Recent Progress in Heme Synthesis and Metabolism

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1 Recent Progress in Heme Synthesis and Metabolism Shigeru Sassa The Rockefeller University, New York, New York, USA Key Words. 6-Aminolevulinic acid synthase 6-Aminolevulinic acid dehydratase Porphobilinogen deaminase Ferrochelatase Heme oxygenase Tissue-specific expression Porphyrias Abstract. Heme serves as the prosthetic group of various hemoproteins that carry out many essential functions for cells. For example, the transport of oxygen is carried out by hemoglobin and myoglobin; electron transport depends on the function of various mitochondrial cytochromes; oxidative metabolism in a number of xenobiotic substances and endogenous steroid hormones, vitamins and fatty acids are catalyzed by the activity of the microsomal cytochrome P450. In addition, heme is involved in the translation of proteins, and is required in certain aspects of cell development and differentiation. Inherited and acquired enzymatic defects in heme biosynthesis result in clinical conditions termed the porphyrias. Patients with porphyria express various difficulties in these functions as neurological disturbances, abnormal drug metabolism, and/or skin photosensitivity. Recent advances in this field have shed much light on the genetic, enzymological and clinical aspects of heme synthesis, catabolism and inherited defects of these enzymes. Introduction Heme, i.e., ferroprotoporphyrin IX, is the prosthetic group of all hemoproteins, which are required for the function of all aerobic cells. It serves as the prosthetic group of hemoglobin, mitochondria1 and microsornal cytochromes, catalase, peroxidases, tryptophan pyrrolase, prostaglandin H synthetase and NO synthase. Heme is involved in the transport of oxygen and electrons, in the oxidative metabolism of various endogenous and exogenous chemicals, in the decomposition of hydrogen peroxide or Correspondence: Dr. Shigeru Sassa, The Rockefeller University, 1230 York Avenue, New York, N.Y , USA. OAlphaMed Press ISBN X activation of peroxides, and in the oxidation of tryptophan. Alterations or defects of enzymatic activities in the heme biosynthetic pathway result in profound disturbances in these functions, which lead to the development of clinical conditions termed the porphyrias. Recent advances in the molecular studies of these enzymes have contributed not only to an understanding of the pathogenesis of these diseases, but also to the elucidation of hitherto unrecognized heme-mediated processes in normal cells. Biosynthesis of heme requires eight enzymes, while catabolism of heme requires four enzymes. As shown in Figure 1, the first step and the last three steps of heme biosynthesis take place in mitochondria, while the first two enzymes in heme catabolism are found in endoplasmic reticulum, and all the remaining steps of reactions occur in cytosol. Genetic deficiencies leading to the development of the porphyrias have been detected in seven out of the eight enzymes in the biosynthetic pathway, while genetic deficiencies of enzymes in the heme catabolic pathway have been found only in the last step: bilirubin-udpglucuronosyltransferase. In this chapter, important progress in heme synthesis and metabolism will be reviewed. Tissue-specific Regulation of Heme Synthesis Recent studies suggest that there is a copsiderable difference in the regulation of heme synthesis between erythroid and non-erythroid cells [ This distinctive tissue-specific regulation of heme synthesis occurs on the level of at least three enzymatic steps in the heme biosynthetic pathway, which are discussed below.

2 Uro'l p, +ljrod Copro'l - ALA 1 ALAD PBG 1 PBGD Uro'lll Copro'lll ALAS Mitochondrion Succinyl COA + Glycine - Heme T +Fe Fe-C Prot ol X t Proto 'Ox + Proto'lX :opro'ox. Recent Progress in Heme Synthesis and Metabolism Cytosol 'Free Heme", Endoplasmic Reticulum +NADPH * Biliverdin - CR-HO Bilirubin ~ glucuronide +NADPH * Bil BR tudp - GT ubin, Fe Excretion into bile Fig. 1. Subcellular localization of enzymes and intermediates in heme biosynthesis and catabolism [23]. Free heme is considered as heme which is either synthesized very recently and not yet bound, as the prosthetic group of hemoproteins, or heme which has just been released from hemoproteins. Free heme turns over very rapidly, and is involved in the repression of the synthesis of ALAS-N and in the induction of HO. ALA: 8-Aminolevulinic acid; ALAS: ALA synthase; ALAD: ALA dehydratase; PBG: Porphobilinogen; PBGD: PBG deaminase; HMB: Hydroxymethylbilane; Uro': Uroporphyrinogen; Uro'CoS: Uro'II1 cosynthase; Copro': Coproporphyrinogen; Copro'Ox: Copro' oxidase; Proto': Protoporphyrinogen; Proto'Ox: Proto' oxidase; Fe-C: Ferrochelatase; CR: NADPH-cytochrome c reductase; HO: Heme oxygenase; BR: Biliverdin reductase; GT: Bilirubin-UDP- Glucuronosyltransferase. b-ai~zinole~~srlitzute Sytzthase (ALAS) (EC In the liver, the rate of heme formation is determined by the level of ALAS, the first enzyme of the heme biosynthetic pathway [4], and the level of ALAS is in turn controlled by intracellular free heme concentration [5]. For example, in the liver, a) only ALAS activity is increased, while the activity of other heme pathway enzymes remains unchanged when heme synthesis is increased [6, 71, and b) heme suppresses the synthesis of hepatic ALAS [5,6]. An excessive amount of free heme in the liver induces microsomal heme oxygenase (HO) resulting in the breakdown of free heme [S], thus serving as a useful control mechanism to maintain a critical free heme concentration [5]. These findings suggest a negative feedback control of ALAS by heme, i.e., the end-product of the biosynthetic pathway. It has generally been believed that the synthesis of heme in non-hepatic cells, including erythroid cells in the bone marrow, may also be regulated in a similar manner. However, recent studies including our own [9-111 suggest that there may be a distinctive regulatory feature of heme biosynthesis in erythroid cells [lo].,

3 Sassa 3 Specifically, in erythroid precursor cells which can undergo erythroid cell differentiation such as murine Friend virus transformed erythroleukemia (MEL) cells, or K562 human erythroleukemia cells, a) there is an up-regulation of all heme pathway enzyme genes during cell differentiation [ 12, 131; b) hemin stimulates, rather than inhibits, the synthesis of ALAS-E [14, 151, ferrochelatase [16], heme [ll], globin mrna [17,18], and hemoglobin [18]; and c) heme oxygenase mrna [19] and its enzyme activity are down-regulated when cells are induced to undergo erythroid differentiation [20]. The positive effect of heme on its own synthesis in erythroid cells is in contrast to that in the liver. In in vitro erythroid colony culture systems, hemin also stimulates the growth and differentiation of erythroid colonies [21, 221. Thus heme up-regulates its own synthesis in erythroid cells, as well as erythroid cell differentiation. Distinctive aspects in the regulation of heme synthesis between hepatic and erythroid cells can be largely accounted for by the existence of tissue-specific ALAS isozymes, which are encoded by two different genes [23]. The original findings on two ALAS genes were made in avian cells [24], and similar findings were made subsequently in mammalian cells [14,25,26]. For example, the transcript encoding the non-specific ALAS (ALAS-N, or ALAS1) in mice was shown to be present in all tissues, while that encoding the erythroid-specific ALAS (ALAS-E, or ALAS2) was expressed only in erythroid cells [27]. During erythroid cell differentiation of MEL cells, ALAS-E mrna was found to increase dramatically [14], while ALAS-N mrna was rapidly down-regulated [12, 191. In humans, the ALAS-E gene spans 22 kb and is organized into 11 exons [28]. As in avian and murine cells, the human ALAS-E gene is expressed exclusively in erythroid cells. An openreading frame of 1,761 nucleotides from the first ATG codon encodes a precursor of 587 amino acids with a M, of 64.6 kda. Nucleotide sequences for ALAS-E and ALAS-N are = 60% similar, with the longest stretch of identical sequence being 21 nucleotides. There is no homology, however, in the amino-terminal region, while there is a high homology (= 73%) after hepatic residue 197 [29]. From their structural organization, it can be speculated that the two human ALAS genes may have evolved by duplication of a common ancestral gene which encoded a primitive catalytic peptide, with subsequent addition of DNA sequences encoding variable functions [28]. The human ALAS-E gene has been assigned to a distal subregion of Xp11.21 [30], while the ALAS-N gene is on chromosome 3p21 [26, 29, 311. The X-chromosomal localization of the erythroid gene is interesting in view of the fact that this enzyme activity is known to be markedly reduced in erythroid cells of patients with sideroblastic anemia [32], which often occurs as an X-linked disorder [33]. Recently, it has been shown in a patient with a pyridoxineresponsive X-linked sideroblastic anemia that there was a single T4714A transition in a highly conserved region of exon 9, resulting in an Ile +-Am substitution [34]. The amino acid substitution occurred in the putative pyridoxal5'- phosphate binding site, and the mutant enzyme which was expressed in a prokaryotic system showed low enzyme activity, which required a higher concentration of pyridoxal5'-phosphate than the wild-type enzyme for maximal activity. Thus the decreased enzyme activity appears to be due to a decreased affinity of the mutant enzyme to this cofactor. The promoter in the human ALAS-E contains several putative erythroid-specific cis-acting elements including both GATA-1 and an NF-E2 binding site [28]. Both GATA-1 and NF-E2 are erythroid-specific transcription factors which have been shown to bind to multiple DNA sites such as the promoter of the human P-globin gene, and the erythroid porphobilinogen deaminase (PBGD) promoter [35,36]. In addition to the transcriptional control described above, ALAS-E expression may also be controlled at the translational level. It is known that the synthesis of ferritin and transferrin receptor, two major proteins involved in iron homeostasis, is regulated post-transcriptionally by iron-responsive elements (IRES), which are located in the untranslated region of their mrna [37, 381. A functional IRE has also been identified [27, 291 in the 5'-untranslated region of the human ALAd-E mrna, but it is absent in ALAS-N mrna. This finding suggests that expression'of ALAS-E, but not that of ALAS-N, may also be translationally controlled by iron, or heme (as an iron donor), during the development of erythroid cells [15, 391.

4 4 Recent Progress in Heme Synthesis and Metabolism Substances or chemicals that induce ALAS activity in erythroid and non-erythroid cells are also distinct. For example, ALAS activity in the liver is strongly induced by treatment of animals [40] or hepatocytes in culture [4, 61 with 3,5-dicarbethoxy- 1,4-dihydrocollidine, while this chemical does not influence ALAS activity in erythroid cells [40]. Conversely, hypoxia or erythropoietin treatment increases ALAS activity in erythroid cells without affecting hepatic enzyme activity [40]. These findings indicate that the genes encoding the two ALAS isozymes are under separate controls and suggest that, while ALAS-N in erythroid cells is under feedback control by heme similar to that in the liver, the ALAS-E gene is up-regulated by heme and is responsible for increased heme synthesis in developing erythroid cells. b-a tn irzolevu /inate De hyd ratase (ALAD) [EC There are two alternative transcripts for ALAD, which contain either exon 1A or 1B, that are spliced to exon 2 where the coding region begins [41]. Exon 1A is transcribed in all tissues, while exon 1B is transcribed only in erythroid tissues. This is an interesting finding since the ALAD proteins encoded by the two-tissue specific transcripts are identical, and yet the regulation of mrna expression is under tissue-specific control. These findings indicate that there are erythroid-specific and non-specific transcripts of ALAD, as in the case of ALAS and PBGD, that they are likely be regulated in a tissue-specific manner, and that up-regulation of the erythroid-specific genes is probably accomplished via the transcription factor, GATA-1. Porplzobilinogeri Deaminase (PBGD) [EC In addition to ALAS, two distinct molecular forms of PBGD have been identified [42, 431. The larger form (44 kda) corresponds to a nonerythroid isoform of PBGD (PBGD-N), while the smaller form (42 kda) represents an erythroid-specific isoform of the enzyme (PBGD- E). Analysis of cell-free translation products directed by mrna from human erythropoietic spleen and from human liver showed that the two isoforms of PBGD were encoded by distinct mrna. Comparison of the sequences from human PBGD-E mrna [44] to that of PBGD-N mrna [43] revealed a 1,320 bp stretch of perfect identity, but with a mismatch in the first exon at their 5 extremities. An additional inframe AUG codon was found 51 bp upstream from the initiating codon of PBGD-E cdna. Thus an additional 17 amino acid residues occurred at the N-terminus of PBGD-N, which accounted for its higher molecular mass. It was also shown that the expression of PBGD-E mrna is exclusive to erythroid cells. Further studies of the cloned human PBGD gene demonstrated that the gene is split into 15 exons spread over 10 kb of DNA [45]. The two distinct mrna were produced through alternative splicing of two primary transcripts arising from two promoters. The upstream promoter was active in all tissues, and thus the enzyme encoded by the larger transcript was termed the housekeeping PBGD. The other promoter, located 3 kb downstream, was active only in erythroid cells. It displayed structural homology with the p- globin gene promoter, suggesting that some common trans-acting factors may coregulate the transcription of these genes during erythroid development [46]. It was found that two erythroid-specific trans-acting factors recognized sequences in the PBGD erythroid promoter [35, 361. One of these factors was GATA-1, which bound three sites located upstream (-180 bp and -70 bp) and downstream (+45 bp) of the initiation site. A sequence which matched the consensus CAC box found upstream of the P-globin gene cap site in multiple species was found twenty bases upstream from the GATA-1 binding site. The second erythroid-specific factor (NF-E2) recognized a sequence located at -160 bp. Point mutation and deletion studies suggested that GATA-1 and NF-E2 are necessary for correct regulation of this promoter in erythroid cells. Tissue specific expression of the molecular defect of acute intermittent porphyria (AIP) has been recognized. Studies of the PBGD gene locus showed that AIP mutations were associated with a different restriction haplotype [47- SO]. In the majority of patients with AIP, PBGD activity is decreased by 50% compared with normals. However, in some families, the enzyme activity is decreased in non-erythroid cells, but not in erythroid cells. In one of these families, a G - A transition was observed at the first position of the first intron. This modified the normal splice consensus sequence CGGTGAGT to CGATGAGT. In another family, a different

5 Sassa 5 point mutation, i.e., a single base substitution (G + T), was found within the non-erythropoietic exon [51], at the last position of exon 1, which also led to a splicing defect. Other Heme Pathway Enzymes Situations with respect to other enzymes in the heme biosynthetic pathway are less clear. No erythroid-specific transcript has been identified for uroporphyrinogen (Uro ) 111 cosynthase and Uro decarboxylase (Uro D). However, the level of Uro D mrna in erythroid cells appears to be higher than in the liver [52]. The human gene encoding ferrochelatase, the terminal enzyme in the heme biosynthetic pathway, contains a potential binding site for Spl, NF-E2 and GATA-1 [53]. Consistent with this finding, some erythroid-specific expression, characterized by the preferential utilization of a polyadenylation signal, has been suggested in MEL cells [54]. Coproporphyrinogen oxidase cdna has recently been cloned from a MEL cell library [55]. A single transcript of the oxidase is expressed both in erythroid and in non-erythroid cells. cdna cloning for the remaining enzyme in the heme biosynthetic pathway, i.e., protoporphyrinogen oxidase, has not been reported. Hepatic Versus Erythropoietic Porphyrias Inherited gene defects of heme pathway enzymes, except ALAS, are collectively called the porphyrias. Porphyrias are classified as either hepatic or erythropoietic, depending on the principal site of expression of the effect of the specific gene defect. Although the ALAS defect is not called a porphyria (since it does not accompany increased synthesis, nor excretion of porphyrins, nor their precursors), a defective ALAS-E gene has been described in patients with X-linked sideroblastic anemia [34]. It has never been sufficiently explained, however, why patients with a hepatic porphyria, e.g., AIP, have normal erythroid heme synthesis. For example, patients with AIP or hereditary coproporphyria had normal hemoglobin synthesis, despite the fact that their hepatic heme synthesis was markedly deranged [7]. As discussed above, many of these distinctive features of heme synthesis can now be ascribed to tissue-specific isozymes and their tissue-specific regulation [23]. Evidence also suggests that expression of the erythroid-specific heme pathway enzyme genes is critical in erythroid heme formation. For example, point mutations in the ALAS-E gene were described in patients with X-linked sideroblastic anemia, which appear to account for the disturbances of heme synthesis only in erythroid cells in this disorder [34]. In MEL cells, the ALAS-E gene is up-regulated, whereas the ALAS-N gene is down-regulated when cells are induced to undergo erythroid cell differentiation. A dimethyl sulfoxide (DMS0)-resistant mutant clone of MEL cells, termed DR, is defective in hemoglobin synthesis and in erythroid cell differentiation [ 141. The DR defect appears to be restricted to the lack of expression of ALAS-E mrna, since other heme pathway enzymes are all expressed and up-regulatable by DMSO [14]. In DR cells, ALAS-N mrna is also expressed, but is down-regulated by DMSO treatment as it is in DMSO-sensitive cells [12, 14, 561. These findings suggest that the expression and the up-regulation of erythroid-specific heme pathway enzymes are critical to increased heme formation during erythroid cell differentiation. The isozymic nature of certain enzymes also suggests that there may be tissue-specific expression of their gene defects. Recently a spontaneous form of erythropoietic protoporphyria (EPP) in the house mouse has been described [57]. The mouse form of EPP is a viable autosomal recessive mutation causing jaundice, hemolytic anemia and photosensitivity. Ferrochelatase activity is % of normal. The availability of an animal model of porphyria which is amenable to various experimental manipulations should be very useful for a trial of gene therapy. The Role of Heme Oxygenase in Heme Catabolism, Host Defense and cgmp Production Enzymes in the heme catabolic pathway consist of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase (CR), microsomal heme oxygenase (HO) and biliverdin reductase (BR). In animal cells, heme is converted to biliverdin IXa by the combined enzymatic actions of CR and HO. Biliverdin IXa is then reduced to bilirubin IXa by BR. This sequence of reactions requires NADPH and molecular oxygen. cdna for all these enzymes have been cloned and characterized. Microsomal heme oxygenase (HO) [EC is the key enzyme in the heme

6 6 Recent Progress in Heme Synthesis and Metabolism catabolic sequence in that it determines the mode of heme cleavage at the a-methene bridge. and it is the rate-limiting enzyme in this sequence [58]. There are two isozymes of HO, i.e., HO-1 and HO-2. HO-1 activity can be induced in many cell types by treatment with hemin, is., the substrate for the enzyme, as well as with non-heme substances [59]. For example. the enzyme is known to be inducible with treatment of animals or cells by various trace metals [60-631, and by oxidative stress such as UVA radiation, hydrogen peroxide and sodium arsenite [64]. cdna for HO-1 has been cloned from a rat spleen expression library by immunoscreening [65]. The primary structure of rat HO-1 deduced from the nucleotide sequence of cdna consisted of a protein with 289 amino acids with a M, of 33,000. Human HO-1 cdna has also been cloned and characterized [66]. Human HO-1 was found to be composed of 288 amino acids with a M, of 32,800. The degree of sequence homology between the rat and the human HO- I was = 80%. Both rat and human HO-1 contained a putative membrane segment at the carboxyl-terminus which was enriched in hydrophobic amino acids. The rat HO-1 gene was composed of 6,830 nucleotides and organized into four introns and five exons [67]. Its 5'-flanking region contained a number of DNA sequences of potential regulatory significance, including a heat shock element (HSE) [68]. By studying the expression of a chimeric gene which contained the promoter of rat HO-1 gene and Escherichia coli gene gpt, Slzibahara et al. [69] demonstrated that the 5'-flanking region bearing a HSE of the rat HO- 1 gene confers heat inducibility on the expression of gpt. In contrast, treatment with hemin did not increase gpt transcript expression, suggesting that heat and hemin may induce HO-1 by two different mechanisms. Although the human HO-1 gene also contained a potential HSE, it was originally thought that the human HO-1 gene was not activated by heat [66]. However, more recently, HO-1 mrna was shown to be inducible in human fibroblasts [64], and in human Hep3B hepatoma cells [70], suggesting that the human HO-1 may behave as a heat shock protein (HSP) in certain human cell lines. In Hep3B cells, but not in HepG2 hepatoma cells that do not show a heat-mediated HO-1 induction, a transcriptional activation of a heat-inducible nuclear factor was demonstrated, which binds specifically to the HSE in the human HO-1 gene [71]. This factor may therefore be involved, via binding to the HSE, in the activation of the human HO-1 gene in response to heat treatment. In addition to the HSE, the 5'-flanking region of the rat HO-1 gene contains GCN4- binding sites. GCN4 is a positive regulator for the gene for amino acid biosynthesis in yeast. It has been shown that there are certain functional similarities among the jun oncoproteins, AP-1 (a mammalian activating protein) and GCN4 [67]. Thus, it is possible that HSE and GCN4-binding sites in the rat HO-1 gene may function in activating transcription during stress reactions including heat shock and amino acid deprivation. In this respect, it is also interesting to note that HO-1 mrna has been shown to increase in human hepatoma cells in response to interleukin-6 (hil-6), an acute-phase inducer [70, 721. A nuclear factor was also demonstrated both in untreated and in the hil-6 treated Hep3B cells, which binds specifically to the IL-6 responsive element (IL6-RE) of the human HO- 1 gene. In human hepatoma cells, DMSO also elicited an acute-phase-like reaction [73], followed by an induction of HO-1 mrna [70]. These findings suggest that HO-1 is a positive acute-phase reactant which may be involved in host defense mechanisms [72, 741, and that the nuclear factor specific to the IL6-RE may be involved in the activation of the HO-1 gene during acute inflammation. In support of this conclusion, NFkB and AP-2-like binding sites, important regulatory sites in inflammation, have recently been reported in the 5'-untranslated region of the human HO-1 gene [75]. In contrast to the liver-derived cells, HO-1 mrna in erythroid cells, such as differentiating MEL cells, was found to rapidly decrease after DMSO treatment. Since differentiated MEL cells contain a considerable amount of heme, this finding may suggest that the regulation of HO-1 in erythroid cells is different. The downregulation of HO-1 mrna, as well as HSP70 mrna, another housekeeping protein, proceeded significantly earlier than the up-regulation of erythroid-specific genes [19]. Thus it was speculated that HO-1 may be involved in the cellular events that determine the onset of terminal cell differentiation [19].,,

7 Sassa 7 HO-2 cdna has also been isolated and characterized. The cdna for HO-2 encoded a protein with 315 amino acids corresponding to a M, of 35,757 and was reported to show a high HO activity when expressed in E. coli [76,77]. One of the products of the heme oxygenase reaction is carbon monoxide (CO), and this enzymatic reaction is the only known source of CO production in mammalian cells [78]. CO has recently been proposed to be a potential regulator of cyclic guanosine monophosphate (cgmp) production in the brain [79]. This hypothesis is based on a) the chemical interaction of CO with the heme moiety of guanylate cyclase, the enzyme which produces cgmp upon binding of its heme moiety to NO (or possibly to CO), b) the colocalization of guanylate cyclase with HO-1 and HO-2, c) the correlation between HO activity and cgmp levels, and d) the ability of CO to modulate cgmp levels [80]. Although this hypothesis has yet to be tested, it is interesting to note that CO may act as a retrograde message for long-term potentiation (LTP) in the hippocampus [81, 821. Thus it is reasonable to suggest that the role of HO is not only for the catabolism of heme, but also for the regulation of other fundamental functions such as heat-shock and acute-phase responses and, perhaps, cgmp production in the brain. Conclusion Recent progress in the molecular biology of heme pathway enzyme genes has elucidated a number of new aspects in the regulation of these genes in normal cells and in abnormal conditions such as the porphyrias. The availability of molecular probes, both normal and abnormal, has not only contributed significantly to a better understanding of the pathogenesis of the human porphyrias, but also opened up the possibility of gene therapy of the inherited porphyrias. Acknowledgments This work was supported in part by grants from U.S.P.H.S. DK and DK References 1 Sassa S. Heme stimulation of cellular growth and differentiation. Semin Hematol 1988;25: Abraham NG, Levere RD, Lutton JD. Eclectic mechanism of heme regulation of hematopoiesis. Int J Cell Cloning 1991;9: Ponka P, Schulman HM. Regulation of heme biosynthesis: distinct regulatory features in erythroid cells. Stem Cells 1993;11(suppl 1): Granick S. The induction in vitro of the synthesis of 6-aminolevulinic acid synthetase in chemical porphyria: a response to certain drugs, sex hormones, and foreign chemicals. J Biol Chem 1966;241: Granick S, Sinclair P, Sassa S, Grieninger G. Effects by heme, insulin, and serum albumin on heme and protein synthesis in chick embryo liver cells cultured in a chemically defined medium, and a spectrofluorometric assay for porphyrin composition. J Biol Chem 1975;250: Sassa S, Granick S. Induction of 6-aminolevulinic acid synthetase in chick embryo liver cells in culture. Proc Natl Acad Sci USA 1970;67: Kappas A, Sassa S, Galbraith RA, Nordmann Y. The Porphyrias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease. Ed 6. New York: McGraw- Hill Book Co., 1989: Sassa S, Kappas A, Bernstein SE, Alvares AP. Heme biosynthesis and drug metabolism in mice with hereditary hemolytic anemia. J Biol Chem 1979;254: Sassa S, Takaku F, Nakao K. Regulation of erythropoiesis in the Friend leukemia mouse. Blood 1968;31: Sassa S. Control of heme biosynthesis in erythroid cells. In: Rossi GB, ed. In Vitro and In Vivo Erythropoiesis: The Friend System. Amsterdam: Elsevier/North-Holland Biochem Press, 1980: Granick JL, Sassa S. Hemin control of heme biosynthesis in mouse Friend virus-transformed erythroleukemia cells in culture. J Biol Chem 1978;253: Fujita H, Yamamoto M, Yamagami T, Hayashi N, Bishop TR, de Verneuil H, Yoshinaga T, Shibahara S, Morimoto R, Sassa S. Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Biochim Biophys Acta 1991;1090: Hoffman R, Ibrahim N, Murnane MJ, Diamond A, Forget BG, Levere RD. Hemin control of heme biosynthesis and catabolism in a human leukemia cell line. Blood 1980;56:

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