Deferoxamine photosensitizes cancer cells in vitro

Effect of the iron chelator deferoxamine (DF) on the production of endogenous porphyrins was studied in adenocarcinoma WiDr cells and erythroid K562 cells in vitro. Porphyrin fluorescence was observed in the cells in vitro incubated with DF. The fluorescence spectra recorded in the cells were similar to that of protoporphyrin IX (PpIX). The amount of PpIX generated by DF was around 5% of the ALA effect. Around 90% of the WiDr cells incubated in vitro with DF (0.5 mM, 24 h) and then exposed to light (400-460 nm, 20 min) were photodynamically inactivated. In conclusion, the present study describes a novel approach of using iron chelating agents without 5-aminolevulinic acid (ALA) to photosensitize cancer cells.     

Regulation of ferrochelatase gene expression by hypoxia

Ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, catalyzes the insertion of iron into protoporphyrin to form heme. This pathway provides heme for hemoglobin and other essential hemoproteins. The regulatory role of oxygen in the pathway has not been clearly established. In this study, we examined whether FECH gene expression is upregulated during hypoxia by a mechanism which involves the hypoxia-inducible factor 1 (HIF-1). Two HIF-1 binding motifs were identified within the -150 bp FECH minimal promoter sequence. Exposure of HEL, K562, and Hep-G2 cells to hypoxia for 18 hours resulted in a significant increase in FECH mRNA expression (p < 0.05). Hypoxia also transactivated the minimal promoter for the FECH gene in the cells. Transient co-expression of wild-type HIF-1alpha or a dominant negative HIF-1alpha with the FECH minimal promoter luciferase construct stimulated or blocked FECH promoter activity, respectively. Expression of the von Hippel-Lindau (VHL) tumor suppressor factor blocked the expression of both FECH mRNA and HIF-1alpha protein during normoxic culture of renal carcinoma cell line (RCC4). The results suggest that the FECH gene is a target for HIF-1 during hypoxia.     

Roles of SIRT1 in the acute and restorative phases following induction of inflammation

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that in vivo endotoxin triggers a rapid and transient decline in ATP concentration in human peripheral blood leukocytes and murine peripheral blood leukocytes and liver, which is associated with a brief increase in expression of the autophagy indicator LC3-II. In both of these tissues, the ATP concentration reaches a nadir, and autophagy is induced between 2 and 4 h post-endotoxin infusion, and homeostasis is restored within 12 h. Mouse liver SIRT1 and AMP-activated protein kinase (AMPK) protein expression levels decline precipitously within 10 min and remain below detection levels for up to 12 h post-endotoxin administration. In marked contrast, the expression of HIF-1α is induced within 90 min and remains elevated for up to 12 h. The ATP recovery is delayed, and the increases in both HIF-1α expression and autophagy are prolonged in endotoxin-challenged SIRT1 liver knock-out mice. Resveratrol prevents the decline in ATP concentration and SIRT1 expression, as well as the increase in HIF-1α expression and autophagy in liver of endotoxin-challenged wild type mice but not in SIRT1 liver knock-out mice. These results provide novel insight into the state of both cellular bioenergetics and metabolic networks during the acute phase of systemic inflammation and suggest a role for SIRT1 in acute metabolic decline, as well as the restoration of metabolic homeostasis during an inflammatory challenge.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.     

Enzymes of heme biosynthesis

 Heme is a necessary component in a variety of oxygen-binding proteins and electron-transfer proteins, and as such it occupies a central role in cellular and organismal metabolism. With only rare exceptions, organisms that utilize heme possess the entire biosynthetic pathway to produce this tetrapyrrole compound. The enzymes involved catalyze a variety of interesting reactions and utilize both common and unique cofactors and metals. Aminolevulinate dehydratase from all organisms and ferrochelatase from higher animals are both metalloenzymes, while 5-aminolevulinate synthase contains pyridoxal phosphate, and porphobilinogen deaminase possesses a unique dipyrrole cofactor. Two pathway enzymes catalyze multiple decarboxylations and yet have no cofactors, and one enzyme catalyzes a six-electron oxidation with a single FAD. To add additional scientific interest there exist biochemically and clinically distinct human genetic diseases for every step in this pathway. Received: 12 March 1997 / Accepted: 8 May 1997     

Systematic analysis of molecular defects in the ferrochelatase gene from patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP; MIM 177000) is an inherited disorder caused by partial deficiency of ferrochelatase (FECH), the last enzyme in the heme biosynthetic pathway. In EPP patients, the FECH deficiency causes accumulation of free protoporphyrin in the erythron, associated with a painful skin photosensitivity. In rare cases, the massive accumulation of protoporphyrin in hepatocytes may lead to a rapidly progressive liver failure. The mode of inheritance in EPP is complex and can be either autosomal dominant with low clinical penetrance, as it is in most cases, or autosomal recessive. To acquire an in-depth knowledge of the genetic basis of EPP, we conducted a systematic mutation analysis of the FECH gene, following a procedure that combines the exon-by-exon denaturing-gradient-gel-electrophoresis screening of the FECH genomic DNA and direct sequencing. Twenty different mutations, 15 of which are newly described here, have been characterized in 26 of 29 EPP patients of Swiss and French origin. All the EPP patients, including those with liver complications, were heterozygous for the mutations identified in the FECH gene. The deleterious effect of all missense mutations has been assessed by bacterial expression of the respective FECH cDNAs generated by site-directed mutagenesis. Mutations leading to a null allele were a common feature among three EPP pedigrees with liver complications. Our systematic molecular study has resulted in a significant enlargement of the mutation repertoire in the FECH gene and has shed new light on the hereditary behavior of EPP.     

Screening for ferrochelatase mutations: molecular heterogeneity of erythropoietic protoporphyria

The DNA of 21 patients from 19 unrelated families with erythropoietic protoporphyria (EPP) were screened for the 6 ferrochelatase point mutations so far described. The mutation previously described by us (A ? t transversion at position ?3 of the donor site of intron 10, causing exon 10 skipping) was detected in two additional unrelated EPP patients: in these patients, cDNA lacking exon 10 was also detected. The mutation described by Nakahashi et al. as responsible for exon 2 skipping (C ? T transition at position ?23 of the acceptor site of intron 1), although also observed in some normal individuals, was invariably observed in all EPP patients tested and may thus play some role in the pathognesis of EPP. Thus, it does not appear that this mutation is the primary mechanism underlying exon 2 skipping. None of the other four previously described mutations were detected. These data demonstrate the heterogeneity of the ferrochelatase locus and of the genetic defect in EPP.     

Melatonin and Environmental Lighting Regulate ALA‐S Gene Expression and So Porphyrin Biosynthesis in the Rat Harderian Gland

The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day‐night PPIX levels as well as day‐night activity and mode of expression of the porphyrinogenic enzymes δ‐aminolevulinate synthase (ALA‐S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630‐ to 670‐fold higher than in the liver, without a day‐night difference, which is the consequence of both low Fech gene expression (5‐ to 7‐fold lower than in the liver) and ALA‐S over‐expression (4‐ to 7‐fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day‐night variation, whereas HG ALA‐S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA‐S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day‐night ALA‐S variation in the HG that is observed under standard 12 L∶12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA‐S gene expression in the rat HG.