Maintenance by the endothelium of a semi-

Mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of a ferrous ion into protoporphyrin and contains a labile [2Fe22S] cluster center at the C-terminus. To clarify the roles of the iron±sulfur cluster in the expression of mammalian ferrochelatase, enzyme activity in human erythroleukemia K562 cells under iron-depleted conditions was examined. Treatment of cells with an iron chelator, desferrioxamine, resulted in a decrease in enzyme activity, in a dose-and time-dependent manner. Heme content decreased during desferrioxamine treatment of the cells. Addition of ferric ion-nitrilotriacetate [Fe (III)NTA] to desferrioxamine-containing cultures led to restoration of the reduction in the enzyme activity. While RNA blots showed that the amount of ferrochelatase mRNA remained unchanged during these treatments, the amount of ferrochelatase decreased with a concomitant decrease in enzyme activity. When full-length human ferrochelatase was expressed in Cos7 cells, the activity was found mainly in the mitochondria and was decreased markedly by treatment with desferrioxamine. The activity in Cos7 cells expressing human ferrochelatase in cytoplasm decreased with desferrioxamine, but to a lesser extent. When Escherichia coli ferrochelatase, which lacks the iron±sulfur cluster, was expressed in Cos7 cells, the activity did not change following any treatment. Conversely, the addition of Fe (III)NTA to the culture of K562 and Cos7 cells led to an increase in ferrochelatase activity. These results indicate that the expression of mammalian ferrochelatase is regulated by intracellular iron levels, via the iron±sulfur cluster center at the C-terminus, and this contributes to the regulation of the biosynthesis of heme at the terminal step.Keywords: desferrioxamine; ferrochelatase; iron±sulfur cluster; iron; K562 cells.As the terminal enzyme of the heme biosynthetic pathway, ferrochelatase catalyzes the insertion of a ferrous ion into protoporphyrin IX to form protoheme, and the animal enzyme is located at the inner membrane of the mitochondria [1]. Both cDNA and genes for ferrochelatase from various species including mouse, human, yeast, plant, Escherichia coli and Bacillus subtilis have been isolated [2,3]. The mammalian enzyme is nuclear encoded, as a precursor form (48 kDa) and translocated into the mitochondrion where the enzyme is processed proteolytically to its mature size of 41±42 kDa [2]. The deduced amino-acid sequences from various species show 10±88% identity to the human enzyme [2,3]. Heterologous overexpression of ferrochelatase demonstrated that a conserved and essential histidine residue is involved in the binding of the metal substrate [4] and a conserved glutamate residue may play a role in the catalytic reaction [5]. The mammalian enzyme contains a [2Fe22S] cluster in the C-terminal region. However, yeast and plant ferrochelatase do not contain this motif, or alternatively the C-terminus of prokaryotic ferrochelatase is < 30 amino acids shorter than that of the m...

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We have determined the structure of the human ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. This gene was assigned to human chromosornc 18 at region q21.3, by fluorescent in situ hybridization. The gene contains a total of 11 exons and has a minimum size of about 45 kb. The exonlintron boundary sequences conform to consensus acceptor (GTn) and donor (nAG) sequences, and the exons in the gene appear to encode functional protcin domains. A major site of the transcription initiation, determined by S1 nuclease mapping, was assigned to an adcnine base 89 bases upstream from the adenine base of the translation initiation ATG. The promoter region contains a potential binding site for Spl, NF-E2 and erythroidspecific transcriptional factor GATA-1, but not a typical TATAA or CCAAT sequence. Analysis of primer extension showed that the transcription starts at the same position between hepatoma HepG2 and erythroleukemia K562 cell mRNA, thereby suggesting that there can be a single transcript in erythroid and non-erythroid cells.Ferrochelatase (protoheme lerro-lyase) is the terminal enzyme of the heme biosynthetic pathway and catalyzes the insertion of fcrrous ion into protoporphyrin 1X to form protoheme. The enzyme is located in the innermembrane ofmilochondria of various tissues including liver [l, 21, heart [3], kidney [4] and erythroid cells [4, 51. A genetic ferrochelatase deficiency in infants results in crythropoietic protoporphyria with excessive accumulation and excretion of protoporphyrin [6]. Protoporphyria in humans is a dominantly inherited disease. The enzyme activity decreases to about 50% compared with normal levels in all tissues and isolated cell preparations, including bone marrow [7], liver [8] and cultured skin fibroblasts [9]. The molecular basis of the defect has apparently not been reported.Regulation of the heme synthetic pathway differs between erythroid and non-erythroid cells. In liver and non-erythroid tissues, the rate of heme synthesis is controlled by the levcl of the first pathway enzyme, 5-aminolevulinic-acid synthase [6]. In differentiating erythroid cells, a different type of 5-aminolevulinic-acid synthase is present and cannot be ratelimiting for heme production [lo]. It has been shown that hemc synthesis increased when mouse erythroleukemia cells were induced to differentiate to cells producing hemoglobin, C'orrespondence to S. Taketani,

Cisplatin is commonly used as a chemotherapeutic agent for hepatocellular carcinoma (HCC). However, it cannot satisfactorily improve the survival rate for patients with advanced HCC due to intrinsic or acquired drug resistance caused by multidrug resistance-associated proteins (MRPs). To clarify whether or not glycyrrhizin and lamivudine have modulator effects on HCC treated with cisplatin, we established a cisplatin-resistant Huh7 HCC cell line and analyzed the mRNA expression of MRPs in the resistant cells. The resistant cells showed 14.1-fold higher resistance to cisplatin, and they expressed higher levels of MRP2 (6.29-fold), MRP3 (3.2-fold), MRP4 (11.3-fold) and MRP5 (3.39-fold) mRNAs than the wild-type cells by using real-time PCR. However, MRP1, MDR1 and GST-π mRNA were not induced. Compared with the treatment of the resistant cells with cisplatin only, co-treatment with cisplatin and glycyrrhizin or lamivudine significantly decreased the cell viability to 76.8% and 79.5%, respectively. Co-treatment with cisplatin and both glycyrrhizin and lamivudine further decreased the cell viability to 65.1%. Intracellular concentration of cisplatin in the resistant cells decreased to 36.4% of that of the wild-type cells while it increased to 47.7% or 48.4% when glycyrrhizin or lamivudine were added separately, or 60% when they were added together. Our findings indicate the following: i) high expression of MRP2, MRP3, MRP4 and MRP5 decreases cisplatin accumulation in cisplatin-resistant HCC cells and contributes to cisplatin resistance; ii) glycyrrhizin and/or lamivudine accumulate cisplatin in resistant cells by inhibiting the cisplatin efflux from the cells; and iii) glycyrrhizin and lamivudine both act as modulators and have the effect of reversing cisplatin resistance, and co-treatment with glycyrrhizin and lamivudine enhances modulator activity in reversing the cisplatin resistance.