Hiroyoshi Fujita

Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells

Changes in the level of transcripts encoding enzymes of the heme biosynthetic pathway as well as those encoding ubiquitous proteins were examined in murine Friend virus-transformed erythroleukemia cells during erythroid cell differentiation induced by chemicals including dimethyl sulfoxide (DMSO). Early changes following DMSO treatment were marked decreases in mRNAs for three ubiquitous proteins, i.e., a 70 kDa heat shock protein (less than 6 h), heme oxygenase and nonspecific delta-aminolevulinate synthase (ALAS) (less than 12 h). These changes were followed by sequential increases in mRNAs for enzymes in the heme biosynthetic pathway. Namely, mRNAs for the erythroid-specific ALAS, delta-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase started to increase at 12, 18, 18-24 and 24 h, respectively. Nuclear runoff studies revealed that these changes are largely transcriptional. Treatments with other inducers of erythroid differentiation, e.g., hexamethylene bisacetamide, n-butyric acid and N-methylnicotinamide, also showed similar effects on mRNAs as those following DMSO. These findings suggest that both suppression of ubiquitous genes and activation of heme pathway enzyme genes are associated with erythroid differentiation, and the former occurs preceding changes in the latter.

Heat shock induction of heme oxygenase mRNA in human Hep 3B hepatoma cells

Heat shock treatment of human Hep 3B hepatoma cells led to the induction of mRNA for microsomal heme oxygenase. The maximum induction of heme oxygenase mRNA (5----7-fold) was observed with treatment of cells at 43.5 degrees C, for 60 min. The heat-mediated induction of heme oxygenase mRNA was blocked by simultaneous treatment of cells with actinomycin D or cycloheximide. In contrast to Hep 3B cells, cells of another human hepatoma line, Hep G2, showed little induction of heme oxygenase mRNA by heat treatment. These findings suggest that heat shock treatment induces heme oxygenase mRNA in certain human hepatoma cells, but not in others.

Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells

Effects of various stresses were examined on the accumulation of mRNA for microsomal heme oxygenase and a heat shock protein, hsp70, in three human hepatoma cell lines. By heat shock, hsp70 mRNA was induced in all three hepatoma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was increased only in Hep 3B. Time-courses of the heat shock induction of both mRNAs in Hep 3B were similar. Arsenite caused induction of both mRNAs in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and heme oxygenase are heat shock proteins, the mode of induction of mRNAs for these proteins is different.

The rapid and decremental change in haem oxygenase mRNA during erythroid differentiation of murine erythroleukaemia cells.

Changes in mRNA for haem oxygenase (HO), the rate‐limiting enzyme for haem catabolism, were examined in murine Friend‐virus transformed erythroleukaemia (MEL) cells while they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulfoxide (DMSO). When MEL cells were treated with 1.5% (v/v) DMSO, a rapid decrease in HO mRNA content was observed (< 12 h) which reached the lowest value at 18 h (18% of the untreated control). HO mRNA levels remained at substantially lower levels (< 50%) than those in untreated controls thereafter. A rapid decline in HO mRNA may be involved in the cellular events that determine the onset of erythroid differentiation.

Dimethyl sulphoxide and haemin induce ferrochelatase mRNA by different mechanisms in murine erythroleukaemia cells

The level of mRNA encoding ferrochelatase (FeC), the terminal enzyme of the haem biosynthetic pathway, was examined in murine erythroleukaemia (MEL) cells when they were induced to undergo erythroid cell differentiation by treatment with dimethyl sulphoxide (DMSO), or haemin. FeC mRNA increased within 12 h after DMSO or haemin treatment of MEL cells, and its level continued to increase for 48 h. Treatment of cells with succinylacetone (SA), a potent inhibitor of haem synthesis, suppressed a DMSO‐mediated increase in FeC mRNA, and haemin treatment reversed a SA‐mediated decrease in FeC mRNA. Nuclear runoff analyses showed that, while DMSO increased the rate of transcription of FeC mRNA, haemin did not. These results indicate that the induction of FeC mRNA by DMSO is largely transcriptional, while that by haemin is post‐transcriptional.

Message amplification phenotyping of an inherited δ-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the probands cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the probands cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.

Haem is necessary for a continued increase in ferrochelatase mRNA in murine erythroleukaemia cells during erythroid differentiation

Summary The level of mRNA encoding ferrochelatase (FeC) was examined in two murine erythroleukaemia (MEL) clones, DS and DR, a DMSO‐sensitive, and a DMSO‐resistant clone, respectively. DS cells undergo erythroid differentiation by DMSO treatment with a marked increase in haem synthesis, while DR cells fail to do so due to the lack of the erythroid‐specific δ‐aminolaevulinate synthase (ALAS‐E). Both DS and DR cells showed an increase in the level of FeC mRNA within 18 h of DMSO treatment. The level of FeC mRNA in DR cells was then decreased, while that in DS cells continued to increase for 72 h. Treatment with haemin significantly increased FeC mRNA in DR cells. When cells were treated with both DMSO and haemin, the level of FeC mRNA in DR cells increased to a level comparable to that in DS cells. These findings suggest that the failure to maintain increased FeC mRNA DR cells after DMSO treatment may be due to a deficiency of haem in these cells.

Hereditary Hepatic Porphyria Due To Homozygous Delta-aminolevulinic-acid Dehydratase Deficiency - Studies in Lymphocytes and Erythrocytes

Abstract. Activities of δ‐aminolevulinic acid (ALA) dehydratase and porphobilinogen (PBG) deaminase, and haem content were determined in EB‐virus transformed lymphocytes from two patients with homozygous ALA dehydratase deficiency, and their family members to determine the expression of the specific gene defect in this cell type. ALA dehydratase activity, but not PBG deaminase activity or haem content, was markedly decreased in lymphocyte preparations from both patients with homozygous enzyme deficiency, and moderately decreased in subjects heterozygous for enzyme deficiency. Immunochemical quantitation of erythrocyte ALA dehydratase suggested the presence of a cross‐reactive material in a patient with a late‐onset of acute hepatic porphyria due to the homozygous enzyme deficiency.

Regulation of heme synthesis in HepG2 human hepatoma cells by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) treatment of human HepG2 hepatoma cells increases the activity and the concentration of delta-aminolevulinic acid dehydratase (ALAD) to a comparable degree. Results of experiments with transcriptional inhibitors suggest that the increase in ALAD reflects de novo synthesis of the enzyme resulting from transcriptional activation. Commitment to increased ALAD activity in HepG2 cells is seen after 18 hr and complete by 48 hr. In contrast to the effects on ALAD, DMSO decreases the activities of porphobilinogen deaminase and uroporphyrinogen decarboxylase.

Genetic Regulation of the Heme Pathway

Genetic regulation of the heme pathway can best be illustrated by the porphyrias. The porphyrias are inherited and acquired disorders characterized by overproduction of porphyrins and their precursors, and are invariably associated with abnormalities in the activities of specific enzymes of the heme biosynthetic pathway. These diseases are classified as either hepatic or erythropoietic depending on the principal site of expression of the enzymatic defect in each disorder. Because the bone marrow and liver are the two major organs involved in heme biosynthesis, it is perhaps not surprising that porphyrias primarily express themselves in these organs. In almost ail porphyrias, environmental factors, such as lead, polyhalogenated aromatic hydrocarbons, and nonheritable metabolic factors, play a significant role in determining the clinical expression of the primary gene defect. In this respect, porphyrias are the paradigm of disorders in which the interactions between genetic factors and environmental chemicals are critically involved in the pathogenesis of human disease. Because it is not possible to discuss the genetic regulation of all enzymes in the heme pathway within the limited space available, we will describe the recent findings from our laboratory on the genetic and chemical modulation of uroporphyrinogen decarboxylase, and its implications in porphyria cutanea tarda (PCT). In this study, we examined both the enzyme activity and the enzyme protein content in erythrocytes from normal subjects, patients with PCT, and patients with hepatoerythropoietic porphyria (HEP) using a monospecific antibody against normal uroporphyrinogen decarboxylase. In addition, we examined a spectrum of polychlorinated biphenyls (PCBs), a group of toxic environmental chemicals that cause experimental PCT in animals and in isolated chick embryo hepatocyte cultures, and analyzed the structure-activity relationships of PCB and uroporphyrin accumulation.