR1 mRNA expression in post-mortem human and fresh-frozen Sprague-Dawley rat tissues by using radioactive oligonucleotide probe in situ hybridization. We report MDR1 mRNA in human and rat endothelial cells of small vessels in the brain and pia mater. Mdr1 mRNA is also expressed in the blood vessel walls of rat sensory dorsal root and sympathetic ganglia. In peripheral tissues, we have observed MDR1 mRNA in human and rat liver and renal tubules and in human adrenal cortex and the epithelial lining of rat intestine. In female and male reproductive tissues of rat, strong gene activity has been found in steroid-hormone-synthesizing cells. Quantification of MDR1 mRNA in human striatum has revealed reduced levels in Parkinson patients compared with control individuals. The high expression of MDR1 mRNA in blood vessels of the nervous system, in tissues involved in absorption and excretion, and in tissues forming barriers to the environment support the physiological role of MDR1 as a regulator of intracellular levels of endogenous and exogenous compounds.     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

Pneumocystis carinii: growth variables and estimates in the A549 and WI-38 VA13 human cell lines

Recent studies indicate that rat Pneumocystis carinii can be propagated in the A549 cell line, an alveolar epithelioid cell line derived from human lung carcinoma. In the present study, growth of P. carinii was compared in the A549 cell line and the WI-38 VA13 subline 2RA, an SV40 transformed derivative of the human fetal fibroblast cell line with epithelioid morphology. Similar P. carinii growth occurred in both cell lines under optimal conditions, but the WI-38 VA13 cell line was usually more sensitive to changes in the culture system. Growth of P. carinii was affected by temperature, environmental gas mixture, motion of the cultures, and source and concentration of serum additives, but not by the presence of antibodies in the medium. A technique was developed for quantitating P. carinii in the lung inoculum which permitted analysis of P. carinii growth during the first 24 hr of culture. Inverted microscope and oil immersion phase-contrast microscopy were very helpful in monitoring the organism's stages of development and viability. Thus, this culture system should be helpful in establishing standard methodology for in vitro work with P. carinii.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b₅₆₁ family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g_max = 3.7 corresponding to a highly anisotropic species, and another at g_max = 3.18; these species are similar to those observed in other cytochromes of the b₅₆₁ family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g_max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b₅₆₁ of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Dancing with the sterols: Critical roles for ABCG1, ABCA1, miRNAs, and nuclear and cell surface receptors in controlling cellular sterol homeostasis

ATP binding cassette (ABC) transporters represent a large and diverse family of proteins that transport specific substrates across a membrane. The importance of these transporters is illustrated by the finding that inactivating mutations within 17 different family members are known to lead to specific human diseases. Clinical data from humans and/or studies with mice lacking functional transporters indicate that ABCA1, ABCG1, ABCG4, ABCG5 and ABCG8 are involved in cholesterol and/or phospholipid transport. This review discusses the multiple mechanisms that control cellular sterol homeostasis, including the roles of microRNAs, nuclear and cell surface receptors and ABC transporters, with particular emphasis on recent findings that have provided insights into the role(s) of ABCG1. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).